WO2023278502A1 - Renal health determination and uses thereof - Google Patents

Renal health determination and uses thereof Download PDF

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WO2023278502A1
WO2023278502A1 PCT/US2022/035416 US2022035416W WO2023278502A1 WO 2023278502 A1 WO2023278502 A1 WO 2023278502A1 US 2022035416 W US2022035416 W US 2022035416W WO 2023278502 A1 WO2023278502 A1 WO 2023278502A1
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twenty
thirty
protein
level
measuring
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PCT/US2022/035416
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French (fr)
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Laura SAMPSON
Missy SIMPSON
Rachel Ostroff
Jeremy PRIMUS
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Somalogic Operating Co., Inc.
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Publication of WO2023278502A1 publication Critical patent/WO2023278502A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present application relates generally to the detection of biomarkers and methods of evaluating the current state of kidney function in an individual and, more specifically, to one or more biomarkers, methods, devices, reagents, systems, and kits used to evaluate an individual’s kidney health based on glomerular filtration rate (GFR).
  • GFR glomerular filtration rate
  • Chronic kidney disease is defined as having abnormalities of kidney structure or function present for > 3 months (Table 1) and affects approximately 13% of adults in the US; risk factors for the disease are heterogeneous and include genetic and demographic predisposition and diabetes.
  • Kidneys serve three primary functions: they filter metabolic byproducts from the blood, produce urine and, in so doing, help to regulate blood pressure and fluid and electrolyte balance, and they have important endocrine functions.
  • the eGFR is used to screen for early kidney damage, as one of the diagnostic modalities in chronic kidney disease (CKD), and to monitor kidney status. It is recommended that an eGFR be calculated every time a creatinine blood test is done.
  • Serum creatinine is ordered frequently as part of routine blood chemistry and it can vary because of many factors including hydration status, muscle damage, and kidney health. Because serum creatinine originates from extra-renal factors, two low eGFR values ( ⁇ 60 ml/min/1.73 m 2 ) a few months apart are more indicative of impaired renal function then an isolated low value.
  • eGFR The standard means to monitor kidney function in the clinical setting is eGFR, which reflects the amount of blood that passes through the kidneys each minute.
  • eGFR The standard means to monitor kidney function in the clinical setting.
  • the present application discloses biomarkers, methods, devices, reagents, systems, and kits to evaluate an individual’s renal health based on eGFR or a determination of glomerular filtration rate (GFR).
  • the current state kidney function test disclosed herein is intended to estimate or determine glomerular filtration rate (GFR) from a blood sample measurement and provide current status of kidney health.
  • the current state kidney function test can be used as part of a broader liquid health check as a convenient way to monitor kidney function and identify potential signs of early functional impairment (i.e. potential chronic kidney disease).
  • the current state kidney function test provides a convenient estimate of kidney function as part of a liquid health check and serves as a tool for health care providers to identify candidates for additional screening for potential functional impairment.
  • An additional advantage of the current state kidney function test disclosed herein is that it does not require patient characteristics such as sex, age, and race as inputs.
  • a method comprising: a) measuring the level of TMEDA protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonu
  • a method comprising: a) measuring the level of ARMEL protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonu
  • a method comprising: a) measuring the level of CAC02 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonucle
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of NAGPA protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHR1, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribon
  • GFR glomerular filtration rate
  • Cystatin C and ARMEL TMEDA and ARMEL, DSC2 and ARMEL, HPRT and ARMEL,
  • FABP and ARMEL PPIC and ARMEL, GBRAP and ARMEL, ISK7 and ARMEL, FSH and ARMEL, RBP and ARMEL, HE4 and ARMEL, C01A1 and ARMEL, PGRP-L and ARMEL, ERBBl and ARMEL, Testican-2 and ARMEL, FHR1 and ARMEL, SRC A and ARMEL, CAC02 and ARMEL, NAGPA and ARMEL, DLK1 and ARMEL, SLIT2 and ARMEL, HCC-1 and ARMEL, CDON and ARMEL, COFA1 and ARMEL, PEAR1 and ARMEL, Ribonuclease UK1 14 and ARMEL, Activin A and ARMEL, Heparin cofactor II and ARMEL, SUMF1 and ARMEL, MP2K4 and ARMEL, SELW and ARMEL, HYALl and ARMEL, a 1 -Antitrypsin and ARMEL,
  • MP2K4 and CAC02 SELW and CAC02, HYALl and CAC02, al -Antitrypsin and CAC02, MMP-7 and CAC02, QSOX2 and CAC02, IGDC4 and CAC02, S100A6 and CAC02, ATS 13 and CAC02.
  • Cystatin C and NAGPA TMEDA and NAGPA, DSC2 and NAGPA, HPRT and NAGPA,
  • FABP and NAGPA PPIC and NAGPA, GBRAP and NAGPA, ISK7 and NAGPA, ARMEL and NAGPA, FSH and NAGPA, RBP and NAGPA, HE4 and NAGPA, COl Al and NAGPA, PGRP-L and NAGPA, ERBBl and NAGPA, Testican-2 and NAGPA, FHRl and NAGPA, SRCA and NAGPA, CAC02 and NAGPA, DLK1 and NAGPA, SLIT2 and NAGPA, HCC-1 and NAGPA, CDON and NAGPA, COFA1 and NAGPA, PEARl and NAGPA, Ribonuclease UK1 14 and NAGPA, Activin A and NAGPA, Heparin cofactor II and NAGPA, SUMFl and NAGPA, MP2K4 and NAGPA, SELW and NAGPA, HYALl and NAGPA, al -Antitrypsin and NAGPA, MMP-7 and
  • TMEDA and ARMEL and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2,
  • TMEDA and CAC02 and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1,
  • TMEDA and NAGPA at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1,
  • ARMEL and CAC02 and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1,
  • ARMEL and NAGPA and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1,
  • CAC02 and NAGPA and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, DLK1,
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising TMEDA protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA,
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising ARMEL protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHRl, S
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising CAC02 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, F
  • UK114 Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising NAGPA protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2,
  • Cystatin C and ARMEL TMEDA and ARMEL, DSC2 and ARMEL, HPRT and ARMEL,
  • FABP and ARMEL PPIC and ARMEL, GBRAP and ARMEL, ISK7 and ARMEL, FSH and ARMEL, RBP and ARMEL, HE4 and ARMEL, COIAI and ARMEL, PGRP-L and ARMEL, ERBB1 and ARMEL, Testican-2 and ARMEL, FHR1 and ARMEL, SRC A and ARMEL, CAC02 and ARMEL, NAGPA and ARMEL, DLK1 and ARMEL, SLIT2 and ARMEL, HCC-1 and ARMEL, CDON and ARMEL, COFA1 and ARMEL, PEAR1 and ARMEL, Ribonuclease UK1 14 and ARMEL, Activin A and ARMEL, Heparin cofactor II and ARMEL, SUMF1 and ARMEL, MP2K4 and ARMEL, SELW and ARMEL, HYALl and ARMEL, a 1 -Antitrypsin and ARMEL, M
  • MP2K4 and CAC02 SELW and CAC02, HYALl and CAC02, al -Antitrypsin and CAC02, MMP-7 and CAC02, QSOX2 and CAC02, IGDC4 and CAC02, S100A6 and CAC02, ATS 13 and CAC02.
  • Cystatin C and NAGPA TMEDA and NAGPA, DSC2 and NAGPA, HPRT and NAGPA,
  • FABP and NAGPA PPIC and NAGPA, GBRAP and NAGPA, ISK7 and NAGPA, ARMEL and NAGPA, FSH and NAGPA, RBP and NAGPA, HE4 and NAGPA, COIAI and NAGPA, PGRP-L and NAGPA, ERBB1 and NAGPA, Testican-2 and NAGPA, FHR1 and NAGPA, SRCA and NAGPA, CAC02 and NAGPA, DLK1 and NAGPA, SLIT2 and NAGPA, HCC-1 and NAGPA, CDON and NAGPA, COFA1 and NAGPA, PEAR1 and NAGPA, Ribonuclease UK1 14 and NAGPA, Activin A and NAGPA, Heparin cofactor II and NAGPA, SUMFl and NAGPA, MP2K4 and NAGPA, SELW and NAGPA, HYALl and NAGPA, al -Antitrypsin and NAGPA, MMP-7 and
  • TMEDA and ARMEL and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2,
  • TMEDA and CAC02 and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1,
  • TMEDA and NAGPA at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1,
  • ARMEL and CAC02 and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1,
  • ARMEL and NAGPA and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1,
  • CAC02 and NAGPA and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, DLK1, SLIT2,
  • a method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a ARMEL protein; and b) measuring the level of each protein with the two capture reagents.
  • a method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a CAC02 protein; and b) measuring the level of each protein with the two capture reagents.
  • a method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
  • a method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a ARMEL protein and the second capture reagent has affinity for a CAC02 protein; and b) measuring the level of each protein with the two capture reagents.
  • a method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a ARMEL protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
  • a method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a CAC02 protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
  • any one of aspects 38 to 56 further comprising measuring the level of a Testican-2 protein with a capture reagent having affinity for the Testican-2 protein.
  • [0067] 58 The method of any one of aspects 38 to 57, further comprising measuring the level of a FHR1 protein with a capture reagent having affinity for the FHR1 protein.
  • a method comprising: a) measuring the level of TMEDA and ARMEL in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and ARMEL.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of TMEDA and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and CAC02.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of TMEDA and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and NAGPA.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of ARMEL and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and CAC02.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of ARMEL and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and NAGPA.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of CAC02 and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of CAC02 and NAGPA.
  • GFR glomerular filtration rate
  • [00130] 120 The method of any one of aspects 79 to 119, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
  • 121 A method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from TMEDA, ARMEL and CAC02; and b) measuring the level of each protein with the three capture reagents.
  • a method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from TMEDA, ARMEL and NAGPA; and b) measuring the level of each protein with the three capture reagents.
  • a method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from ARMEL, CAC02 and NAGPA; and b) measuring the level of each protein with the three capture reagents.
  • [00135] 125 The method of any one of aspects 121 to 124, further comprising measuring the level of a DSC2 protein with a capture reagent having affinity for the DSC2 protein.
  • 131 The method of any one of aspects 121 to 130, further comprising measuring the level of a FSH protein with a capture reagent having affinity for the FSH protein.
  • 132 The method of any one of aspects 121 to 131, further comprising measuring the level of a RBP protein with a capture reagent having affinity for the RBP protein.
  • 133 The method of any one of aspects 121 to 132, further comprising measuring the level of a HE4 protein with a capture reagent having affinity for the HE4 protein.
  • 134 The method of any one of aspects 121 to 133, further comprising measuring the level of a COl A1 protein with a capture reagent having affinity for the COl A1 protein.
  • [00160] 150 The method of any one of aspects 121 to 149, further comprising measuring the level of a MP2K4 protein with a capture reagent having affinity for the MP2K4 protein.
  • [00161] 151 The method of any one of aspects 121 to 150, further comprising measuring the level of a SELW protein with a capture reagent having affinity for the SELW protein.
  • [00162] 152 The method of any one of aspects 121 to 151, further comprising measuring the level of a HYAL1 protein with a capture reagent having affinity for the HYAL1 protein.
  • a method compri sing : a) measuring the level of TMEDA, ARMEL and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA, ARMEL and CAC02.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of TMEDA, ARMEL and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA, ARMEL and NAGPA.
  • GFR glomerular filtration rate
  • a method comprising: a) measuring the level of ARMEL, CAC02 and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL, CAC02 and NAGPA.
  • GFR glomerular filtration rate
  • [00175] 165 The method of any one of aspects 159 to 164, further comprising measuring the level of a FABP protein.
  • a method comprising: a) measuring the level of at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty-five, thirty-six, thirty- seven, thirty-eight proteins, or thirty-nine proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PG
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, thirty-eight proteins, or thirty-nine proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC,
  • GBRAP GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
  • a method comprising: a) measuring the level of Cystatin C protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC- 1, CDON, COFA1, PEAR
  • a method comprising: a) measuring the level of DSC2 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C , TMEDA, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl
  • a method comprising: a) measuring the level of HPRT protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR
  • a method comprising: a) measuring the level of FABP protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl,
  • a method comprising: a) measuring the level of PPIC protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl
  • a method comprising: a) measuring the level of GBRAP protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ri
  • a method comprising: a) measuring the level of ISK7 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising Cystatin C protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1,
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising DSC2 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C , TMEDA, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, F
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising HPRT protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, S
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising FABP protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising PPIC protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, F
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising GBRAP protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA
  • a method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising ISK7 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, F
  • estimating or determining GFR for the human subject is based on input of the level of each protein measured in a statistical model.
  • model output is an estimation or determination of GFR as a continuous integer value from 5 to 100 ml/min/1.73 m 2 , inclusive.
  • GFR glomerular filtration rate
  • the model estimates or determines glomerular filtration rate (GFR) for the human subject based on the level of each protein measured selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYAL1, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
  • GFR glomerular filtration rate
  • Figure 1 shows a concordance plot of true eGFR values vs. estimated values of validation data.
  • Figure 2 illustrates an exemplary computer system for use with various computer-implemented methods described herein.
  • Figure 3 is a flowchart for a method of estimating or determining glomerular filtration rate in accordance with one embodiment.
  • the term “about” represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.
  • Adaptive Normalization by Maximum Likelihood refers to a process for normalizing analytes to mitigate site bias.
  • the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.
  • the present application includes biomarkers, methods, devices, reagents, systems, and kits for the estimation or determination of the current state of kidney function.
  • Progressive Chronic Renal Insufficiency or “PCRI” or “renal insufficiency” means a composite endpoint which is treated as a classification endpoint (yes/no within a given time frame) as defined by the development of at least one of the following within the time frame from test results:
  • End Stage Renal Disease or “ESRD” means that at least one of the following conditions are met: glomerular filtration rate is less than 15 ml/min/1.73 m 2 , chronic renal dialysis is needed, or kidney transplantation is needed.
  • Relative risk means the risk for developing PCRI in a given time frame as compared to the average risk in a reference population.
  • the range for relative risk is 0.01-3.24.
  • relative risk can be calculated
  • RR — q wherein p* is the probability that an individual develops PCRI within 4 years and q is the probability for the baseline individual in a training cohort.
  • Biological sample “sample”, and “test sample” are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained or otherwise derived from an individual. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, huffy coat, plasma, and serum), dried blood spots (e.g., obtained from infants), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid.
  • blood including whole blood, leukocytes, peripheral blood mononuclear cells, huffy coat, plasma, and serum
  • dried blood spots e
  • a blood sample can be fractionated into serum, plasma or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes).
  • a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample.
  • biological sample also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example.
  • biological sample also includes materials derived from a tissue culture or a cell culture.
  • exemplary methods include, e.g., phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsy procedure.
  • tissue susceptible to fine needle aspiration include lymph node, lung, lung washes, BAL (bronchoalveolar lavage), thyroid, breast, pancreas and liver.
  • Samples can also be collected, e.g., by micro dissection (e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)), bladder wash, smear (e.g., a PAP smear), or ductal lavage.
  • micro dissection e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)
  • LMD laser micro dissection
  • bladder wash e.g., smear, a PAP smear
  • smear e.g., a PAP smear
  • ductal lavage
  • a “biological sample” obtained or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual. [00261] Further, it should be realized that a biological sample can be derived by taking biological samples from a number of individuals and pooling them or pooling an aliquot of each individual’s biological sample.
  • the biological sample can be urine.
  • Urine samples provide certain advantages over blood or serum samples. Collecting blood or plasma samples through venipuncture is more complex than is desirable, can deliver variable volumes, can be worrisome for the patient, and involves some (small) risk of infection. Also, phlebotomy requires skilled personnel. The simplicity of collecting urine samples can lead to more widespread application of the subject methods.
  • the phrase “data attributed to a biological sample from an individual” is intended to mean that the data in some form derived from, or were generated using, the biological sample of the individual.
  • the data may have been reformatted, revised, or mathematically altered to some degree after having been generated, such as by conversion from units in one measurement system to units in another measurement system; but, the data are understood to have been derived from, or were generated using, the biological sample.
  • Target refers to any molecule of interest that may be present in a biological sample.
  • a “molecule of interest” includes any minor variation of a particular molecule, such as, in the case of a protein, for example, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule.
  • a “target molecule”, “target”, or “analyte” is a set of copies of one type or species of molecule or multi-molecular structure.
  • Target molecules refer to more than one such set of molecules.
  • exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affybodies, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing.
  • polypeptide As used herein, “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • Polypeptides can be single chains or associated chains. Also included within the definition are preproteins and intact mature proteins; peptides or polypeptides derived from a mature protein; fragments of a protein; splice variants; recombinant forms of a protein; protein variants with amino acid modifications, deletions, or substitutions; digests; and post-translational modifications, such as glycosylation, acetylation, phosphorylation, and the like.
  • marker and “biomarker” and “feature” are used interchangeably to refer to a target molecule that indicates or is a sign of a normal or abnormal process in an individual or of a disease or other condition in an individual. More specifically, a “marker” or “biomarker” or “feature” is an anatomic, physiologic, biochemical, or molecular parameter associated with the presence of a specific physiological state or process, whether normal or abnormal, and, if abnormal, whether chronic or acute. Biomarkers are detectable and measurable by a variety of methods including laboratory assays and medical imaging.
  • a biomarker is a protein
  • a feature is an analyte/ SOMAmer reagent of other predictors in a statistical model.
  • biomarker value As used herein, “biomarker value”, “value”, “biomarker level”, “feature level” and “level” are used interchangeably to refer to a measurement that is made using any analytical method for detecting the biomarker in a biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, a level, an expression level, a ratio of measured levels, or the like, of, for, or corresponding to the biomarker in the biological sample.
  • the exact nature of the “value” or “level” depends on the specific design and components of the particular analytical method employed to detect the biomarker.
  • biomarker indicates or is a sign of an abnormal process or a disease or other condition in an individual
  • that biomarker is generally described as being either over expressed or under-expressed as compared to an expression level or value of the biomarker that indicates or is a sign of a normal process or an absence of a disease or other condition in an individual.
  • Up-regulation “up-regulated”, “over-expression”, “over-expressed”, and any variations thereof are used interchangeably to refer to a value or level of a biomarker in a biological sample that is greater than a value or level (or range of values or levels) of the biomarker that is typically detected in similar biological samples from healthy or normal individuals.
  • the terms may also refer to a value or level of a biomarker in a biological sample that is greater than a value or level (or range of values or levels) of the biomarker that may be detected at a different stage of a particular disease.
  • Down-regulation “down-regulated”, “under-expression”, “under-expressed”, and any variations thereof are used interchangeably to refer to a value or level of a biomarker in a biological sample that is less than a value or level (or range of values or levels) of the biomarker that is typically detected in similar biological samples from healthy or normal individuals.
  • the terms may also refer to a value or level of a biomarker in a biological sample that is less than a value or level (or range of values or levels) of the biomarker that may be detected at a different stage of a particular disease.
  • a biomarker that is either over-expressed or under-expressed can also be referred to as being “differentially expressed” or as having a “differential level” or “differential value” as compared to a “normal” expression level or value of the biomarker that indicates or is a sign of a normal process or an absence of a disease or other condition in an individual.
  • “differential expression” of a biomarker can also be referred to as a variation from a “normal” expression level of the biomarker.
  • differential gene expression and “differential expression” are used interchangeably to refer to a gene (or its corresponding protein expression product) whose expression is activated to a higher or lower level in a subject suffering from a specific disease or condition, relative to its expression in a normal or control subject.
  • the terms also include genes (or the corresponding protein expression products) whose expression is activated to a higher or lower level at different stages of the same disease or condition. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product.
  • Differential gene expression may include a comparison of expression between two or more genes or their gene products; or a comparison of the ratios of the expression between two or more genes or their gene products; or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease; or between various stages of the same disease.
  • Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
  • “individual” refers to a test subject or patient.
  • the individual can be a mammal or a non-mammal.
  • the individual is a mammal.
  • a mammalian individual can be a human or non-human.
  • the individual is a human.
  • a healthy or normal individual is an individual in which the disease or condition of interest (including, for example, renal insufficiency) is not detectable by conventional diagnostic methods.
  • Diagnose refers to the detection, determination, or recognition of a health status or condition of an individual on the basis of one or more signs, symptoms, data, or other information pertaining to that individual.
  • the health status of an individual can be diagnosed as healthy / normal (i.e., a diagnosis of the absence of a disease or condition) or diagnosed as ill / abnormal (i.e., a diagnosis of the presence, or an assessment of the characteristics, of a disease or condition).
  • diagnosis encompass, with respect to a particular disease or condition, the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of disease response after the administration of a treatment or therapy to the individual.
  • Mean Absolute Error refers to the mean of the absolute values of the prediction error on all instances of a dataset.
  • NRMSE Normalized Root Mean Square Error
  • RMSE Root Mean Square Error
  • a prediction regarding the current state of kidney function can be an estimation of glomerular filtration rate.
  • Prognose refers to the prediction of a future course of a disease or condition in an individual who has the disease or condition (e.g., predicting patient survival), and such terms encompass the evaluation of disease or condition response after the administration of a treatment or therapy to the individual.
  • R 2 refers to the proportion of the variance in outcome that can be explained by a model.
  • “Evaluate”, “evaluating”, “evaluation”, and variations thereof encompass both “diagnose” and “prognose” and also encompass determinations or estimations about the current or future course of a disease or condition in an individual who may or may not have the disease as well as determinations or estimations regarding the risk that a disease or condition will recur in an individual who apparently has been cured of the disease or has had the condition resolved.
  • the term “evaluate” also encompasses assessing an individual’s response to a therapy, such as, for example, determining whether an individual is likely to respond favorably to a therapeutic agent or is unlikely to respond to a therapeutic agent (or will experience toxic or other undesirable side effects, for example), selecting a therapeutic agent for administration to an individual, or monitoring or determining an individual’s response to a therapy that has been administered to the individual.
  • renal insufficiency can include, for example, any of the following: determining an individual’s response to a renal insufficiency treatment or selecting a renal insufficiency treatment to administer to an individual based upon a determination of the biomarker values derived from the individual’s biological sample.
  • Evaluation renal insufficiency can include embodiments such as the assessment of renal insufficiency on a continuous scale, or classification of renal insufficiency in escalating classifications. Classification of insufficiency includes, for example, classification into two or more classifications such as “No renal insufficiency” and “renal insufficiency.”
  • additional biomedical information refers to one or more evaluations of an individual, other than using any of the biomarkers described herein, that are associated current state of kidney health.
  • “Additional biomedical information” includes any of the following: physical descriptors of an individual, including the height and/or weight of an individual; the age of an individual; the gender of an individual; change in weight; the ethnicity of an individual; occupational history; family history of renal insufficiency; the presence of a genetic marker(s) correlating with a higher risk of renal insufficiency in the individual; clinical symptoms such as abdominal pain, weight gain or loss gene expression values; physical descriptors of an individual, including physical descriptors observed by radiologic imaging; smoking status; alcohol use history; occupational history; dietary habits - salt, saturated fat and cholesterol intake; caffeine consumption; and imaging information.
  • Testing of biomarker levels in combination with an evaluation of any additional biomedical information may, for example, improve sensitivity, specificity, and/or AUC for estimation or determination of current state of kidney health as compared to biomarker testing alone or evaluating any particular item of additional biomedical information alone (e.g., carotid intima thickness imaging alone).
  • Additional biomedical information can be obtained from an individual using routine techniques known in the art, such as from the individual themselves by use of a routine patient questionnaire or health history questionnaire, etc., or from a medical practitioner, etc.
  • Testing of biomarker levels in combination with an evaluation of any additional biomedical information may, for example, improve sensitivity, specificity, and/or thresholds for estimation or determination of the current state of kidney function as compared to biomarker testing alone or evaluating any particular item of additional biomedical information alone (e.g., CT imaging alone).
  • detecting or “determining” with respect to a biomarker value includes the use of both the instrument required to observe and record a signal corresponding to a biomarker value and the material/s required to generate that signal.
  • the biomarker value is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.
  • Solid support refers herein to any substrate having a surface to which molecules may be attached, directly or indirectly, through either covalent or non-covalent bonds.
  • a “solid support” can have a variety of physical formats, which can include, for example, a membrane; a chip (e.g., a protein chip); a slide (e.g., a glass slide or coverslip); a column; a hollow, solid, semi-solid, pore- or cavity- containing particle, such as, for example, a bead; a gel; a fiber, including a fiber optic material; a matrix; and a sample receptacle.
  • Exemplary sample receptacles include sample wells, tubes, capillaries, vials, and any other vessel, groove or indentation capable of holding a sample.
  • a sample receptacle can be contained on a multi sample platform, such as a microtiter plate, slide, microfluidics device, and the like.
  • a support can be composed of a natural or synthetic material, an organic or inorganic material. The composition of the solid support on which capture reagents are attached generally depends on the method of attachment (e.g., covalent attachment).
  • Other exemplary receptacles include microdroplets and microfluidic controlled or bulk oil/aqueous emulsions within which assays and related manipulations can occur.
  • Suitable solid supports include, for example, plastics, resins, polysaccharides, silica or silica-based materials, functionalized glass, modified silicon, carbon, metals, inorganic glasses, membranes, nylon, natural fibers (such as, for example, silk, wool and cotton), polymers, and the like.
  • the material composing the solid support can include reactive groups such as, for example, carboxy, amino, or hydroxyl groups, which are used for attachment of the capture reagents.
  • Polymeric solid supports can include, e.g., polystyrene, polyethylene glycol tetraphthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile, polymethyl methacrylate, polytetrafluoroethylene, butyl rubber, styrenebutadiene rubber, natural rubber, polyethylene, polypropylene, (poly)tetrafluoroethylene, (poly)vinylidenefluoride, polycarbonate, and polymethylpentene.
  • Suitable solid support particles that can be used include, e.g., encoded particles, such as Luminex®-type encoded particles, magnetic particles, and glass particles.
  • adaptive normalization by maximum likelihood means a process for normalizing the analytes to mitigate site bias.
  • analyte is the protein target of a capture reagent.
  • the capture reagent is an aptamer.
  • the capture reagent is a SOMAmer.
  • “Lin’s Concordance correlation coefficient” or “Lin’s CCC” means concordance correlation coefficient which measures the concordance between a new test and an existing test that is considered the gold standard.
  • test means a set of samples and clinical data that are analyzed to derive the test.
  • training dataset means a subset of data from a study used to fit a model.
  • validation dataset means a final subset of data used to assess the performance of a final model developed on a verification dataset.
  • “verification dataset” means a separate subset of data used to provide an unbiased evaluation of a model fit on the training dataset while tuning model parameters.
  • the term “need” or “needed” refers to a judgement made by a health care provider regarding treatment of a patient which is considered by the health care provider to be beneficial to the health status of the patient.
  • a test that reflects the current state of kidney health using estimated glomerular filtration rate (eGFR) as the truth standard.
  • eGFR estimated glomerular filtration rate
  • the test can be used in adults and provides an eGFR result which may be used to screen for early kidney damage and to monitor kidney status.
  • the test for kidney health was developed using two cohorts: The Chronic Renal Insufficiency Cohort (CRIC) and Covance, both of which were split into training (70%), verification (15%), and validation datasets (15%).
  • the minimum performance requirement for a useful test was set at an R 2 of 0.8 for estimating or determining eGFR endpoint as calculated by the CKD epi equation. (Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150:604-612.)
  • the model was developed using linear regression and contains 39 features.
  • the minimum performance requirement for a useful test is an R 2 value > 0.65, 0.7, 0.75, or 0.8.
  • the model output is an estimation or determination of GFR as a continuous integer value of from 5 to 100 ml/min/1.73 m 2 , inclusive. Values ⁇ 5 ml/min/1.73 m 2 and > 100 ml/min/1.73 m 2 will be reported as ⁇ 5 ml/min/1.73 m 2 and > 100 ml/min/1.73 m 2 , respectively. Validation exceeds the performance metric of an R 2 > 0.8 (Table 2).
  • Table 2 R 2 metrics for training, verification, and validation for the final model
  • the testing methods disclosed herein provide convenience for health care providers for estimating or determining glomerular filtration rate (GFR) from a blood sample measurement and providing current status of kidney health [00297] Indications for use include but are not limited to those of Table 3.
  • the benefits and risks pertain to decision making in research studies for participant monitoring, stratification, and enrichment.
  • Benefits of the current state kidney function test disclosed herein include: a convenient reflection of kidney function that does not require health care providers to calculate eGFR, which requires age, gender and self-identified race as inputs along with serum creatinine; identification of patients with potentially decreased kidney function to identify candidates for additional diagnostics and treatment for suspicion of early stages of CKD; and earlier identification of potential kidney disease increases the ability to slow the progression of disease and minimize the symptoms that a patient may experience.
  • the test disclosed herein provides a convenient method for health care providers to assess and monitor kidney function and may help to identify patients who are at risk for loss of kidney function and require referral for additional diagnostics.
  • a further benefit of the test disclosed herein is that it can be bundled with other tests which measure protein levels in a sample as part of a liquid health check thus enabling an assessment of general health using one modality and a single blood draw.
  • test disclosed herein can be used in conjunction with additional assessments including but not limited to health status assessments, including evaluations of comorbid conditions such as diabetes, additional laboratory tests including but not limited to measurement of serum creatinine, urine albumin, clinical pathology, renal imaging, and histology.
  • additional assessments including but not limited to health status assessments, including evaluations of comorbid conditions such as diabetes, additional laboratory tests including but not limited to measurement of serum creatinine, urine albumin, clinical pathology, renal imaging, and histology.
  • one or more biomarkers are provided for use either alone or in various combinations to evaluate the current state of kidney function.
  • exemplary embodiments include the biomarkers provided in Table 5, which were identified using a multiplex SOMAmer-based assay.
  • the model has 39 features (Table 5) and estimates or determines glomerular filtration rate (GFR).
  • the model output is a continuous integer between the values of 5 and 100 ml/min/1.73m 2 .
  • the number of biomarkers useful for a biomarker subset or panel is based on a selection of biomarkers with non-zero coefficients as a measure of estimation power for GFR.
  • biomarkers to be used in a subset or panel of biomarkers Another factor that can affect the number of biomarkers to be used in a subset or panel of biomarkers is the procedures used to obtain biological samples from individuals who are being assessed for risk of renal insufficiency. In a carefully controlled sample procurement environment, the number of biomarkers necessary to meet desired eGFR predictive power will be lower than in a situation where there can be more variation in sample collection, handling and storage. Exemplary Uses of Biomarkers
  • biomarker levels can also be done in conjunction with determination of SNPs or other genetic lesions or variability that are indicative of increased risk of susceptibility of disease or condition. (See, e.g., Amos et ah, Nature Genetics 40, 616-622 (2009)).
  • biomarker levels can also be used in conjunction with screening methods, including renal imaging techniques, and more specifically, radiologic screening. Biomarker levels can also be used in conjunction with relevant symptoms or genetic testing. Detection of any of the biomarkers described herein may be useful to evaluate and/or to guide appropriate clinical care of the individual, whether the individual has healthy renal function, renal insufficiency.
  • biomarkers can also be evaluated in conjunction with other types of data, particularly data that indicates an individual’s current state of kidney health (e.g., patient clinical history, symptoms, family history, history of smoking or alcohol use, risk factors such as the presence of a genetic marker(s), and/or status of other biomarkers, etc.).
  • data that indicates an individual’s current state of kidney health (e.g., patient clinical history, symptoms, family history, history of smoking or alcohol use, risk factors such as the presence of a genetic marker(s), and/or status of other biomarkers, etc.).
  • biomarker levels in conjunction with radiologic screening in high risk individuals e.g., assessing biomarker levels in conjunction with blockage detected in a coronary angiogram
  • information regarding the biomarkers can also be evaluated in conjunction with other types of data, particularly data that indicates an individual’s current state of kidney health (e.g., patient clinical history, symptoms, family history of renal disease, risk factors such as whether or not the individual is a smoker, heavy alcohol user and/or status of other biomarkers, etc.).
  • data that indicates an individual’s current state of kidney health e.g., patient clinical history, symptoms, family history of renal disease, risk factors such as whether or not the individual is a smoker, heavy alcohol user and/or status of other biomarkers, etc.
  • These various data can be assessed by automated methods, such as a computer program/software, which can be embodied in a computer or other apparatus/device.
  • any of the described biomarkers may also be used in imaging tests.
  • an imaging agent can be coupled to any of the described biomarkers, which can be used to aid in estimating or determining the glomerular filtration rate (eGFR) and also the presence of absence of renal insufficiency, to monitor response to therapeutic interventions, to select for target populations in a clinical trial among other uses. Detection and Determination of Biomarkers and Biomarker Values
  • a biomarker value for the biomarkers described herein can be detected using any of a variety of known analytical methods.
  • a biomarker value is detected using a capture reagent.
  • a “capture agent” or “capture reagent” refers to a molecule that is capable of binding specifically to a biomarker.
  • the capture reagent can be exposed to the biomarker in solution or can be exposed to the biomarker while the capture reagent is immobilized on a solid support.
  • the capture reagent contains a feature that is reactive with a secondary feature on a solid support.
  • the capture reagent can be exposed to the biomarker in solution, and then the feature on the capture reagent can be used in conjunction with the secondary feature on the solid support to immobilize the biomarker on the solid support.
  • the capture reagent is selected based on the type of analysis to be conducted.
  • Capture reagents include but are not limited to SOMAmers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, an F(ab')2 fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand binding receptor, affybodies, nanobodies, imprinted polymers, avimers, peptidomimetics, a hormone receptor, a cytokine receptor, and synthetic receptors, and modifications and fragments of these.
  • a biomarker value is detected using a biomarker/capture reagent complex.
  • the biomarker value is derived from the biomarker/capture reagent complex and is detected indirectly, such as, for example, as a result of a reaction that is subsequent to the biomarker/capture reagent interaction, but is dependent on the formation of the biomarker/capture reagent complex.
  • the biomarker value is detected directly from the biomarker in a biological sample.
  • the biomarkers are detected using a multiplexed format that allows for the simultaneous detection of two or more biomarkers in a biological sample.
  • capture reagents are immobilized, directly or indirectly, covalently or non-covalently, in discrete locations on a solid support.
  • a multiplexed format uses discrete solid supports where each solid support has a unique capture reagent associated with that solid support, such as, for example quantum dots.
  • an individual device is used for the detection of each one of multiple biomarkers to be detected in a biological sample. Individual devices can be configured to permit each biomarker in the biological sample to be processed simultaneously. For example, a microtiter plate can be used such that each well in the plate is used to uniquely analyze one of multiple biomarkers to be detected in a biological sample.
  • a fluorescent tag can be used to label a component of the biomarker/capture complex to enable the detection of the biomarker value.
  • the fluorescent label can be conjugated to a capture reagent specific to any of the biomarkers described herein using known techniques, and the fluorescent label can then be used to detect the corresponding biomarker value.
  • Suitable fluorescent labels include rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, allophycocyanin, PBXL-3, Qdot 605, Lissamine, phycoerythrin, Texas Red, and other such compounds.
  • the fluorescent label is a fluorescent dye molecule.
  • the fluorescent dye molecule includes at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance.
  • the dye molecule includes an AlexFluor molecule, such as, for example, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700.
  • the dye molecule includes a first type and a second type of dye molecule, such as, e.g., two different AlexaFluor molecules.
  • the dye molecule includes a first type and a second type of dye molecule, and the two dye molecules have different emission spectra.
  • Fluorescence can be measured with a variety of instrumentation compatible with a wide range of assay formats.
  • spectrofluorimeters have been designed to analyze microtiter plates, microscope slides, printed arrays, cuvettes, etc. See Principles of Fluorescence Spectroscopy, by J.R. Lakowicz, Springer Science + Business Media, Inc., 2004. See Bioluminescence & Chemiluminescence: Progress & Current Applications; Philip E. Stanley and Larry J. Kricka editors, World Scientific Publishing Company, January 2002.
  • a chemiluminescence tag can optionally be used to label a component of the biomarker/capture complex to enable the detection of a biomarker value.
  • Suitable chemiluminescent materials include any of oxalyl chloride, Rodamin 6G, Ru(bipy)32+ , TMAE (tetrakis(dimethylamino)ethylene), Pyrogallol (1,2,3-trihydroxibenzene), Lucigenin, peroxyoxalates, Aryl oxalates, Acridinium esters, dioxetanes, and others.
  • the detection method includes an enzyme/substrate combination that generates a detectable signal that corresponds to the biomarker value.
  • the enzyme catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques, including spectrophotometry, fluorescence, and chemiluminescence.
  • Suitable enzymes include, for example, luciferases, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta- galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucose-6- phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase beta- galactosidase
  • glucoamylase lysozyme
  • glucose oxidase galactose oxidase
  • glucose-6- phosphate dehydrogenase uricase
  • xanthine oxidase lactoperoxidase
  • microperoxidase and the like
  • the detection method can be a combination of fluorescence, chemiluminescence, radionuclide or enzyme/substrate combinations that generate a measurable signal.
  • Multimodal signaling could have unique and advantageous characteristics in biomarker assay formats.
  • biomarker values for the biomarkers described herein can be detected using known analytical methods including, singleplex SOMAmer assays, multiplexed SOMAmer assays, singleplex or multiplexed immunoassays, mRNA expression profiling, miRNA expression profiling, mass spectrometric analysis, histological/cytological methods, etc. as detailed below.
  • Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other samples are important tools in scientific research and in the health care field.
  • One class of such assays involves the use of a microarray that includes one or more aptamers immobilized on a solid support.
  • the aptamers are each capable of binding to a target molecule in a highly specific manner and with very high affinity. See, e.g., U.S. Patent No. 5,475,096 entitled “Nucleic Acid Ligands”; see also, e.g., U.S. Patent No. 6,242,246, U.S. Patent No. 6,458,543, and U.S. Patent No.
  • an “aptamer” refers to a nucleic acid that has a specific binding affinity for a target molecule. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other components in a test sample.
  • An “aptamer” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence.
  • An aptamer can include any suitable number of nucleotides, including any number of chemically modified nucleotides.
  • “Aptamers” refers to more than one such set of molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers can be DNA or RNA or chemically modified nucleic acids and can be single stranded, double stranded, or contain double stranded regions, and can include higher ordered structures. An aptamer can also be a photoaptamer, where a photoreactive or chemically reactive functional group is included in the aptamer to allow it to be covalently linked to its corresponding target. Any of the aptamer methods disclosed herein can include the use of two or more aptamers that specifically bind the same target molecule. As further described below, an aptamer may include a tag. If an aptamer includes a tag, all copies of the aptamer need not have the same tag. Moreover, if different aptamers each include a tag, these different aptamers can have either the same tag or a different tag.
  • An aptamer can be identified using any known method, including the SELEX process. Once identified, an aptamer can be prepared or synthesized in accordance with any known method, including chemical synthetic methods and enzymatic synthetic methods.
  • a “SOMAmer” or Slow Off-Rate Modified Aptamer refers to an aptamer having improved off-rate characteristics. SOMAmers can be generated using the improved SELEX methods described in U.S. Publication No. 2009/0004667, entitled “Method for Generating Aptamers with Improved Off-Rates.”
  • SELEX and “SELEX process” are used interchangeably herein to refer generally to a combination of (1) the selection of aptamers that interact with a target molecule in a desirable manner, for example binding with high affinity to a protein, with (2) the amplification of those selected nucleic acids.
  • the SELEX process can be used to identify aptamers with high affinity to a specific target or biomarker.
  • SELEX generally includes preparing a candidate mixture of nucleic acids, binding of the candidate mixture to the desired target molecule to form an affinity complex, separating the affinity complexes from the unbound candidate nucleic acids, separating and isolating the nucleic acid from the affinity complex, purifying the nucleic acid, and identifying a specific aptamer sequence.
  • the process may include multiple rounds to further refine the affinity of the selected aptamer.
  • the process can include amplification steps at one or more points in the process. See, e.g., U.S. Patent No. 5,475,096, entitled “Nucleic Acid Ligands”.
  • the SELEX process can be used to generate an aptamer that covalently binds its target as well as an aptamer that non-covalently binds its target. See, e.g., U.S. Patent No. 5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”
  • the SELEX process can be used to identify high-affinity aptamers containing modified nucleotides that confer improved characteristics on the aptamer, such as, for example, improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified aptamers containing modified nucleotides are described in U.S. Patent No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides", which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5’- and 2’-positions of pyrimidines. U.S. Patent No.
  • Patent Application Publication 20090004667 entitled “Method for Generating Aptamers with Improved Off-Rates”, which describes improved SELEX methods for generating aptamers that can bind to target molecules.
  • these slow off-rate aptamers are known as “SOMAmers.”
  • Methods for producing aptamers or SOMAmers and photoaptamers or SOMAmers having slower rates of dissociation from their respective target molecules are described. The methods involve contacting the candidate mixture with the target molecule, allowing the formation of nucleic acid-target complexes to occur, and performing a slow off-rate enrichment process wherein nucleic acid-target complexes with fast dissociation rates will dissociate and not reform, while complexes with slow dissociation rates will remain intact. Additionally, the methods include the use of modified nucleotides in the production of candidate nucleic acid mixtures to generate aptamers or SOMAmers with improved off-rate performance.
  • a variation of this assay employs aptamers that include photoreactive functional groups that enable the aptamers to covalently bind or “photocrosslink” their target molecules. See, e.g., U.S. Patent No. 6,544,776 entitled “Nucleic Acid Ligand Diagnostic Biochip”. These photoreactive aptamers are also referred to as photoaptamers. See, e.g., U.S. Patent No. 5,763,177, U.S. Patent No. 6,001,577, and U.S. Patent No.
  • the aptamers or SOMAmers are immobilized on the solid support prior to being contacted with the sample. Under certain circumstances, however, immobilization of the aptamers or SOMAmers prior to contact with the sample may not provide an optimal assay. For example, pre-immobilization of the aptamers or SOMAmers may result in inefficient mixing of the aptamers or SOMAmers with the target molecules on the surface of the solid support, perhaps leading to lengthy reaction times and, therefore, extended incubation periods to permit efficient binding of the aptamers or SOMAmers to their target molecules.
  • the solid support may tend to scatter or absorb the light used to effect the formation of covalent bonds between the photoaptamers or photoSOMAmers and their target molecules.
  • detection of target molecules bound to their aptamers or photoSOMAmers can be subject to imprecision, since the surface of the solid support may also be exposed to and affected by any labeling agents that are used.
  • immobilization of the aptamers or SOMAmers on the solid support generally involves an aptamer or SOMAmer-preparation step (i.e., the immobilization) prior to exposure of the aptamers or SOMAmers to the sample, and this preparation step may affect the activity or functionality of the aptamers or SOMAmers.
  • SOMAmer assays that permit a SOMAmer to capture its target in solution and then employ separation steps that are designed to remove specific components of the SOMAmer-target mixture prior to detection have also been described (see U.S. Patent Application Publication 20090042206, entitled “Multiplexed Analyses of Test Samples”).
  • the described SOMAmer assay methods enable the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., a SOMAmer).
  • the described methods create a nucleic acid surrogate (i.e, the SOMAmer) for detecting and quantifying a non-nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, including protein targets.
  • a nucleic acid surrogate i.e, the SOMAmer
  • SOMAmers can be constructed to facilitate the separation of the assay components from a SOMAmer biomarker complex (or photoSOMAmer biomarker covalent complex) and permit isolation of the SOMAmer for detection and/or quantification.
  • these constructs can include a cleavable or releasable element within the SOMAmer sequence.
  • additional functionality can be introduced into the SOMAmer, for example, a labeled or detectable component, a spacer component, or a specific binding tag or immobilization element.
  • the SOMAmer can include a tag connected to the SOMAmer via a cleavable moiety, a label, a spacer component separating the label, and the cleavable moiety.
  • a cleavable element is a photocleavable linker.
  • the photocleavable linker can be attached to a biotin moiety and a spacer section, can include an NHS group for derivatization of amines, and can be used to introduce a biotin group to a SOMAmer, thereby allowing for the release of the SOMAmer later in an assay method.
  • the molecular capture reagents can be an aptamer (e.g., modified aptamer or SOMAmer reagent) or an antibody or the like and the specific target would be a biomarker as in Table 5.
  • a method for signal generation takes advantage of anisotropy signal change due to the interaction of a fluorophore-labeled capture reagent with its specific biomarker target.
  • the labeled capture reagent reacts with its target, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower changing the anisotropy value.
  • binding events may be used to quantitatively measure the biomarkers in solutions.
  • Other methods include fluorescence polarization assays, molecular beacon methods, time resolved fluorescence quenching, chemiluminescence, fluorescence resonance energy transfer, and the like.
  • An exemplary solution-based SOMAmer assay that can be used to detect a biomarker value corresponding to a biomarker in a biological sample includes the following: (a) preparing a mixture by contacting the biological sample with a SOMAmer that includes a first tag and has a specific affinity for the biomarker, wherein a SOMAmer affinity complex is formed when the biomarker is present in the sample; (b) exposing the mixture to a first solid support including a first capture element, and allowing the first tag to associate with the first capture element; (c) removing any components of the mixture not associated with the first solid support; (d) attaching a second tag to the biomarker component of the SOMAmer affinity complex; (e) releasing the SOMAmer affinity complex from the first solid support; (f) exposing the released SOMAmer affinity complex to a second solid support that includes a second capture element and allowing the second tag to associate with the second capture element; (g) removing any non-complexed SOMAmer from the mixture by
  • any means known in the art can be used to detect a biomarker value by detecting the SOMAmer component of a SOMAmer affinity complex.
  • a number of different detection methods can be used to detect the SOMAmer component of an affinity complex, such as, for example, hybridization assays, mass spectroscopy, or QPCR.
  • nucleic acid sequencing methods can be used to detect the SOMAmer component of a SOMAmer affinity complex and thereby detect a biomarker value. Briefly, a test sample can be subjected to any kind of nucleic acid sequencing method to identify and quantify the sequence or sequences of one or more SOMAmers present in the test sample.
  • the sequence includes the entire SOMAmer molecule or any portion of the molecule that may be used to uniquely identify the molecule.
  • the identifying sequencing is a specific sequence added to the SOMAmer; such sequences are often referred to as “tags,” “barcodes,” or “zipcodes.”
  • the sequencing method includes enzymatic steps to amplify the SOMAmer sequence or to convert any kind of nucleic acid, including RNA and DNA that contain chemical modifications to any position, to any other kind of nucleic acid appropriate for sequencing.
  • the sequencing method includes one or more cloning steps. In other embodiments the sequencing method includes a direct sequencing method without cloning.
  • the sequencing method includes a directed approach with specific primers that target one or more SOMAmers in the test sample. In other embodiments, the sequencing method includes a shotgun approach that targets all SOMAmers in the test sample.
  • the sequencing method includes enzymatic steps to amplify the molecule targeted for sequencing. In other embodiments, the sequencing method directly sequences single molecules.
  • An exemplary nucleic acid sequencing-based method that can be used to detect a biomarker value corresponding to a biomarker in a biological sample includes the following: (a) converting a mixture of SOMAmers that contain chemically modified nucleotides to unmodified nucleic acids with an enzymatic step; (b) shotgun sequencing the resulting unmodified nucleic acids with a massively parallel sequencing platform such as, for example, the 454 Sequencing System (454 Life Sciences/Roche), the Illumina Sequencing System (Illumina), the ABI SOLiD Sequencing System (Applied Biosystems), the Heli Scope Single Molecule Sequencer (Helicos Biosciences), or the Pacific Biosciences Real Time Single- Molecule Sequencing System (Pacific BioSciences) or the Polonator G Sequencing System (Dover), the 454 Sequencing
  • Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format.
  • monoclonal antibodies are often used because of their specific epitope recognition.
  • Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies.
  • Immunoassays have been designed for use with a wide range of biological sample matrices. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.
  • Quantitative results are generated through the use of a standard curve created with known concentrations of the specific analyte to be detected.
  • the response or signal from an unknown sample is plotted onto the standard curve, and a quantity or value corresponding to the target in the unknown sample is established.
  • ELISA or EIA can be quantitative for the detection of an analyte. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly or indirectly, an enzyme. ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (1125) or fluorescence.
  • Additional techniques include, for example, agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assay, and others (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition).
  • Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays.
  • procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.
  • Methods of detecting and/or quantifying a detectable label or signal generating material depend on the nature of the label.
  • the products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light.
  • detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.
  • Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 384 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.
  • Measuring mRNA in a biological sample may be used as a surrogate for detection of the level of the corresponding protein in the biological sample.
  • any of the biomarkers or biomarker panels described herein can also be detected by detecting the appropriate RNA.
  • mRNA expression levels are measured by reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR). RT-PCR is used to create a cDNA from the mRNA. The cDNA may be used in a qPCR assay to produce fluorescence as the DNA amplification process progresses. By comparison to a standard curve, qPCR can produce an absolute measurement such as number of copies of mRNA per cell.
  • miRNA molecules are small RNAs that are non-coding but may regulate gene expression. Any of the methods suited to the measurement of mRNA expression levels can also be used for the corresponding miRNA. Recently many laboratories have investigated the use of miRNAs as biomarkers for disease. Many diseases involve wide-spread transcriptional regulation, and it is not surprising that miRNAs might find a role as biomarkers. The connection between miRNA concentrations and disease is often even less clear than the connections between protein levels and disease, yet the value of miRNA biomarkers might be substantial.
  • RNA biomarkers have similar requirements, although many potential protein biomarkers are secreted intentionally at the site of pathology and function, during disease, in a paracrine fashion. Many potential protein biomarkers are designed to function outside the cells within which those proteins are synthesized.
  • any of the described biomarkers may also be used in molecular imaging tests.
  • an imaging agent can be coupled to any of the described biomarkers, which can be used to aid in estimation of determination of the current state of kidney function, to monitor response to therapeutic interventions, to select a population for clinical trials among other uses.
  • In vivo imaging technologies provide non-invasive methods for determining the state of a particular disease or condition in the body of an individual. For example, entire portions of the body, or even the entire body, may be viewed as a three dimensional image, thereby providing valuable information concerning morphology and structures in the body. Such technologies may be combined with the detection of the biomarkers described herein to provide information concerning the renal health status of an individual.
  • in vivo molecular imaging technologies are expanding due to various advances in technology. These advances include the development of new contrast agents or labels, such as radiolabels and/or fluorescent labels, which can provide strong signals within the body; and the development of powerful new imaging technology, which can detect and analyze these signals from outside the body, with sufficient sensitivity and accuracy to provide useful information.
  • the contrast agent can be visualized in an appropriate imaging system, thereby providing an image of the portion or portions of the body in which the contrast agent is located.
  • the contrast agent may be bound to or associated with a capture reagent, such as a SOMAmer or an antibody, for example, and/or with a peptide or protein, or an oligonucleotide (for example, for the detection of gene expression), or a complex containing any of these with one or more macromolecules and/or other particulate forms.
  • a capture reagent such as a SOMAmer or an antibody, for example, and/or with a peptide or protein, or an oligonucleotide (for example, for the detection of gene expression), or a complex containing any of these with one or more macromolecules and/or other particulate forms.
  • the contrast agent may also feature a radioactive atom that is useful in imaging. Suitable radioactive atoms include technetium-99m or iodine-123 for scintigraphic studies.
  • MRI magnetic resonance imaging
  • iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • Standard imaging techniques include but are not limited to magnetic resonance imaging, computed tomography scanning (coronary calcium score), positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography angiography, and the like.
  • a given contrast agent such as a given radionuclide and the particular biomarker that it is used to target (protein, mRNA, and the like).
  • the radionuclide chosen typically has a type of decay that is detectable by a given type of instrument.
  • its half-life should be long enough to enable detection at the time of maximum uptake by the target tissue but short enough that deleterious radiation of the host is minimized.
  • Exemplary imaging techniques include but are not limited to PET and SPECT, which are imaging techniques in which a radionuclide is synthetically or locally administered to an individual. The subsequent uptake of the radiotracer is measured over time and used to obtain information about the targeted tissue and the biomarker. Because of the high-energy (gamma- ray) emissions of the specific isotopes employed and the sensitivity and sophistication of the instruments used to detect them, the two-dimensional distribution of radioactivity may be inferred from outside of the body.
  • PET and SPECT are imaging techniques in which a radionuclide is synthetically or locally administered to an individual. The subsequent uptake of the radiotracer is measured over time and used to obtain information about the targeted tissue and the biomarker. Because of the high-energy (gamma- ray) emissions of the specific isotopes employed and the sensitivity and sophistication of the instruments used to detect them, the two-dimensional distribution of radioactivity may be inferred from outside of the body.
  • Commonly used positron-emitting nuclides in PET include, for example, carbon- 11, nitrogen-13, oxygen-15, and fluorine-18.
  • Isotopes that decay by electron capture and/or gamma-emission are used in SPECT and include, for example iodine-123 and technetium-99m.
  • An exemplary method for labeling amino acids with technetium-99m is the reduction of pertechnetate ion in the presence of a chelating precursor to form the labile technetium-99m- precursor complex, which, in turn, reacts with the metal binding group of a bifunctionally modified chemotactic peptide to form a technetium-99m-chemotactic peptide conjugate.
  • Antibodies are frequently used for such in vivo imaging diagnostic methods.
  • the preparation and use of antibodies for in vivo diagnosis is well known in the art.
  • Labeled antibodies which specifically bind any of the biomarkers in Table 5 can be injected into an individual suspected of having renal insufficiency, detectable according to the particular biomarker used, for the purpose of diagnosing or evaluating the disease status or condition of the individual.
  • the label used will be selected in accordance with the imaging modality to be used, as previously described. Localization of the label permits determination of the tissue damage or other indications related to renal insufficiency.
  • the amount of label within an organ or tissue also allows determination of the involvement of the current state of kidney health biomarkers in that organ or tissue.
  • SOMAmers may be used for such in vivo imaging diagnostic methods.
  • a SOMAmer that was used to identify a particular biomarker described in Table 5 (and therefore binds specifically to that particular biomarker) may be appropriately labeled and injected into an individual being evaluated for renal insufficiency, detectable according to the particular biomarker, for the purpose of diagnosing or evaluating the levels of tissue damage, components of inflammatory response and other factors associated with the renal insufficiency in the individual.
  • the label used will be selected in accordance with the imaging modality to be used, as previously described. Localization of the label permits determination of the site of the processes leading to increased risk.
  • SOMAmer- directed imaging agents could have unique and advantageous characteristics relating to tissue penetration, tissue distribution, kinetics, elimination, potency, and selectivity as compared to other imaging agents.
  • Such techniques may also optionally be performed with labeled oligonucleotides, for example, for detection of gene expression through imaging with antisense oligonucleotides. These methods are used for in situ hybridization, for example, with fluorescent molecules or radionuclides as the label. Other methods for detection of gene expression include, for example, detection of the activity of a reporter gene.
  • optical imaging Another general type of imaging technology is optical imaging, in which fluorescent signals within the subject are detected by an optical device that is external to the subject. These signals may be due to actual fluorescence and/or to bioluminescence. Improvements in the sensitivity of optical detection devices have increased the usefulness of optical imaging for in vivo diagnostic assays.
  • in vivo molecular biomarker imaging is increasing, including for clinical trials, for example, to more rapidly measure clinical efficacy in trials for new disease or condition therapies and/or to avoid prolonged treatment with a placebo for those diseases, such as multiple sclerosis, in which such prolonged treatment may be considered to be ethically questionable.
  • mass spectrometers can be used to detect biomarker values.
  • Several types of mass spectrometers are available or can be produced with various configurations.
  • a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument- control system, and a data system. Difference in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities.
  • an inlet can be a capillary-column liquid chromatography source or can be a direct probe or stage such as used in matrix-assisted laser desorption.
  • Common ion sources are, for example, electrospray, including nanospray and microspray or matrix-assisted laser desorption.
  • Common mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time-of-flight mass analyzer. Additional mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, New York (2000)).
  • Protein biomarkers and biomarker values can be detected and measured by any of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI- MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflex III TOF/TOF, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)N, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS), APP
  • Sample preparation strategies are used to label and enrich samples before mass spectroscopic characterization of protein biomarkers and determination biomarker values.
  • Labeling methods include but are not limited to isobaric tag for relative and absolute quantitation (iTRAQ) and stable isotope labeling with amino acids in cell culture (SILAC).
  • Capture reagents used to selectively enrich samples for candidate biomarker proteins prior to mass spectroscopic analysis include but are not limited to SOMAmers, antibodies, nucleic acid probes, chimeras, small molecules, an F(ab’)2 fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand-binding receptor, affybodies, nanobodies, ankyrins, domain antibodies, alternative antibody scaffolds (e.g.
  • a proximity ligation assay can be used to determine biomarker values. Briefly, a test sample is contacted with a pair of affinity probes that may be a pair of antibodies or a pair of SOMAmers, with each member of the pair extended with an oligonucleotide.
  • the targets for the pair of affinity probes may be two distinct determinates on one protein or one determinate on each of two different proteins, which may exist as homo- or hetero-multimeric complexes. When probes bind to the target determinates, the free ends of the oligonucleotide extensions are brought into sufficiently close proximity to hybridize together.
  • oligonucleotide extensions The hybridization of the oligonucleotide extensions is facilitated by a common connector oligonucleotide which serves to bridge together the oligonucleotide extensions when they are positioned in sufficient proximity. Once the oligonucleotide extensions of the probes are hybridized, the ends of the extensions are joined together by enzymatic DNA ligation.
  • Each oligonucleotide extension comprises a primer site for PCR amplification. Once the oligonucleotide extensions are ligated together, the oligonucleotides form a continuous DNA sequence which, through PCR amplification, reveals information regarding the identity and amount of the target protein, as well as, information regarding protein-protein interactions where the target determinates are on two different proteins. Proximity ligation can provide a highly sensitive and specific assay for real-time protein concentration and interaction information through use of real-time PCR. Probes that do not bind the determinates of interest do not have the corresponding oligonucleotide extensions brought into proximity and no ligation or PCR amplification can proceed, resulting in no signal being produced.
  • the foregoing assays enable the detection of biomarker values that are useful in methods for determining or estimating glomerular filtration rate (eGFR) and/or renal insufficiency, where the methods comprise detecting, in a biological sample from an individual, biomarker values that each correspond to a biomarker selected from the group consisting of the biomarkers provided in Table 5, wherein an assessment, as described in detail below, using the biomarker values indicates the current state of kidney health in the individual. While certain of the described renal biomarkers are useful alone for estimating or determining current state of kidney health, methods are also described herein for the grouping of multiple subsets of the renal health biomarkers that are each useful as a panel of three or more biomarkers.. In accordance with any of the methods described herein, biomarker values can be detected and evaluated individually or they can be detected and evaluated collectively, as for example in a multiplex assay format.
  • a biomarker “signature” for a given diagnostic or predictive test contains a set of markers, each marker having different levels in the populations of interest. Different levels, in this context, may refer to different means of the marker levels for the individuals in two or more groups, or different variances in the two or more groups, or a combination of both.
  • markers can be used to assign an unknown sample from an individual into one of two groups, either renal insufficiency or not.
  • classification The assignment of a sample into one of two or more groups is known as classification, and the procedure used to accomplish this assignment is known as a classifier or a classification method. Classification methods may also be referred to as scoring methods.
  • classification methods There are many classification methods that can be used to construct a diagnostic classifier from a set of biomarker values.
  • classification methods are most easily performed using supervised learning techniques where a data set is collected using samples obtained from individuals within two (or more, for multiple classification states) distinct groups one wishes to distinguish. Since the class (group or population) to which each sample belongs is known in advance for each sample, the classification method can be trained to give the desired classification response. It is also possible to use unsupervised learning techniques to produce a diagnostic classifier.
  • training data includes samples from the distinct groups (classes) to which unknown samples will later be assigned.
  • samples collected from individuals in a control population and individuals in a particular disease, condition or event population can constitute training data to develop a classifier that can classify unknown samples (or, more particularly, the individuals from whom the samples were obtained) as either having the disease, condition or elevated risk of an event or being free from the disease, condition or elevated risk of an event.
  • the development of the classifier from the training data is known as training the classifier. Specific details on classifier training depend on the nature of the supervised learning technique (see, e.g., Pattern Classification, R.O.
  • Over-fitting occurs when a statistical model describes random error or noise instead of the underlying relationship. Over-fitting can be avoided in a variety of ways, including, for example, by limiting the number of markers used in developing the classifier, by assuming that the marker responses are independent of one another, by limiting the complexity of the underlying statistical model employed, and by ensuring that the underlying statistical model conforms to the data.
  • PCA Principal Component Analysis
  • biomarkers can be analyzed for those components of difference between samples which were specific to the separation between the control samples and early event samples.
  • One method that may be employed is the use of DSGA (Bair,E. and Tibshirani,R. (2004) Semi-supervised methods to predict patient survival from gene expression data. PLOS Biol., 2, 511-522) to remove (deflate) the first three principal component directions of variation between the samples in the control set.
  • DSGA Air,E. and Tibshirani,R. (2004) Semi-supervised methods to predict patient survival from gene expression data. PLOS Biol., 2, 511-522) to remove (deflate) the first three principal component directions of variation between the samples in the control set.
  • the dimensionality reduction is performed on the control set to discover, both the samples in the control and the samples from the early event samples are run through the PCA. Separation of cases from early events can be observed along the horizontal axis.
  • Cross validated selection of proteins relevant to renal function estimation or determination [00377] In order to avoid over-fitting of protein predictive power to idiosyncratic features of a particular selection of samples, a cross-validation and dimensional reduction approach can be taken. Cross-validation involves the multiple selection of sets of samples to determine the association of risk by protein combined with the use of the unselected samples to monitor the ability of the method to apply to samples which were not used in producing the model of risk (The Elements of Statistical Learning - Data Mining, Inference, and Prediction, T. Hastie, et ah, editors, Springer Science+Business Media, LLC, 2nd edition, 2009). We applied the supervised PCA method of Tibshirani et al (Bair,E.
  • the supervised PCA (SPCA) method inolves the univariate selection of a set of proteins statistically associated with the observed event hazard in the data and the determination of the correlated component which combines information from all of these proteins. This determination of the correlated component is a dimensionality reduction step which not only combines information across proteins, but also mitigates the likelihood of overfitting by reducing the number of independent variables from the full protein menu of over 1000 proteins down to a few principal components (in this work, we only examined the first principal component).
  • Cox proportional hazard model (Cox, David R (1972). "Regression Models and Life-Tables". Journal of the Royal Statistical Society. Series B (Methodological) 34 (2): 187-220.)) is widely used in medical statistics. Cox regression avoids fitting a specific function of time to the cumulative survival, and instead employs a model of relative risk referred to a baseline hazard function (which may vary with time).
  • the baseline hazard function describes the common shape of the survival time distribution for all individuals, while the relative risk gives the level of the hazard for a set of covariate values (such as a single individual or group), as a multiple of the baseline hazard.
  • the relative risk is constant with time in the Cox model.
  • any combination of the biomarkers of Table 5 can be detected using a suitable kit, such as for use in performing the methods disclosed herein.
  • any kit can contain one or more detectable labels as described herein, such as a fluorescent moiety, etc.
  • a kit includes (a) one or more capture reagents (such as, for example, at least one SOMAmer or antibody) for detecting one or more biomarkers in a biological sample, wherein the biomarkers include any of the biomarkers set forth in Table 5 and optionally (b) one or more software or computer program products for computing current state of kidney health.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • kits The combination of a solid support with a corresponding capture reagent having a signal generating material is referred to herein as a “detection device” or “kit”.
  • the kit can also include instructions for using the devices and reagents, handling the sample, and analyzing the data. Further the kit may be used with a computer system or software to analyze and report the result of the analysis of the biological sample.
  • kits can also contain one or more reagents (e.g., solubilization buffers, detergents, washes, or buffers) for processing a biological sample.
  • reagents e.g., solubilization buffers, detergents, washes, or buffers
  • Any of the kits described herein can also include, e.g., buffers, blocking agents, mass spectrometry matrix materials, antibody capture agents, positive control samples, negative control samples, software and information such as protocols, guidance and reference data.
  • kits for the analysis of current state of kidney health include PCR primers for one or more SOMAmers specific to biomarkers selected from Table 5.
  • the kit may further include instructions for use and correlation of the biomarkers with an estimation or determination of current state of kidney health.
  • the kit may also include a DNA array containing the complement of one or more of the aptamers or SOMAmer reagents specific for the biomarkers selected from Table 5, reagents, and/or enzymes for amplifying or isolating sample DNA.
  • the kits may include reagents for real-time PCR, for example, TaqMan probes and/or primers, and enzymes.
  • a kit can comprise (a) reagents comprising at least capture reagent for quantifying one or more biomarkers in a test sample, wherein said biomarkers comprise the biomarkers set forth in Table 5, or any other biomarkers or biomarkers panels described herein, and optionally (b) one or more algorithms or computer programs for performing the steps of comparing the amount of each biomarker quantified in the test sample to one or more predetermined cutoffs and assigning a score for each biomarker quantified based on said comparison, combining the assigned scores for each biomarker quantified to obtain a total score, comparing the total score with a predetermined score, and using said comparison to determine whether an individual has renal insufficiency.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • a method for diagnosing an individual can comprise the following: 1) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; 3) perform any data normalization or standardization required for the method used to collect biomarker values; 4) calculate the marker score; 5) combine the marker scores to obtain a total diagnostic or predictive score; and 6) report the individual’s diagnostic or predictive score.
  • the diagnostic or predictive score may be a single number determined from the sum of all the marker calculations that is compared to a preset threshold value that is an indication of the presence or absence of disease.
  • the diagnostic or predictive score may be a series of bars that each represent a biomarker value and the pattern of the responses may be compared to a pre-set pattern for determination of the presence or absence of disease, condition or the increased risk (or not) of an event.
  • FIG. 1 An example of a computer system 100 is shown in Figure 2.
  • system 100 is shown comprised of hardware elements that are electrically coupled via bus 108, including a processor 101, input device 102, output device 103, storage device 104, computer-readable storage media reader 105a, communications system 106, processing acceleration (e.g., DSP or special-purpose processors) 107 and memory 109.
  • processing acceleration e.g., DSP or special-purpose processors
  • Computer-readable storage media reader 105a is further coupled to computer-readable storage media 105b, the combination comprehensively representing remote, local, fixed and/or removable storage devices plus storage media, memory, etc.
  • System 100 for temporarily and/or more permanently containing computer-readable information, which can include storage device 104, memory 109 and/or any other such accessible system 100 resource.
  • System 100 also comprises software elements (shown as being currently located within working memory 191) including an operating system 192 and other code 193, such as programs, data and the like.
  • system 100 has extensive flexibility and configurability.
  • a single architecture might be utilized to implement one or more servers that can be further configured in accordance with currently desirable protocols, protocol variations, extensions, etc.
  • embodiments may well be utilized in accordance with more specific application requirements.
  • one or more system elements might be implemented as sub-elements within a system 100 component (e.g., within communications system 106).
  • Customized hardware might also be utilized and/or particular elements might be implemented in hardware, software or both.
  • connection to other computing devices such as network input/output devices (not shown) may be employed, it is to be understood that wired, wireless, modem, and/or other connection or connections to other computing devices might also be utilized.
  • the system can comprise a database containing features of biomarkers characteristic of estimating or determining renal health.
  • the biomarker data (or biomarker information) can be utilized as an input to the computer for use as part of a computer implemented method.
  • the biomarker data can include the data as described herein.
  • the system further comprises one or more devices for providing input data to the one or more processors.
  • the system further comprises a memory for storing a data set of ranked data elements.
  • the device for providing input data comprises a detector for detecting the characteristic of the data element, e.g., such as a mass spectrometer or gene chip reader.
  • the system additionally may comprise a database management system.
  • User requests or queries can be formatted in an appropriate language understood by the database management system that processes the query to extract the relevant information from the database of training sets.
  • the system may be connectable to a network to which a network server and one or more clients are connected.
  • the network may be a local area network (LAN) or a wide area network (WAN), as is known in the art.
  • the server includes the hardware necessary for running computer program products (e.g., software) to access database data for processing user requests.
  • the system may include an operating system (e.g., UNIX or Linux) for executing instructions from a database management system.
  • the operating system can operate on a global communications network, such as the internet, and utilize a global communications network server to connect to such a network.
  • the system may include one or more devices that comprise a graphical display interface comprising interface elements such as buttons, pull down menus, scroll bars, fields for entering text, and the like as are routinely found in graphical user interfaces known in the art.
  • Requests entered on a user interface can be transmitted to an application program in the system for formatting to search for relevant information in one or more of the system databases.
  • Requests or queries entered by a user may be constructed in any suitable database language.
  • the graphical user interface may be generated by a graphical user interface code as part of the operating system and can be used to input data and/or to display inputted data. The result of processed data can be displayed in the interface, printed on a printer in communication with the system, saved in a memory device, and/or transmitted over the network or can be provided in the form of the computer readable medium.
  • the system can be in communication with an input device for providing data regarding data elements to the system (e.g., expression values).
  • the input device can include a gene expression profiling system including, e.g., a mass spectrometer, gene chip or array reader, and the like.
  • the methods and apparatus for analyzing current state of kidney health via an estimation or determination of GFR with biomarker information may be implemented in any suitable manner, for example, using a computer program operating on a computer system.
  • a conventional computer system comprising a processor and a random access memory, such as a remotely-accessible application server, network server, personal computer or workstation may be used.
  • Additional computer system components may include memory devices or information storage systems, such as a mass storage system and a user interface, for example a conventional monitor, keyboard and tracking device.
  • the computer system may be a stand-alone system or part of a network of computers including a server and one or more databases.
  • the current state of kidney health via the estimation or determination of GFR with the biomarker analysis system can provide functions and operations to complete data analysis, such as data gathering, processing, analysis, reporting and/or diagnosis.
  • the computer system can execute the computer program that may receive, store, search, analyze, and report information relating to the current state of kidney health biomarkers.
  • the computer program may comprise multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate a current state of kidney health status.
  • Calculation of current state of kidney health may optionally comprise generating or collecting any other information, including additional biomedical information, regarding the condition of the individual relative to the disease, condition or event, identifying whether further tests may be desirable, or otherwise evaluating the health status of the individual.
  • biomarker information can be retrieved for an individual.
  • the biomarker information can be retrieved from a computer database, for example, after testing of the individual’s biological sample is performed.
  • the biomarker information can comprise biomarker values that each correspond to one or more of the biomarkers of Table 5.
  • a computer can be utilized to perform a computation with each of the biomarker values.
  • an estimation or determination can be made of glomerular filtration rate.
  • the indication can be output to a display or other indicating device so that it is viewable by a person.
  • a display or other indicating device so that it is viewable by a person.
  • it can be displayed on a display screen of a computer or other output device.
  • a computer program product may include a computer readable medium having computer readable program code embodied in the medium for causing an application program to execute on a computer with a database.
  • a “computer program product” refers to an organized set of instructions in the form of natural or programming language statements that are contained on a physical media of any nature (e.g., written, electronic, magnetic, optical or otherwise) and that may be used with a computer or other automated data processing system. Such programming language statements, when executed by a computer or data processing system, cause the computer or data processing system to act in accordance with the particular content of the statements.
  • Computer program products include without limitation: programs in source and object code and/or test or data libraries embedded in a computer readable medium.
  • the computer program product that enables a computer system or data processing equipment device to act in pre-selected ways may be provided in a number of forms, including, but not limited to, original source code, assembly code, object code, machine language, encrypted or compressed versions of the foregoing and any and all equivalents.
  • a computer program product for the estimation or determination of glomerular filtration rate.
  • the computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises biomarker values that each correspond to one or more of the biomarkers of Table 5; and code that executes a computational method that indicates current state of kidney health of the individual as a function of the biomarker values.
  • a computer program product for indicating a likelihood of renal insufficiency.
  • the computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises a biomarker value corresponding to one or more of the biomarkers of Table 5; and code that executes a computational method that indicates current state of kidney health as a function of the biomarker value.
  • the embodiments may be embodied as code stored in a computer-readable memory of virtually any kind including, without limitation, RAM, ROM, magnetic media, optical media, or magneto-optical media. Even more generally, the embodiments could be implemented in software, or in hardware, or any combination thereof including, but not limited to, software running on a general purpose processor, microcode, PLAs, or ASICs.
  • embodiments could be accomplished as computer signals embodied in a carrier wave, as well as signals (e.g., electrical and optical) propagated through a transmission medium.
  • signals e.g., electrical and optical
  • the various types of information discussed above could be formatted in a structure, such as a data structure, and transmitted as an electrical signal through a transmission medium or stored on a computer readable medium.
  • biomarker identification process The biomarker identification process, the utilization of the biomarkers disclosed herein, and the various methods for determining biomarker values are described in detail above with respect to evaluating an individual’s eGFR and whether that individual may have renal insufficiency. However, the application of the process, the use of identified biomarkers, and the methods for determining biomarker values are fully applicable to other specific types of diseases or medical conditions, or to the identification of individuals who may or may not be benefited by an ancillary medical treatment.
  • Example 1 Model Specification [00410] 1.1 Model result description. Estimated glomerular filtration rate (eGFR) reported in ml/min/1.73m 2 with values from 5 to 100 ml/min/1.73 m 2 .
  • eGFR estimated glomerular filtration rate
  • the final model is a linear model with 39 features with non-zero coefficients.
  • the model was trained on a loglO transformed eGFR endpoint. This transformation means that final model estimation or determinations must be back-transformed (i.e., io estimatlon ) to deliver eGFR determinations in the scale familiar to clinicians.
  • the intercept 1.677504887 and the feature names and coefficients are shown in Table 5.
  • the feature level is the RFU (relative fluorescence units) as measured in a sample by a proteomic assay, for example, an aptamer-based assay.
  • R 2 values are provided in Tables 6a- 6e for selected Table 5 features and combinations of Table 5 features.
  • CRIC Development and validation cohort(s).
  • CRIC is a multi-site observational study initiated to explore the relationship between chronic renal insufficiency and cardiovascular disease and has since expanded to measure many outcomes that are thought to be associated with renal insufficiency such as cognitive decline and frailty.
  • CRIC enrolled patients ages 21 to 74 years of age, half of whom have diabetes mellitus. Participants had annual in-person follow up visits (where urine and plasma were collected and stored) and telephone interviews every 6 months, where study outcomes and general health status were ascertained. Study recruitment began in 2003 and recruitment lasted for about 2.5 years at 13 clinical sites in the United States; investigators continue to monitor this cohort.
  • the SomaLogic CRIC dataset includes clinical data and second annual visit samples (collected July 2003 through December 2009) for 3413 participants with kidney disease who were not yet experiencing end stage renal disease by the second annual visit.
  • the Covance study is a cross-sectional study designed to measure baseline information from normal individuals based on clinical labs, lifestyle factors, and health history.
  • Table 7 Summary statistics of clinical covariates in model training and verification data splits
  • Table 8 Summary statistics for relevant covariates in model validation dataset
  • Models developed in refinement used the training and verification dataset(s), as combined from the respective CRIC and Covance splits.
  • the final model was developed using a loglO transformation on the endpoint. This was found to reduce consistent underprediction of eGFR for those with most severe chronic kidney disease (stage 4 and stage 5), as well as retain homoscedasticity in the model residuals. Only features with greater than 0.75 correlation between assay versions 4.0 and 4.1 were used in model development.
  • Initial feature selection using stability selection yielded 73 features, which was further pared down to a final feature list of 39 features by repeated training of elastic net models. Five repeats of 10-fold cross validation were used in model training.
  • RMSE was normalized (NRMSE) to the average eGFR in each respective disease stage.
  • NRMSE as a relative error demonstrates that the model performs sufficiently well across the meaningful range of eGFR.
  • the refinement process also included assessments for robustness.
  • the proposed model passes all checks for robust performance. Given some observed bias in the residuals for those with eGFR > 100 ml/min/1.73m 2 , it is recommended that values over 100 be reported as ‘greater than 100’ for LDT use. This will avoid reporting otherwise healthy values that may be unusually high in the clinical setting, while preserving the ability to distinguish values in the unhealthy range. Threshold values other than 100 with greater clinical significance could be used for mapping as well.
  • Table 11 Imputation for out-of-range RFU values.

Abstract

The present disclosure includes biomarkers, methods, devices, reagents, systems, and kits for the evaulation of the current state of kidney function, including estimating or determining glomerular filtration rate. In one aspect, the disclosure provides biomarkers that can be used alone or in various combinations to estimate or determine glomerular filtration rate. In another aspect, methods are provided for estimation or determination of glomerular filtration rate in an individual, where the methods include detecting, in a biological sample from an individual, at least one biomarker value corresponding to at least one biomarker selected from the group of biomarkers provided in Table 5.

Description

Renal Health Determination and Uses Thereof
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority of US Provisional
Application No. 63/217,151, filed June 30, 2021, which is incorporated by reference herein in its entirety for any purpose.
FIELD OF THE INVENTION
[0002] The present application relates generally to the detection of biomarkers and methods of evaluating the current state of kidney function in an individual and, more specifically, to one or more biomarkers, methods, devices, reagents, systems, and kits used to evaluate an individual’s kidney health based on glomerular filtration rate (GFR).
BACKGROUND
[0003] The following description provides a summary of information relevant to the present application and is not an admission that any of the information provided or publications referenced herein is prior art to the present application.
[0004] Chronic kidney disease (CKD) is defined as having abnormalities of kidney structure or function present for > 3 months (Table 1) and affects approximately 13% of adults in the US; risk factors for the disease are heterogeneous and include genetic and demographic predisposition and diabetes. Kidneys serve three primary functions: they filter metabolic byproducts from the blood, produce urine and, in so doing, help to regulate blood pressure and fluid and electrolyte balance, and they have important endocrine functions. The eGFR is used to screen for early kidney damage, as one of the diagnostic modalities in chronic kidney disease (CKD), and to monitor kidney status. It is recommended that an eGFR be calculated every time a creatinine blood test is done. Serum creatinine is ordered frequently as part of routine blood chemistry and it can vary because of many factors including hydration status, muscle damage, and kidney health. Because serum creatinine originates from extra-renal factors, two low eGFR values (< 60 ml/min/1.73 m2) a few months apart are more indicative of impaired renal function then an isolated low value.
Table 1: Criteria for CKD (either of the following present for > 3 months)
Figure imgf000003_0001
_
Figure imgf000004_0001
[0005] The standard means to monitor kidney function in the clinical setting is eGFR, which reflects the amount of blood that passes through the kidneys each minute. There are several equations to calculate eGFR but they all use a combination of patient characteristics (sex, age, and race), serum creatinine, and cystatin-c when available.
[0006] Accordingly, a need exists for biomarkers, methods, devices, reagents, systems, and kits to evaluate an individual’s renal health based on estimated glomerular filtration rate (eGFR) which do not require health care providers to calculate eGFR which requires age, gender and self-identified race as inputs along with serum creatinine.
SUMMARY OF THE INVENTION
[0007] The present application discloses biomarkers, methods, devices, reagents, systems, and kits to evaluate an individual’s renal health based on eGFR or a determination of glomerular filtration rate (GFR). In certain aspects, the current state kidney function test disclosed herein is intended to estimate or determine glomerular filtration rate (GFR) from a blood sample measurement and provide current status of kidney health. The current state kidney function test can be used as part of a broader liquid health check as a convenient way to monitor kidney function and identify potential signs of early functional impairment (i.e. potential chronic kidney disease).
[0008] The current state kidney function test provides a convenient estimate of kidney function as part of a liquid health check and serves as a tool for health care providers to identify candidates for additional screening for potential functional impairment. An additional advantage of the current state kidney function test disclosed herein is that it does not require patient characteristics such as sex, age, and race as inputs.
[0009] The following numbered paragraphs [0010] - [00244] contain statements of broad combinations of the inventive technical features herein disclosed:
[0010] 1. A method comprising: a) measuring the level of TMEDA protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[0011] 2. A method comprising: a) measuring the level of ARMEL protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMFl, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[0012] 3. A method comprising: a) measuring the level of CAC02 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II,
SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of CAC02 and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[0013] 4. A method comprising: a) measuring the level of NAGPA protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHR1, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II,
SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of NAGPA and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[0014] 5. The method of aspect 1, wherein the method comprises measuring
Cystatin C and TMEDA, DSC2 and TMEDA, HPRT and TMEDA, FABP and TMEDA, PPIC and TMEDA, GBRAP and TMEDA, ISK7 and TMEDA, ARMEL and TMEDA, FSH and TMEDA, RBP and TMEDA, HE4 and TMEDA, COIAI and TMEDA, PGRP-L and TMEDA, ERBBl and TMEDA, Testican-2 and TMEDA, FHRl and TMEDA, SRCA and TMEDA, CAC02 and TMEDA, NAGPA and TMEDA, DLK1 and TMEDA, SLIT2 and TMEDA, HCC-1 and TMEDA, CDON and TMEDA, COFA1 and TMEDA, PEARl and TMEDA, Ribonuclease UK114 and TMEDA, Activin A and TMEDA, Heparin cofactor II and TMEDA, SUMF1 and TMEDA, MP2K4 and TMEDA, SELW and TMEDA, HYALl and TMEDA, al -Antitrypsin and TMEDA, MMP-7 and TMEDA, QSOX2 and TMEDA, IGDC4 and TMEDA, S100A6 and
TMEDA, ATS 13 and TMEDA.
[0015] 6. The method of aspect 2, wherein the method comprises measuring
Cystatin C and ARMEL, TMEDA and ARMEL, DSC2 and ARMEL, HPRT and ARMEL,
FABP and ARMEL, PPIC and ARMEL, GBRAP and ARMEL, ISK7 and ARMEL, FSH and ARMEL, RBP and ARMEL, HE4 and ARMEL, C01A1 and ARMEL, PGRP-L and ARMEL, ERBBl and ARMEL, Testican-2 and ARMEL, FHR1 and ARMEL, SRC A and ARMEL, CAC02 and ARMEL, NAGPA and ARMEL, DLK1 and ARMEL, SLIT2 and ARMEL, HCC-1 and ARMEL, CDON and ARMEL, COFA1 and ARMEL, PEAR1 and ARMEL, Ribonuclease UK1 14 and ARMEL, Activin A and ARMEL, Heparin cofactor II and ARMEL, SUMF1 and ARMEL, MP2K4 and ARMEL, SELW and ARMEL, HYALl and ARMEL, a 1 -Antitrypsin and ARMEL, MMP-7 and ARMEL, QSOX2 and ARMEL, IGDC4 and ARMEL, S100A6 and ARMEL, ATS 13 and ARMEL.
[0016] 7. The method of aspect 3, wherein the method comprises measuring
Cystatin C and CAC02, TMEDA and CAC02, DSC2 and CAC02, HPRT and CAC02, FABP and CAC02, PPIC and CAC02, GBRAP and CAC02, ISK7 and CAC02, ARMEL and CAC02, FSH and CAC02, RBP and CAC02, HE4 and CAC02, C01A1 and CAC02, PGRP-L and CAC02, ERBBl and CAC02, Testican-2 and CAC02, FHRl and CAC02, SRCA and CAC02, NAGPA and CAC02, DLK1 and CAC02, SLIT2 and CAC02, HCC-1 and CAC02, CDON and CAC02, COFA1 and CAC02, PEARl and CAC02, Ribonuclease UK114 and CAC02, Activin A and CAC02, Heparin cofactor II and CAC02, SUMF1 and CAC02,
MP2K4 and CAC02, SELW and CAC02, HYALl and CAC02, al -Antitrypsin and CAC02, MMP-7 and CAC02, QSOX2 and CAC02, IGDC4 and CAC02, S100A6 and CAC02, ATS 13 and CAC02.
[0017] 8. The method of aspect 4, wherein the method comprises measuring
Cystatin C and NAGPA, TMEDA and NAGPA, DSC2 and NAGPA, HPRT and NAGPA,
FABP and NAGPA, PPIC and NAGPA, GBRAP and NAGPA, ISK7 and NAGPA, ARMEL and NAGPA, FSH and NAGPA, RBP and NAGPA, HE4 and NAGPA, COl Al and NAGPA, PGRP-L and NAGPA, ERBBl and NAGPA, Testican-2 and NAGPA, FHRl and NAGPA, SRCA and NAGPA, CAC02 and NAGPA, DLK1 and NAGPA, SLIT2 and NAGPA, HCC-1 and NAGPA, CDON and NAGPA, COFA1 and NAGPA, PEARl and NAGPA, Ribonuclease UK1 14 and NAGPA, Activin A and NAGPA, Heparin cofactor II and NAGPA, SUMFl and NAGPA, MP2K4 and NAGPA, SELW and NAGPA, HYALl and NAGPA, al -Antitrypsin and NAGPA, MMP-7 and NAGPA, QSOX2 and NAGPA, IGDC4 and NAGPA, S100A6 and NAGPA, ATS 13 and NAGPA. [0018] 9. The method of aspect 1, wherein the method comprises measuring
TMEDA and ARMEL, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0019] 10. The method of aspect 1, wherein the method comprises measuring
TMEDA and CAC02, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0020] 11. The method of aspect 1, wherein the method comprises measuring
TMEDA and NAGPA, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0021] 12. The method of aspect 2, wherein the method comprises measuring
ARMEL and CAC02, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0022] 13. The method of aspect 2, wherein the method comprises measuring
ARMEL and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0023] 14. The method of aspect 3, wherein the method comprises measuring
CAC02 and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0024] 15. The method of any one of aspects 1 to 14, wherein the estimated or determined GFR is between 5 and 100 ml/min/1.73m2.
[0025] 16. The method of any one of aspects 1 to 15, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay. [0026] 17. The method of any one of aspects 1 to 16, wherein the sample is selected from blood, plasma, serum or urine.
[0027] 18. The method of any one of aspects 1 to 17, wherein the human subject is determined to have renal insufficiency.
[0028] 19. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising TMEDA protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[0029] 20. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising ARMEL protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[0030] 21. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising CAC02 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease
UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[0031] 22. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising NAGPA protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMFl, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[0032] 23. The method of aspect 19, wherein the method comprises measuring
Cystatin C and TMEDA, DSC2 and TMEDA, HPRT and TMEDA, FABP and TMEDA, PPIC and TMEDA, GBRAP and TMEDA, ISK7 and TMEDA, ARMEL and TMEDA, FSH and TMEDA, RBP and TMEDA, HE4 and TMEDA, COIAI and TMEDA, PGRP-L and TMEDA, ERBBl and TMEDA, Testican-2 and TMEDA, FHRl and TMEDA, SRCA and TMEDA, CAC02 and TMEDA, NAGPA and TMEDA, DLK1 and TMEDA, SLIT2 and TMEDA, HCC-1 and TMEDA, CDON and TMEDA, COFA1 and TMEDA, PEARl and TMEDA, Ribonuclease UK114 and TMEDA, Activin A and TMEDA, Heparin cofactor II and TMEDA, SUMFl and TMEDA, MP2K4 and TMEDA, SELW and TMEDA, HYALl and TMEDA, al -Antitrypsin and TMEDA, MMP-7 and TMEDA, QSOX2 and TMEDA, IGDC4 and TMEDA, S100A6 and TMEDA, ATS 13 and TMEDA.
[0033] 24. The method of aspect 20, wherein the method comprises measuring
Cystatin C and ARMEL, TMEDA and ARMEL, DSC2 and ARMEL, HPRT and ARMEL,
FABP and ARMEL, PPIC and ARMEL, GBRAP and ARMEL, ISK7 and ARMEL, FSH and ARMEL, RBP and ARMEL, HE4 and ARMEL, COIAI and ARMEL, PGRP-L and ARMEL, ERBB1 and ARMEL, Testican-2 and ARMEL, FHR1 and ARMEL, SRC A and ARMEL, CAC02 and ARMEL, NAGPA and ARMEL, DLK1 and ARMEL, SLIT2 and ARMEL, HCC-1 and ARMEL, CDON and ARMEL, COFA1 and ARMEL, PEAR1 and ARMEL, Ribonuclease UK1 14 and ARMEL, Activin A and ARMEL, Heparin cofactor II and ARMEL, SUMF1 and ARMEL, MP2K4 and ARMEL, SELW and ARMEL, HYALl and ARMEL, a 1 -Antitrypsin and ARMEL, MMP-7 and ARMEL, QSOX2 and ARMEL, IGDC4 and ARMEL, S100A6 and ARMEL, ATS 13 and ARMEL.
[0034] 25. The method of aspect 21, wherein the method comprises measuring
Cystatin C and CAC02, TMEDA and CAC02, DSC2 and CAC02, HPRT and CAC02, FABP and CAC02, PPIC and CAC02, GBRAP and CAC02, ISK7 and CAC02, ARMEL and CAC02, FSH and CAC02, RBP and CAC02, HE4 and CAC02, COIAI and CAC02, PGRP-L and CAC02, ERBB1 and CAC02, Testican-2 and CAC02, FHR1 and CAC02, SRCA and CAC02, NAGPA and CAC02, DLK1 and CAC02, SLIT2 and CAC02, HCC-1 and CAC02, CDON and CAC02, COFA1 and CAC02, PEAR1 and CAC02, Ribonuclease UK114 and CAC02, Activin A and CAC02, Heparin cofactor II and CAC02, SUMF1 and CAC02,
MP2K4 and CAC02, SELW and CAC02, HYALl and CAC02, al -Antitrypsin and CAC02, MMP-7 and CAC02, QSOX2 and CAC02, IGDC4 and CAC02, S100A6 and CAC02, ATS 13 and CAC02.
[0035] 26. The method of aspect 22, wherein the method comprises measuring
Cystatin C and NAGPA, TMEDA and NAGPA, DSC2 and NAGPA, HPRT and NAGPA,
FABP and NAGPA, PPIC and NAGPA, GBRAP and NAGPA, ISK7 and NAGPA, ARMEL and NAGPA, FSH and NAGPA, RBP and NAGPA, HE4 and NAGPA, COIAI and NAGPA, PGRP-L and NAGPA, ERBB1 and NAGPA, Testican-2 and NAGPA, FHR1 and NAGPA, SRCA and NAGPA, CAC02 and NAGPA, DLK1 and NAGPA, SLIT2 and NAGPA, HCC-1 and NAGPA, CDON and NAGPA, COFA1 and NAGPA, PEAR1 and NAGPA, Ribonuclease UK1 14 and NAGPA, Activin A and NAGPA, Heparin cofactor II and NAGPA, SUMFl and NAGPA, MP2K4 and NAGPA, SELW and NAGPA, HYALl and NAGPA, al -Antitrypsin and NAGPA, MMP-7 and NAGPA, QSOX2 and NAGPA, IGDC4 and NAGPA, S100A6 and NAGPA, ATS 13 and NAGPA.
[0036] 27. The method of aspect 19, wherein the method comprises measuring
TMEDA and ARMEL, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0037] 28. The method of aspect 19, wherein the method comprises measuring
TMEDA and CAC02, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0038] 29. The method of aspect 19, wherein the method comprises measuring
TMEDA and NAGPA, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0039] 30. The method of aspect 20, wherein the method comprises measuring
ARMEL and CAC02, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0040] 31. The method of aspect 20, wherein the method comprises measuring
ARMEL and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0041] 32. The method of aspect 21, wherein the method comprises measuring
CAC02 and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[0042] 33. The method of any one of aspects 19 to 32, wherein the protein levels are used to estimate or determine glomerular filtration rate (GFR) for the human subject.
[0043] 34. The method of any one of aspects 19 to 33, wherein the estimated or determined GFR is between 5 and 100 ml/min/1.73m2.
[0044] 35. The method of any one of aspects 19 to 34, wherein the set of capture reagents is selected from aptamers, antibodies and a combinations of aptamers and antibodies. [0045] 36. The method of any one of aspects 19 to 35, wherein the sample is selected from blood, plasma, serum or urine.
[0046] 37. The method of any one of aspects 19 to 36, wherein the human subject is determined to have renal insufficiency.
[0047] 38. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a ARMEL protein; and b) measuring the level of each protein with the two capture reagents.
[0048] 39. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a CAC02 protein; and b) measuring the level of each protein with the two capture reagents.
[0049] 40. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
[0050] 41. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a ARMEL protein and the second capture reagent has affinity for a CAC02 protein; and b) measuring the level of each protein with the two capture reagents.
[0051] 42. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a ARMEL protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
[0052] 43. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a CAC02 protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
[0053] 44. The method of any one of aspects 38 to 43, further comprising measuring the level of a Cystatin C protein with a capture reagent having affinity for the Cystatin C protein. [0054] 45. The method of any one of aspects 38 to 44, further comprising measuring the level of a DSC2 protein with a capture reagent having affinity for the DSC2 protein.
[0055] 46. The method of any one of aspects 38 to 45, further comprising measuring the level of a HPRT protein with a capture reagent having affinity for the HPRT protein.
[0056] 47. The method of any one of aspects 38 to 46, further comprising measuring the level of a FABP protein with a capture reagent having affinity for the FABP protein.
[0057] 48. The method of any one of aspects 38 to 47, further comprising measuring the level of a PPIC protein with a capture reagent having affinity for the PPIC protein.
[0058] 49. The method of any one of aspects 38 to 48, further comprising measuring the level of a GBRAP protein with a capture reagent having affinity for the GBRAP protein. [0059] 50. The method of any one of aspects 38 to 49, further comprising measuring the level of a ISK7 protein with a capture reagent having affinity for the ISK7 protein.
[0060] 51. The method of any one of aspects 38 to 50, further comprising measuring the level of a FSH protein with a capture reagent having affinity for the FSH protein.
[0061] 52. The method of any one of aspects 38 to 51, further comprising measuring the level of a RBP protein with a capture reagent having affinity for the RBP protein.
[0062] 53. The method of any one of aspects 38 to 52, further comprising measuring the level of a HE4 protein with a capture reagent having affinity for the HE4 protein.
[0063] 54. The method of any one of aspects 38 to 53, further comprising measuring the level of a COl A1 protein with a capture reagent having affinity for the COl A1 protein. [0064] 55. The method of any one of aspects 38 to 54, further comprising measuring the level of a PGRP-L protein with a capture reagent having affinity for the PGRP-L protein. [0065] 56. The method of any one of aspects 38 to 55, further comprising measuring the level of a ERBBl protein with a capture reagent having affinity for the ERBBl protein. [0066] 57. The method of any one of aspects 38 to 56, further comprising measuring the level of a Testican-2 protein with a capture reagent having affinity for the Testican-2 protein. [0067] 58. The method of any one of aspects 38 to 57, further comprising measuring the level of a FHR1 protein with a capture reagent having affinity for the FHR1 protein.
[0068] 59. The method of any one of aspects 38 to 58, further comprising measuring the level of a SRCA protein with a capture reagent having affinity for the SRCA protein.
[0069] 60. The method of any one of aspects 38 to 59, further comprising measuring the level of a DLK1 protein with a capture reagent having affinity for the DLK1 protein.
[0070] 61. The method of any one of aspects 38 to 60, further comprising measuring the level of a SLIT2 protein with a capture reagent having affinity for the SLIT2 protein.
[0071] 62. The method of any one of aspects 38 to 61, further comprising measuring the level of a HCC-1 protein with a capture reagent having affinity for the HCC-1 protein.
[0072] 63. The method of any one of aspects 38 to 62, further comprising measuring the level of a CDON protein with a capture reagent having affinity for the CDON protein.
[0073] 64. The method of any one of aspects 38 to 63, further comprising measuring the level of a COFA1 protein with a capture reagent having affinity for the COFA1 protein. [0074] 65. The method of any one of aspects 38 to 64, further comprising measuring the level of a PEAR1 protein with a capture reagent having affinity for the PEARl protein. [0075] 66. The method of any one of aspects 38 to 65, further comprising measuring the level of a Ribonuclease UK114 protein with a capture reagent having affinity for the Ribonuclease UK114 protein.
[0076] 67. The method of any one of aspects 38 to 66, further comprising measuring the level of a Activin A protein with a capture reagent having affinity for the Activin A protein. [0077] 68. The method of any one of aspects 38 to 67, further comprising measuring the level of a Heparin cofactor II protein with a capture reagent having affinity for the Heparin cofactor II protein.
[0078] 69. The method of any one of aspects 38 to 68, further comprising measuring the level of a SUMF1 protein with a capture reagent having affinity for the SUMF1 protein. [0079] 70. The method of any one of aspects 38 to 69, further comprising measuring the level of a MP2K4 protein with a capture reagent having affinity for the MP2K4 protein. [0080] 71. The method of any one of aspects 38 to 70, further comprising measuring the level of a SELW protein with a capture reagent having affinity for the SELW protein.
[0081] 72. The method of any one of aspects 38 to 71, further comprising measuring the level of a HYALl protein with a capture reagent having affinity for the HYALl protein. [0082] 73. The method of any one of aspects 38 to 72, further comprising measuring the level of a al -Antitrypsin protein with a capture reagent having affinity for the al -Antitrypsin protein.
[0083] 74. The method of any one of aspects 38 to 73, further comprising measuring the level of a MMP-7 protein with a capture reagent having affinity for the MMP-7 protein.
[0084]
[0085] 75. The method of any one of aspects 38 to 74, further comprising measuring the level of a QSOX2 protein with a capture reagent having affinity for the QSOX2 protein. [0086] 76. The method of any one of aspects 38 to 75, further comprising measuring the level of a IGDC4 protein with a capture reagent having affinity for the IGDC4 protein.
[0087] 77. The method of any one of aspects 38 to 76, further comprising measuring the level of a S100A6 protein with a capture reagent having affinity for the S100A6 protein. [0088] 78. The method of any one of aspects 38 to 77, further comprising measuring the level of a ATS 13 protein with a capture reagent having affinity for the ATS 13 protein.
[0089] 79. A method comprising: a) measuring the level of TMEDA and ARMEL in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and ARMEL.
[0090] 80. A method comprising: a) measuring the level of TMEDA and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and CAC02.
[0091] 81. A method comprising: a) measuring the level of TMEDA and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and NAGPA.
[0092] 82. A method comprising: a) measuring the level of ARMEL and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and CAC02.
[0093] 83. A method comprising: a) measuring the level of ARMEL and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and NAGPA.
[0094] 84. A method comprising: a) measuring the level of CAC02 and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of CAC02 and NAGPA.
[0095] 85. The method of any one of aspects 79 to 84, further comprising measuring the level of a Cystatin C protein.
[0096] 86. The method of any one of aspects 79 to 85, further comprising measuring the level of a DSC2 protein.
[0097] 87. The method of any one of aspects 79 to 86, further comprising measuring the level of a HPRT protein.
[0098] 88. The method of any one of aspects 79 to 87, further comprising measuring the level of a FABP protein.
[0099] 89. The method of any one of aspects 79 to 88, further comprising measuring the level of a PPIC protein.
[00100] 90. The method of any one of aspects 79 to 89, further comprising measuring the level of a GBRAP protein.
[00101] 91. The method of any one of aspects 79 to 90, further comprising measuring the level of a ISK7 protein.
[00102] 92. The method of any one of aspects 79 to 91, further comprising measuring the level of a FSH protein.
[00103] 93. The method of any one of aspects 79 to 92, further comprising measuring the level of a RBP protein.
[00104] 94. The method of any one of aspects 79 to 93, further comprising measuring the level of a HE4 protein.
[00105] 95. The method of any one of aspects 79 to 94, further comprising measuring the level of a COIAI protein.
[00106] 96. The method of any one of aspects 79 to 95, further comprising measuring the level of a PGRP-L protein.
[00107] 97. The method of any one of aspects 79 to 96, further comprising measuring the level of a ERBB 1 protein.
[00108] 98. The method of any one of aspects 79 to 97, further comprising measuring the level of a Testican-2 protein.
[00109] 99. The method of any one of aspects 79 to 98, further comprising measuring the level of a FHR1 protein.
[00110] 100. The method of any one of aspects 79 to 99, further comprising measuring the level of a SRCA protein. [00111] 101. The method of any one of aspects 79 to 100, further comprising measuring the level of a DLK1 protein.
[00112] 102. The method of any one of aspects 79 to 101, further comprising measuring the level of a SLIT2 protein.
[00113] 103. The method of any one of aspects 79 to 102, further comprising measuring the level of a HCC-1 protein.
[00114] 104. The method of any one of aspects 79 to 103, further comprising measuring the level of a CDON protein.
[00115] 105. The method of any one of aspects 79 to 104, further comprising measuring the level of a COFA1 protein.
[00116] 106. The method of any one of aspects 79 to 105, further comprising measuring the level of a PEAR1 protein.
[00117] 107. The method of any one of aspects 79 to 106, further comprising measuring the level of a Ribonuclease UK114 protein.
[00118] 108. The method of any one of aspects 79 to 107, further comprising measuring the level of a Activin A protein.
[00119] 109. The method of any one of aspects 79 to 108, further comprising measuring the level of a Heparin cofactor II protein.
[00120] 110. The method of any one of aspects 79 to 109, further comprising measuring the level of a SUMF1 protein.
[00121] 111. The method of any one of aspects 79 to 110, further comprising measuring the level of a MP2K4 protein.
[00122] 112. The method of any one of aspects 79 to 111, further comprising measuring the level of a SELW protein.
[00123] 113. The method of any one of aspects 79 to 112, further comprising measuring the level of a HYALl protein.
[00124] 114. The method of any one of aspects 79 to 113, further comprising measuring the level of a al -Antitrypsin protein.
[00125] 115. The method of any one of aspects 79 to 114, further comprising measuring the level of a MMP-7 protein.
[00126] 116. The method of any one of aspects 79 to 115, further comprising measuring the level of a QSOX2 protein.
[00127] 117. The method of any one of aspects 79 to 116, further comprising measuring the level of a IGDC4 protein. [00128] 118. The method of any one of aspects 79 to 117, further comprising measuring the level of a S100A6 protein.
[00129] 119. The method of any one of aspects 79 to 118, further comprising measuring the level of a ATS 13 protein.
[00130] 120. The method of any one of aspects 79 to 119, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay. [00131] 121. A method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from TMEDA, ARMEL and CAC02; and b) measuring the level of each protein with the three capture reagents.
[00132] 122. A method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from TMEDA, ARMEL and NAGPA; and b) measuring the level of each protein with the three capture reagents.
[00133] 123. A method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from ARMEL, CAC02 and NAGPA; and b) measuring the level of each protein with the three capture reagents.
[00134] 124. The method of any one of aspects 121 to 123, further comprising measuring the level of a Cystatin C protein with a capture reagent having affinity for the Cystatin C protein.
[00135] 125. The method of any one of aspects 121 to 124, further comprising measuring the level of a DSC2 protein with a capture reagent having affinity for the DSC2 protein.
[00136] 126. The method of any one of aspects 121 to 125, further comprising measuring the level of a HPRT protein with a capture reagent having affinity for the HPRT protein.
[00137] 127. The method of any one of aspects 121 to 126, further comprising measuring the level of a FABP protein with a capture reagent having affinity for the FABP protein. [00138] 128. The method of any one of aspects 121 to 127, further comprising measuring the level of a PPIC protein with a capture reagent having affinity for the PPIC protein.
[00139] 129. The method of any one of aspects 121 to 128, further comprising measuring the level of a GBRAP protein with a capture reagent having affinity for the GBRAP protein.
[00140] 130. The method of any one of aspects 121 to 129, further comprising measuring the level of a ISK7 protein with a capture reagent having affinity for the ISK7 protein.
[00141] 131. The method of any one of aspects 121 to 130, further comprising measuring the level of a FSH protein with a capture reagent having affinity for the FSH protein. [00142] 132. The method of any one of aspects 121 to 131, further comprising measuring the level of a RBP protein with a capture reagent having affinity for the RBP protein. [00143] 133. The method of any one of aspects 121 to 132, further comprising measuring the level of a HE4 protein with a capture reagent having affinity for the HE4 protein. [00144] 134. The method of any one of aspects 121 to 133, further comprising measuring the level of a COl A1 protein with a capture reagent having affinity for the COl A1 protein.
[00145] 135. The method of any one of aspects 121 to 134, further comprising measuring the level of a PGRP-L protein with a capture reagent having affinity for the PGRP-L protein.
[00146] 136. The method of any one of aspects 121 to 135, further comprising measuring the level of a ERBBl protein with a capture reagent having affinity for the ERBBl protein.
[00147] 137. The method of any one of aspects 121 to 136, further comprising measuring the level of a Testican-2 protein with a capture reagent having affinity for the Testican-2 protein.
[00148] 138. The method of any one of aspects 121 to 137, further comprising measuring the level of a FHR1 protein with a capture reagent having affinity for the FHR1 protein.
[00149] 139. The method of any one of aspects 121 to 138, further comprising measuring the level of a SRCA protein with a capture reagent having affinity for the SRCA protein. [00150] 140. The method of any one of aspects 121 to 139, further comprising measuring the level of a DLK1 protein with a capture reagent having affinity for the DLK1 protein.
[00151] 141. The method of any one of aspects 121 to 140, further comprising measuring the level of a SLIT2 protein with a capture reagent having affinity for the SLIT2 protein.
[00152] 142. The method of any one of aspects 121 to 141, further comprising measuring the level of a HCC-1 protein with a capture reagent having affinity for the HCC-1 protein.
[00153] 143. The method of any one of aspects 121 to 142, further comprising measuring the level of a CDON protein with a capture reagent having affinity for the CDON protein.
[00154] 144. The method of any one of aspects 121 to 143, further comprising measuring the level of a COFA1 protein with a capture reagent having affinity for the COFA1 protein.
[00155] 145. The method of any one of aspects 121 to 144, further comprising measuring the level of a PEAR1 protein with a capture reagent having affinity for the PEARl protein.
[00156] 146. The method of any one of aspects 121 to 145, further comprising measuring the level of a Ribonuclease UK114 protein with a capture reagent having affinity for the Ribonuclease UK114 protein.
[00157] 147. The method of any one of aspects 121 to 146, further comprising measuring the level of a Activin A protein with a capture reagent having affinity for the Activin A protein.
[00158] 148. The method of any one of aspects 121 to 147, further comprising measuring the level of a Heparin cofactor II protein with a capture reagent having affinity for the Heparin cofactor II protein.
[00159] 149. The method of any one of aspects 121 to 148, further comprising measuring the level of a SUMF1 protein with a capture reagent having affinity for the SUMF1 protein.
[00160] 150. The method of any one of aspects 121 to 149, further comprising measuring the level of a MP2K4 protein with a capture reagent having affinity for the MP2K4 protein. [00161] 151. The method of any one of aspects 121 to 150, further comprising measuring the level of a SELW protein with a capture reagent having affinity for the SELW protein.
[00162] 152. The method of any one of aspects 121 to 151, further comprising measuring the level of a HYAL1 protein with a capture reagent having affinity for the HYAL1 protein.
[00163] 153. The method of any one of aspects 121 to 152, further comprising measuring the level of a al -Antitrypsin protein with a capture reagent having affinity for the al- Antitrypsin protein.
[00164] 154. The method of any one of aspects 121 to 153, further comprising measuring the level of a MMP-7 protein with a capture reagent having affinity for the MMP-7 protein.
[00165] 155. The method of any one of aspects 121 to 154, further comprising measuring the level of a QSOX2 protein with a capture reagent having affinity for the QSOX2 protein.
[00166] 156. The method of any one of aspects 121 to 155, further comprising measuring the level of a IGDC4 protein with a capture reagent having affinity for the IGDC4 protein.
[00167] 157. The method of any one of aspects 121 to 156, further comprising measuring the level of a S100A6 protein with a capture reagent having affinity for the S100A6 protein.
[00168] 158. The method of any one of aspects 121 to 157, further comprising measuring the level of a ATS 13 protein with a capture reagent having affinity for the ATS 13 protein.
[00169] 159. A method compri sing : a) measuring the level of TMEDA, ARMEL and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA, ARMEL and CAC02.
[00170] 160. A method comprising: a) measuring the level of TMEDA, ARMEL and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA, ARMEL and NAGPA.
[00171] 161. A method comprising: a) measuring the level of ARMEL, CAC02 and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL, CAC02 and NAGPA.
[00172] 162. The method of any one of aspects 159 to 161, further comprising measuring the level of a Cystatin C protein.
[00173] 163. The method of any one of aspects 159 to 162, further comprising measuring the level of a DSC2 protein.
[00174] 164. The method of any one of aspects 159 to 163, further comprising measuring the level of a HPRT protein.
[00175] 165. The method of any one of aspects 159 to 164, further comprising measuring the level of a FABP protein.
[00176] 166. The method of any one of aspects 159 to 165, further comprising measuring the level of a PPIC protein.
[00177] 167. The method of any one of aspects 159 to 166, further comprising measuring the level of a GBRAP protein.
[00178] 168. The method of any one of aspects 159 to 167, further comprising measuring the level of a ISK7 protein.
[00179] 169. The method of any one of aspects 159 to 168, further comprising measuring the level of a FSH protein.
[00180] 170. The method of any one of aspects 159 to 169, further comprising measuring the level of a RBP protein.
[00181] 171. The method of any one of aspects 159 to 170, further comprising measuring the level of a HE4 protein.
[00182] 172. The method of any one of aspects 159 to 171, further comprising measuring the level of a COIAI protein.
[00183] 173. The method of any one of aspects 159 to 172, further comprising measuring the level of a PGRP-L protein.
[00184] 174. The method of any one of aspects 159 to 173, further comprising measuring the level of a ERBB1 protein.
[00185] 175. The method of any one of aspects 159 to 174, further comprising measuring the level of a Testican-2 protein.
[00186] 176. The method of any one of aspects 159 to 175, further comprising measuring the level of a FHR1 protein. [00187] 177. The method of any one of aspects 159 to 176, further comprising measuring the level of a SRCA protein.
[00188] 178. The method of any one of aspects 159 to 177, further comprising measuring the level of a DLK1 protein.
[00189] 179. The method of any one of aspects 159 to 178, further comprising measuring the level of a SLIT2 protein.
[00190] 180. The method of any one of aspects 159 to 179, further comprising measuring the level of a HCC-1 protein.
[00191] 181. The method of any one of aspects 159 to 180, further comprising measuring the level of a CDON protein.
[00192] 182. The method of any one of aspects 159 to 181, further comprising measuring the level of a COFA1 protein.
[00193] 183. The method of any one of aspects 159 to 182, further comprising measuring the level of a PEARl protein.
[00194] 184. The method of any one of aspects 159 to 183, further comprising measuring the level of a Ribonuclease UK114 protein.
[00195] 185. The method of any one of aspects 159 to 184, further comprising measuring the level of a Activin A protein.
[00196] 186. The method of any one of aspects 159 to 185, further comprising measuring the level of a Heparin cofactor II protein.
[00197] 187. The method of any one of aspects 159 to 186, further comprising measuring the level of a SUMF1 protein.
[00198] 188. The method of any one of aspects 159 to 187, further comprising measuring the level of a MP2K4 protein.
[00199] 189. The method of any one of aspects 159 to 188, further comprising measuring the level of a SELW protein.
[00200] 190. The method of any one of aspects 159 to 189, further comprising measuring the level of a HYALl protein.
[00201] 191. The method of any one of aspects 159 to 190, further comprising measuring the level of a al -Antitrypsin protein.
[00202] 192. The method of any one of aspects 159 to 191, further comprising measuring the level of a MMP-7 protein.
[00203] 193. The method of any one of aspects 159 to 192, further comprising measuring the level of a QSOX2 protein. [00204] 194. The method of any one of aspects 159 to 193 further comprising measuring the level of a IGDC4 protein.
[00205] 195. The method of any one of aspects 159 to 194, further comprising measuring the level of a S100A6 protein.
[00206] 196. The method of any one of aspects 159 to 195, further comprising measuring the level of a ATS 13 protein.
[00207] 197. The method of any one of aspects 159 to 196, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay. [00208] 198. A method comprising: a) measuring the level of at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty-five, thirty-six, thirty- seven, thirty-eight proteins, or thirty-nine proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of the at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty- eight or thirty-nine proteins.
[00209] 199. The method of aspect 198, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
[00210] 200. The method of aspect 198 or 199, wherein the sample is selected from blood, plasma, serum or urine.
[00211] 201. The method of any one of aspects 198 to 200, wherein the human subject is determined to have renal insufficiency.
[00212] 202. The method of any one of aspects 198 to 201, further comprising measuring one or more of TMEDA, ARMEL, CAC02 and NAGPA.
[00213] 203. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, thirty-eight proteins, or thirty-nine proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC,
GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[00214] 204. The method of aspect 203, wherein the set of capture reagents are selected from aptamers, antibodies and a combinations of aptamers and antibodies.
[00215] 205. The method of aspect 203 or 204, wherein the sample is selected from blood, plasma, serum or urine.
[00216] 206. The method of any one of aspects 203 to 205, wherein the human subject is determined to have renal insufficiency.
[00217] 207. The method of any one of aspects 203 to 206, further comprising measuring one or more of TMEDA, ARMEL, CAC02 and NAGPA.
[00218] 208. A method comprising: a) measuring the level of Cystatin C protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC- 1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of Cystatin C and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00219] 209. A method comprising: a) measuring the level of DSC2 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C , TMEDA, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of DSC2 and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00220] 210. A method comprising: a) measuring the level of HPRT protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMFl, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of HPRT and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00221] 211. A method comprising: a) measuring the level of FABP protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of FABP and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00222] 212. A method comprising: a) measuring the level of PPIC protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMFl, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of PPIC and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00223] 213. A method comprising: a) measuring the level of GBRAP protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of GBRAP and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00224] 214. A method comprising: a) measuring the level of ISK7 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty -three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty -five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMFl, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ISK7 and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty- seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty -three, thirty -four, thirty- five, thirty-six, thirty-seven, or thirty-eight proteins.
[00225] 215. The method of any one of aspects 208 to 214, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay. [00226] 216. The method of any one of aspects 208 to 215, wherein the sample is selected from blood, plasma, serum or urine.
[00227] 217. The method of any one of aspects 208 to 216, wherein the human subject is determined to have renal insufficiency.
[00228] 218. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising Cystatin C protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[00229] 219. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising DSC2 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C , TMEDA, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[00230] 220. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising HPRT protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[00231] 221. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising FABP protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[00232] 222. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising PPIC protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents. [00233] 223. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising GBRAP protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty- nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
[00234] 224. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising ISK7 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al- Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents
[00235] 225. The method of any one of aspects 218 to 224, wherein the set of capture reagents is selected from aptamers, antibodies and a combinations of aptamers and antibodies. [00236] 226. The method of any one of aspects 218 to 225, wherein the sample is selected from blood, plasma, serum or urine.
[00237] 227. The method of any one of aspects 218 to 226, wherein the human subject is determined to have renal insufficiency. [00238] 228. The method of any one of aspects 19 to 32, 38 to 78, 121 to 197, 203 to 207, and 218 to 227, further comprising estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of each protein measured.
[00239] 229. The method of any one of aspects 1 to 18, 3 to 37, 79 to 120, 198 to 202,
208 to 217, and 228, wherein estimating or determining GFR for the human subject is based on input of the level of each protein measured in a statistical model.
[00240] 230. The method of aspect 229, wherein the model is a linear model for the log of estimated GFR.
[00241] 231. The method of aspect 229 or 230, wherein the R2 value is > 0.65, 0.7, 0.75, or 0 8
[00242] 232. The method of any one of aspects 229 to 231, wherein model output is an estimation or determination of GFR as a continuous integer value from 5 to 100 ml/min/1.73 m2, inclusive.
[00243] 233. The method of any one of aspects 229 to 232, wherein the model estimates or determines glomerular filtration rate (GFR) for the human subject based on the level of each protein measured selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYAL1, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
[00244] 234. The method of any one of aspects 228-233, wherein the level of each protein measure is determined from a relative florescence unit (RFU) or a protein concentration.
BRIEF DESCRIPTION OF THE DRAWINGS
[00245] Figure 1 shows a concordance plot of true eGFR values vs. estimated values of validation data.
[00246] Figure 2 illustrates an exemplary computer system for use with various computer-implemented methods described herein.
[00247] Figure 3 is a flowchart for a method of estimating or determining glomerular filtration rate in accordance with one embodiment.
DETAILED DESCRIPTION
[00248] Reference will now be made in detail to representative embodiments of the invention. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that the invention is not intended to be limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.
[00249] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in and are within the scope of the practice of the present invention. The present invention is in no way limited to the methods and materials described.
[00250] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
[00251] All publications, published patent documents, and patent applications cited in this application are indicative of the level of skill in the art(s) to which the application pertains. All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
[00252] As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.” Thus, reference to “a SOMAmer” includes mixtures of SOMAmers, reference to “a probe” includes mixtures of probes, and the like.
[00253] As used herein, the term “about” represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.
[00254] As used herein, the phrase “Adaptive Normalization by Maximum Likelihood” refers to a process for normalizing analytes to mitigate site bias.
[00255] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter. [00256] The present application includes biomarkers, methods, devices, reagents, systems, and kits for the estimation or determination of the current state of kidney function. [00257] “Progressive Chronic Renal Insufficiency” or “PCRI” or “renal insufficiency” means a composite endpoint which is treated as a classification endpoint (yes/no within a given time frame) as defined by the development of at least one of the following within the time frame from test results:
• a 50% decline in estimated glomerular filtration rate (eGFR),
• a diagnosis that kidney dialysis is needed,
• development of eGFR < 15 ml/min/1.73 m2,
• development of end stage renal disease (ESRD), or
• a diagnosis that a kidney transplantation is needed.
[00258] “End Stage Renal Disease” or “ESRD” means that at least one of the following conditions are met: glomerular filtration rate is less than 15 ml/min/1.73 m2, chronic renal dialysis is needed, or kidney transplantation is needed.
[00259] “Relative risk” means the risk for developing PCRI in a given time frame as compared to the average risk in a reference population. The range for relative risk is 0.01-3.24.
In one aspect, relative risk can be calculated
V *
RR = — q wherein p* is the probability that an individual develops PCRI within 4 years and q is the probability for the baseline individual in a training cohort.
[00260] “Biological sample”, “sample”, and “test sample” are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained or otherwise derived from an individual. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, huffy coat, plasma, and serum), dried blood spots (e.g., obtained from infants), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid. This also includes experimentally separated fractions of all of the preceding. For example, a blood sample can be fractionated into serum, plasma or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes). If desired, a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample. The term “biological sample” also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example. The term “biological sample” also includes materials derived from a tissue culture or a cell culture. Any suitable methods for obtaining a biological sample can be employed; exemplary methods include, e.g., phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsy procedure. Exemplary tissues susceptible to fine needle aspiration include lymph node, lung, lung washes, BAL (bronchoalveolar lavage), thyroid, breast, pancreas and liver. Samples can also be collected, e.g., by micro dissection (e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)), bladder wash, smear (e.g., a PAP smear), or ductal lavage. A “biological sample” obtained or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual. [00261] Further, it should be realized that a biological sample can be derived by taking biological samples from a number of individuals and pooling them or pooling an aliquot of each individual’s biological sample.
[00262] As mentioned above, the biological sample can be urine. Urine samples provide certain advantages over blood or serum samples. Collecting blood or plasma samples through venipuncture is more complex than is desirable, can deliver variable volumes, can be worrisome for the patient, and involves some (small) risk of infection. Also, phlebotomy requires skilled personnel. The simplicity of collecting urine samples can lead to more widespread application of the subject methods.
[00263] “Computation” as used herein refers to any type of mathematical calculation, including arithmetical and non-arithmetical steps.
[00264] For purposes of this specification, the phrase “data attributed to a biological sample from an individual” is intended to mean that the data in some form derived from, or were generated using, the biological sample of the individual. The data may have been reformatted, revised, or mathematically altered to some degree after having been generated, such as by conversion from units in one measurement system to units in another measurement system; but, the data are understood to have been derived from, or were generated using, the biological sample.
[00265] “Target”, “target molecule", and “analyte” are used interchangeably herein to refer to any molecule of interest that may be present in a biological sample. A “molecule of interest” includes any minor variation of a particular molecule, such as, in the case of a protein, for example, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule. A “target molecule", “target”, or “analyte” is a set of copies of one type or species of molecule or multi-molecular structure. “Target molecules", “targets”, and “analytes” refer to more than one such set of molecules. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affybodies, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing.
[00266] As used herein, “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can be single chains or associated chains. Also included within the definition are preproteins and intact mature proteins; peptides or polypeptides derived from a mature protein; fragments of a protein; splice variants; recombinant forms of a protein; protein variants with amino acid modifications, deletions, or substitutions; digests; and post-translational modifications, such as glycosylation, acetylation, phosphorylation, and the like.
[00267] As used herein, “marker” and “biomarker” and “feature” are used interchangeably to refer to a target molecule that indicates or is a sign of a normal or abnormal process in an individual or of a disease or other condition in an individual. More specifically, a “marker” or “biomarker” or “feature” is an anatomic, physiologic, biochemical, or molecular parameter associated with the presence of a specific physiological state or process, whether normal or abnormal, and, if abnormal, whether chronic or acute. Biomarkers are detectable and measurable by a variety of methods including laboratory assays and medical imaging. When a biomarker is a protein, it is also possible to use the expression of the corresponding gene as a surrogate measure of the amount or presence or absence of the corresponding protein biomarker in a biological sample or methylation state of the gene encoding the biomarker or proteins that control expression of the biomarker. In certain aspects, a feature is an analyte/ SOMAmer reagent of other predictors in a statistical model.
[00268] As used herein, “biomarker value", “value”, “biomarker level", “feature level” and “level” are used interchangeably to refer to a measurement that is made using any analytical method for detecting the biomarker in a biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, a level, an expression level, a ratio of measured levels, or the like, of, for, or corresponding to the biomarker in the biological sample. The exact nature of the “value” or “level” depends on the specific design and components of the particular analytical method employed to detect the biomarker.
[00269] When a biomarker indicates or is a sign of an abnormal process or a disease or other condition in an individual, that biomarker is generally described as being either over expressed or under-expressed as compared to an expression level or value of the biomarker that indicates or is a sign of a normal process or an absence of a disease or other condition in an individual. “Up-regulation", “up-regulated", “over-expression", “over-expressed", and any variations thereof are used interchangeably to refer to a value or level of a biomarker in a biological sample that is greater than a value or level (or range of values or levels) of the biomarker that is typically detected in similar biological samples from healthy or normal individuals. The terms may also refer to a value or level of a biomarker in a biological sample that is greater than a value or level (or range of values or levels) of the biomarker that may be detected at a different stage of a particular disease.
[00270] "Down-regulation", “down-regulated", “under-expression", “under-expressed", and any variations thereof are used interchangeably to refer to a value or level of a biomarker in a biological sample that is less than a value or level (or range of values or levels) of the biomarker that is typically detected in similar biological samples from healthy or normal individuals. The terms may also refer to a value or level of a biomarker in a biological sample that is less than a value or level (or range of values or levels) of the biomarker that may be detected at a different stage of a particular disease.
[00271] Further, a biomarker that is either over-expressed or under-expressed can also be referred to as being “differentially expressed” or as having a “differential level” or “differential value” as compared to a “normal” expression level or value of the biomarker that indicates or is a sign of a normal process or an absence of a disease or other condition in an individual. Thus, “differential expression” of a biomarker can also be referred to as a variation from a “normal” expression level of the biomarker.
[00272] The term “differential gene expression” and “differential expression” are used interchangeably to refer to a gene (or its corresponding protein expression product) whose expression is activated to a higher or lower level in a subject suffering from a specific disease or condition, relative to its expression in a normal or control subject. The terms also include genes (or the corresponding protein expression products) whose expression is activated to a higher or lower level at different stages of the same disease or condition. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product. Such differences may be evidenced by a variety of changes including mRNA levels, surface expression, secretion or other partitioning of a polypeptide. Differential gene expression may include a comparison of expression between two or more genes or their gene products; or a comparison of the ratios of the expression between two or more genes or their gene products; or even a comparison of two differently processed products of the same gene, which differ between normal subjects and subjects suffering from a disease; or between various stages of the same disease. Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
[00273] As used herein, “individual” refers to a test subject or patient. The individual can be a mammal or a non-mammal. In various embodiments, the individual is a mammal. A mammalian individual can be a human or non-human. In various embodiments, the individual is a human. A healthy or normal individual is an individual in which the disease or condition of interest (including, for example, renal insufficiency) is not detectable by conventional diagnostic methods.
[00274] “Diagnose”, “diagnosing”, “diagnosis”, and variations thereof refer to the detection, determination, or recognition of a health status or condition of an individual on the basis of one or more signs, symptoms, data, or other information pertaining to that individual. The health status of an individual can be diagnosed as healthy / normal (i.e., a diagnosis of the absence of a disease or condition) or diagnosed as ill / abnormal (i.e., a diagnosis of the presence, or an assessment of the characteristics, of a disease or condition). The terms “diagnose”, “diagnosing”, “diagnosis”, etc., encompass, with respect to a particular disease or condition, the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of disease response after the administration of a treatment or therapy to the individual.
Determining or estimating the glomerular filtration rate (eGFR) and thus the presence of renal insufficiency and the current state of kidney health.
[00275] As used herein “Mean Absolute Error” or “MAE” refers to the mean of the absolute values of the prediction error on all instances of a dataset.
[00276] As used herein, “Normalized Root Mean Square Error or “NRMSE” refers to the standard deviation of prediction errors (residuals) divided by the mean of the outcome.
[00277] As used herein, “Root Mean Square Error” or “RMSE” refers to the standard deviation of prediction errors (residuals). [00278] As used herein the term “predict” refers to an estimation regarding a state or a condition in the present or in the future. In one aspect, to predict or making a prediction refers to an estimation regarding the current state of kidney function. In certain aspects, a prediction regarding the current state of kidney function can be an estimation of glomerular filtration rate. [00279] “Prognose”, “prognosing”, “prognosis”, and variations thereof refer to the prediction of a future course of a disease or condition in an individual who has the disease or condition (e.g., predicting patient survival), and such terms encompass the evaluation of disease or condition response after the administration of a treatment or therapy to the individual.
[00280] As used herein the term “R2” refers to the proportion of the variance in outcome that can be explained by a model.
[00281] “Evaluate”, “evaluating”, “evaluation”, and variations thereof encompass both “diagnose” and “prognose” and also encompass determinations or estimations about the current or future course of a disease or condition in an individual who may or may not have the disease as well as determinations or estimations regarding the risk that a disease or condition will recur in an individual who apparently has been cured of the disease or has had the condition resolved. The term “evaluate” also encompasses assessing an individual’s response to a therapy, such as, for example, determining whether an individual is likely to respond favorably to a therapeutic agent or is unlikely to respond to a therapeutic agent (or will experience toxic or other undesirable side effects, for example), selecting a therapeutic agent for administration to an individual, or monitoring or determining an individual’s response to a therapy that has been administered to the individual. Thus, “evaluating” the glomerular filtration rate (eGFR) of individuals to determine the current state of kidney health in the individual and thus renal insufficiency, which can include, for example, any of the following: determining an individual’s response to a renal insufficiency treatment or selecting a renal insufficiency treatment to administer to an individual based upon a determination of the biomarker values derived from the individual’s biological sample. Evaluation renal insufficiency can include embodiments such as the assessment of renal insufficiency on a continuous scale, or classification of renal insufficiency in escalating classifications. Classification of insufficiency includes, for example, classification into two or more classifications such as “No renal insufficiency” and “renal insufficiency.”
[00282] As used herein, “additional biomedical information” refers to one or more evaluations of an individual, other than using any of the biomarkers described herein, that are associated current state of kidney health. “Additional biomedical information” includes any of the following: physical descriptors of an individual, including the height and/or weight of an individual; the age of an individual; the gender of an individual; change in weight; the ethnicity of an individual; occupational history; family history of renal insufficiency; the presence of a genetic marker(s) correlating with a higher risk of renal insufficiency in the individual; clinical symptoms such as abdominal pain, weight gain or loss gene expression values; physical descriptors of an individual, including physical descriptors observed by radiologic imaging; smoking status; alcohol use history; occupational history; dietary habits - salt, saturated fat and cholesterol intake; caffeine consumption; and imaging information. Testing of biomarker levels in combination with an evaluation of any additional biomedical information, including other laboratory tests, may, for example, improve sensitivity, specificity, and/or AUC for estimation or determination of current state of kidney health as compared to biomarker testing alone or evaluating any particular item of additional biomedical information alone (e.g., carotid intima thickness imaging alone). Additional biomedical information can be obtained from an individual using routine techniques known in the art, such as from the individual themselves by use of a routine patient questionnaire or health history questionnaire, etc., or from a medical practitioner, etc. Testing of biomarker levels in combination with an evaluation of any additional biomedical information may, for example, improve sensitivity, specificity, and/or thresholds for estimation or determination of the current state of kidney function as compared to biomarker testing alone or evaluating any particular item of additional biomedical information alone (e.g., CT imaging alone).
[00283] As used herein, “detecting” or “determining” with respect to a biomarker value includes the use of both the instrument required to observe and record a signal corresponding to a biomarker value and the material/s required to generate that signal. In various embodiments, the biomarker value is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.
[00284] "Solid support” refers herein to any substrate having a surface to which molecules may be attached, directly or indirectly, through either covalent or non-covalent bonds. A “solid support” can have a variety of physical formats, which can include, for example, a membrane; a chip (e.g., a protein chip); a slide (e.g., a glass slide or coverslip); a column; a hollow, solid, semi-solid, pore- or cavity- containing particle, such as, for example, a bead; a gel; a fiber, including a fiber optic material; a matrix; and a sample receptacle. Exemplary sample receptacles include sample wells, tubes, capillaries, vials, and any other vessel, groove or indentation capable of holding a sample. A sample receptacle can be contained on a multi sample platform, such as a microtiter plate, slide, microfluidics device, and the like. A support can be composed of a natural or synthetic material, an organic or inorganic material. The composition of the solid support on which capture reagents are attached generally depends on the method of attachment (e.g., covalent attachment). Other exemplary receptacles include microdroplets and microfluidic controlled or bulk oil/aqueous emulsions within which assays and related manipulations can occur. Suitable solid supports include, for example, plastics, resins, polysaccharides, silica or silica-based materials, functionalized glass, modified silicon, carbon, metals, inorganic glasses, membranes, nylon, natural fibers (such as, for example, silk, wool and cotton), polymers, and the like. The material composing the solid support can include reactive groups such as, for example, carboxy, amino, or hydroxyl groups, which are used for attachment of the capture reagents. Polymeric solid supports can include, e.g., polystyrene, polyethylene glycol tetraphthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile, polymethyl methacrylate, polytetrafluoroethylene, butyl rubber, styrenebutadiene rubber, natural rubber, polyethylene, polypropylene, (poly)tetrafluoroethylene, (poly)vinylidenefluoride, polycarbonate, and polymethylpentene. Suitable solid support particles that can be used include, e.g., encoded particles, such as Luminex®-type encoded particles, magnetic particles, and glass particles.
[00285] As used herein, “adaptive normalization by maximum likelihood” means a process for normalizing the analytes to mitigate site bias.
[00286] As used herein, “analyte” is the protein target of a capture reagent. In certain aspects, the capture reagent is an aptamer. In certain further aspects, the capture reagent is a SOMAmer.
[00287] As used herein, “Lin’s Concordance correlation coefficient” or “Lin’s CCC” means concordance correlation coefficient which measures the concordance between a new test and an existing test that is considered the gold standard.
[00288] As used herein, “study”, means a set of samples and clinical data that are analyzed to derive the test.
[00289] As used herein, “training dataset”, means a subset of data from a study used to fit a model.
[00290] As used herein, “validation dataset”, means a final subset of data used to assess the performance of a final model developed on a verification dataset.
[00291] As used herein, “verification dataset”, means a separate subset of data used to provide an unbiased evaluation of a model fit on the training dataset while tuning model parameters. [00292] As used herein, the term “need” or “needed” refers to a judgement made by a health care provider regarding treatment of a patient which is considered by the health care provider to be beneficial to the health status of the patient.
[00293] In one aspect, a test is disclosed that reflects the current state of kidney health using estimated glomerular filtration rate (eGFR) as the truth standard. In certain aspects, the test can be used in adults and provides an eGFR result which may be used to screen for early kidney damage and to monitor kidney status.
[00294] In one aspect, the test for kidney health was developed using two cohorts: The Chronic Renal Insufficiency Cohort (CRIC) and Covance, both of which were split into training (70%), verification (15%), and validation datasets (15%). The minimum performance requirement for a useful test was set at an R2 of 0.8 for estimating or determining eGFR endpoint as calculated by the CKD epi equation. (Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009; 150:604-612.) The model was developed using linear regression and contains 39 features. In some aspects, the minimum performance requirement for a useful test is an R2 value > 0.65, 0.7, 0.75, or 0.8. [00295] In certain aspects, the model output is an estimation or determination of GFR as a continuous integer value of from 5 to 100 ml/min/1.73 m2, inclusive. Values < 5 ml/min/1.73 m2 and > 100 ml/min/1.73 m2 will be reported as < 5 ml/min/1.73 m2 and > 100 ml/min/1.73 m2, respectively. Validation exceeds the performance metric of an R2 > 0.8 (Table 2).
Table 2: R2 metrics for training, verification, and validation for the final model
Figure imgf000043_0002
[00296] The testing methods disclosed herein provide convenience for health care providers for estimating or determining glomerular filtration rate (GFR) from a blood sample measurement and providing current status of kidney health [00297] Indications for use include but are not limited to those of Table 3.
Table 3: Indication for Use
Application
Figure imgf000043_0001
Indication for Use Statements
Figure imgf000044_0001
[00298] In certain aspects, in RUO contexts, the benefits and risks pertain to decision making in research studies for participant monitoring, stratification, and enrichment.
[00299] Benefits of the current state kidney function test disclosed herein include: a convenient reflection of kidney function that does not require health care providers to calculate eGFR, which requires age, gender and self-identified race as inputs along with serum creatinine; identification of patients with potentially decreased kidney function to identify candidates for additional diagnostics and treatment for suspicion of early stages of CKD; and earlier identification of potential kidney disease increases the ability to slow the progression of disease and minimize the symptoms that a patient may experience.
[00300] The test disclosed herein provides a convenient method for health care providers to assess and monitor kidney function and may help to identify patients who are at risk for loss of kidney function and require referral for additional diagnostics. In one aspect, a further benefit of the test disclosed herein is that it can be bundled with other tests which measure protein levels in a sample as part of a liquid health check thus enabling an assessment of general health using one modality and a single blood draw.
[00301] The test disclosed herein can be used in conjunction with additional assessments including but not limited to health status assessments, including evaluations of comorbid conditions such as diabetes, additional laboratory tests including but not limited to measurement of serum creatinine, urine albumin, clinical pathology, renal imaging, and histology.
[00302] In certain aspects, the test disclosed herein was evaluated based on a minimum performance requirement of R2=0.8 in estimation or determination of glomerular filtration rate as calculated with the 2009 CKD-epi equation (Eq 1)
Eq 1 : 2009 CKD-epi equation for estimate glomerular filtration rate eGFR = 141 x min(Scr/K, l)a x max(Scr /K, l) 1209 X 0.993 Age X 1.018 [iffemale*] x 1.159 [if Black*]
Where: eGFR (estimated glomerular filtration rate) = mL/min/1.73 m2
Scr (standardized serum creatinine) = mg/dL
K = 0.7 (females) or 0.9 (males) a = -0.329 (females) or -0.411 (males) min = indicates the minimum of Scr/k or 1 max = indicates the maximum of Scr/k or 1 age = years
*If male and/or non-black race then the respective term is not included in the equation [00303] In one aspect, one or more biomarkers are provided for use either alone or in various combinations to evaluate the current state of kidney function. As described in detail below, exemplary embodiments include the biomarkers provided in Table 5, which were identified using a multiplex SOMAmer-based assay.
[00304] In a preferred embodiment, the model has 39 features (Table 5) and estimates or determines glomerular filtration rate (GFR). The model output is a continuous integer between the values of 5 and 100 ml/min/1.73m2.
[00305] In one embodiment, the number of biomarkers useful for a biomarker subset or panel is based on a selection of biomarkers with non-zero coefficients as a measure of estimation power for GFR.
[00306] Another factor that can affect the number of biomarkers to be used in a subset or panel of biomarkers is the procedures used to obtain biological samples from individuals who are being assessed for risk of renal insufficiency. In a carefully controlled sample procurement environment, the number of biomarkers necessary to meet desired eGFR predictive power will be lower than in a situation where there can be more variation in sample collection, handling and storage. Exemplary Uses of Biomarkers
[00307] In various exemplary embodiments, methods are provided for estimating or determining renal health by detecting one or more biomarker values corresponding to one or more biomarkers that are present in the circulation of an individual, such as in serum or plasma, by any number of analytical methods, including any of the analytical methods described herein. [00308] In addition to testing biomarker levels as a stand-alone diagnostic test, biomarker levels can also be done in conjunction with determination of SNPs or other genetic lesions or variability that are indicative of increased risk of susceptibility of disease or condition. (See, e.g., Amos et ah, Nature Genetics 40, 616-622 (2009)).
[00309] In addition to testing biomarker levels as a stand-alone diagnostic test, biomarker levels can also be used in conjunction with screening methods, including renal imaging techniques, and more specifically, radiologic screening. Biomarker levels can also be used in conjunction with relevant symptoms or genetic testing. Detection of any of the biomarkers described herein may be useful to evaluate and/or to guide appropriate clinical care of the individual, whether the individual has healthy renal function, renal insufficiency. In addition to testing biomarker levels in conjunction with relevant symptoms or risk factors, information regarding the biomarkers can also be evaluated in conjunction with other types of data, particularly data that indicates an individual’s current state of kidney health (e.g., patient clinical history, symptoms, family history, history of smoking or alcohol use, risk factors such as the presence of a genetic marker(s), and/or status of other biomarkers, etc.). These various data can be assessed by automated methods, such as a computer program/software, which can be embodied in a computer or other apparatus/device.
[00310] In addition to testing biomarker levels in conjunction with radiologic screening in high risk individuals (e.g., assessing biomarker levels in conjunction with blockage detected in a coronary angiogram), information regarding the biomarkers can also be evaluated in conjunction with other types of data, particularly data that indicates an individual’s current state of kidney health (e.g., patient clinical history, symptoms, family history of renal disease, risk factors such as whether or not the individual is a smoker, heavy alcohol user and/or status of other biomarkers, etc.). These various data can be assessed by automated methods, such as a computer program/software, which can be embodied in a computer or other apparatus/device.
[00311] Any of the described biomarkers may also be used in imaging tests. For example, an imaging agent can be coupled to any of the described biomarkers, which can be used to aid in estimating or determining the glomerular filtration rate (eGFR) and also the presence of absence of renal insufficiency, to monitor response to therapeutic interventions, to select for target populations in a clinical trial among other uses. Detection and Determination of Biomarkers and Biomarker Values
[00312] A biomarker value for the biomarkers described herein can be detected using any of a variety of known analytical methods. In one embodiment, a biomarker value is detected using a capture reagent. As used herein, a “capture agent” or “capture reagent” refers to a molecule that is capable of binding specifically to a biomarker. In various embodiments, the capture reagent can be exposed to the biomarker in solution or can be exposed to the biomarker while the capture reagent is immobilized on a solid support. In other embodiments, the capture reagent contains a feature that is reactive with a secondary feature on a solid support. In these embodiments, the capture reagent can be exposed to the biomarker in solution, and then the feature on the capture reagent can be used in conjunction with the secondary feature on the solid support to immobilize the biomarker on the solid support. The capture reagent is selected based on the type of analysis to be conducted. Capture reagents include but are not limited to SOMAmers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, an F(ab')2 fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand binding receptor, affybodies, nanobodies, imprinted polymers, avimers, peptidomimetics, a hormone receptor, a cytokine receptor, and synthetic receptors, and modifications and fragments of these.
[00313] In some embodiments, a biomarker value is detected using a biomarker/capture reagent complex.
[00314] In other embodiments, the biomarker value is derived from the biomarker/capture reagent complex and is detected indirectly, such as, for example, as a result of a reaction that is subsequent to the biomarker/capture reagent interaction, but is dependent on the formation of the biomarker/capture reagent complex.
[00315] In some embodiments, the biomarker value is detected directly from the biomarker in a biological sample.
[00316] In one embodiment, the biomarkers are detected using a multiplexed format that allows for the simultaneous detection of two or more biomarkers in a biological sample. In one embodiment of the multiplexed format, capture reagents are immobilized, directly or indirectly, covalently or non-covalently, in discrete locations on a solid support. In another embodiment, a multiplexed format uses discrete solid supports where each solid support has a unique capture reagent associated with that solid support, such as, for example quantum dots. In another embodiment, an individual device is used for the detection of each one of multiple biomarkers to be detected in a biological sample. Individual devices can be configured to permit each biomarker in the biological sample to be processed simultaneously. For example, a microtiter plate can be used such that each well in the plate is used to uniquely analyze one of multiple biomarkers to be detected in a biological sample.
[00317] In one or more of the foregoing embodiments, a fluorescent tag can be used to label a component of the biomarker/capture complex to enable the detection of the biomarker value. In various embodiments, the fluorescent label can be conjugated to a capture reagent specific to any of the biomarkers described herein using known techniques, and the fluorescent label can then be used to detect the corresponding biomarker value. Suitable fluorescent labels include rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, allophycocyanin, PBXL-3, Qdot 605, Lissamine, phycoerythrin, Texas Red, and other such compounds.
[00318] In one embodiment, the fluorescent label is a fluorescent dye molecule. In some embodiments, the fluorescent dye molecule includes at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the dye molecule includes an AlexFluor molecule, such as, for example, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700. In other embodiments, the dye molecule includes a first type and a second type of dye molecule, such as, e.g., two different AlexaFluor molecules. In other embodiments, the dye molecule includes a first type and a second type of dye molecule, and the two dye molecules have different emission spectra.
[00319] Fluorescence can be measured with a variety of instrumentation compatible with a wide range of assay formats. For example, spectrofluorimeters have been designed to analyze microtiter plates, microscope slides, printed arrays, cuvettes, etc. See Principles of Fluorescence Spectroscopy, by J.R. Lakowicz, Springer Science + Business Media, Inc., 2004. See Bioluminescence & Chemiluminescence: Progress & Current Applications; Philip E. Stanley and Larry J. Kricka editors, World Scientific Publishing Company, January 2002.
[00320] In one or more of the foregoing embodiments, a chemiluminescence tag can optionally be used to label a component of the biomarker/capture complex to enable the detection of a biomarker value. Suitable chemiluminescent materials include any of oxalyl chloride, Rodamin 6G, Ru(bipy)32+ , TMAE (tetrakis(dimethylamino)ethylene), Pyrogallol (1,2,3-trihydroxibenzene), Lucigenin, peroxyoxalates, Aryl oxalates, Acridinium esters, dioxetanes, and others.
[00321] In yet other embodiments, the detection method includes an enzyme/substrate combination that generates a detectable signal that corresponds to the biomarker value. Generally, the enzyme catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques, including spectrophotometry, fluorescence, and chemiluminescence. Suitable enzymes include, for example, luciferases, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta- galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucose-6- phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like.
[00322] In yet other embodiments, the detection method can be a combination of fluorescence, chemiluminescence, radionuclide or enzyme/substrate combinations that generate a measurable signal. Multimodal signaling could have unique and advantageous characteristics in biomarker assay formats.
[00323] More specifically, the biomarker values for the biomarkers described herein can be detected using known analytical methods including, singleplex SOMAmer assays, multiplexed SOMAmer assays, singleplex or multiplexed immunoassays, mRNA expression profiling, miRNA expression profiling, mass spectrometric analysis, histological/cytological methods, etc. as detailed below.
Determination of Biomarker Values using SOMAmer-Based Assays
[00324] Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other samples are important tools in scientific research and in the health care field. One class of such assays involves the use of a microarray that includes one or more aptamers immobilized on a solid support. The aptamers are each capable of binding to a target molecule in a highly specific manner and with very high affinity. See, e.g., U.S. Patent No. 5,475,096 entitled “Nucleic Acid Ligands"; see also, e.g., U.S. Patent No. 6,242,246, U.S. Patent No. 6,458,543, and U.S. Patent No. 6,503,715, each of which is entitled “Nucleic Acid Ligand Diagnostic Biochip". Once the microarray is contacted with a sample, the aptamers bind to their respective target molecules present in the sample and thereby enable a determination of a biomarker value corresponding to a biomarker.
[00325] As used herein, an “aptamer” refers to a nucleic acid that has a specific binding affinity for a target molecule. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other components in a test sample. An “aptamer” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence. An aptamer can include any suitable number of nucleotides, including any number of chemically modified nucleotides. “Aptamers” refers to more than one such set of molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers can be DNA or RNA or chemically modified nucleic acids and can be single stranded, double stranded, or contain double stranded regions, and can include higher ordered structures. An aptamer can also be a photoaptamer, where a photoreactive or chemically reactive functional group is included in the aptamer to allow it to be covalently linked to its corresponding target. Any of the aptamer methods disclosed herein can include the use of two or more aptamers that specifically bind the same target molecule. As further described below, an aptamer may include a tag. If an aptamer includes a tag, all copies of the aptamer need not have the same tag. Moreover, if different aptamers each include a tag, these different aptamers can have either the same tag or a different tag.
[00326] An aptamer can be identified using any known method, including the SELEX process. Once identified, an aptamer can be prepared or synthesized in accordance with any known method, including chemical synthetic methods and enzymatic synthetic methods.
[00327] As used herein, a “SOMAmer” or Slow Off-Rate Modified Aptamer refers to an aptamer having improved off-rate characteristics. SOMAmers can be generated using the improved SELEX methods described in U.S. Publication No. 2009/0004667, entitled “Method for Generating Aptamers with Improved Off-Rates."
[00328] The terms “SELEX” and “SELEX process” are used interchangeably herein to refer generally to a combination of (1) the selection of aptamers that interact with a target molecule in a desirable manner, for example binding with high affinity to a protein, with (2) the amplification of those selected nucleic acids. The SELEX process can be used to identify aptamers with high affinity to a specific target or biomarker.
[00329] SELEX generally includes preparing a candidate mixture of nucleic acids, binding of the candidate mixture to the desired target molecule to form an affinity complex, separating the affinity complexes from the unbound candidate nucleic acids, separating and isolating the nucleic acid from the affinity complex, purifying the nucleic acid, and identifying a specific aptamer sequence. The process may include multiple rounds to further refine the affinity of the selected aptamer. The process can include amplification steps at one or more points in the process. See, e.g., U.S. Patent No. 5,475,096, entitled “Nucleic Acid Ligands". The SELEX process can be used to generate an aptamer that covalently binds its target as well as an aptamer that non-covalently binds its target. See, e.g., U.S. Patent No. 5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX."
[00330] The SELEX process can be used to identify high-affinity aptamers containing modified nucleotides that confer improved characteristics on the aptamer, such as, for example, improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified aptamers containing modified nucleotides are described in U.S. Patent No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides", which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5’- and 2’-positions of pyrimidines. U.S. Patent No. 5,580,737, see supra, describes highly specific aptamers containing one or more nucleotides modified with 2’-amino (2’-NH2), T - fluoro (2’-F), and/or 2’-0-methyl (2’-OMe). See also, U.S. Patent Application Publication 20090098549, entitled “SELEX and PHOTOSELEX", which describes nucleic acid libraries having expanded physical and chemical properties and their use in SELEX and photoSELEX. [00331] SELEX can also be used to identify aptamers that have desirable off-rate characteristics. See U.S. Patent Application Publication 20090004667, entitled “Method for Generating Aptamers with Improved Off-Rates", which describes improved SELEX methods for generating aptamers that can bind to target molecules. As mentioned above, these slow off-rate aptamers are known as “SOMAmers.” Methods for producing aptamers or SOMAmers and photoaptamers or SOMAmers having slower rates of dissociation from their respective target molecules are described. The methods involve contacting the candidate mixture with the target molecule, allowing the formation of nucleic acid-target complexes to occur, and performing a slow off-rate enrichment process wherein nucleic acid-target complexes with fast dissociation rates will dissociate and not reform, while complexes with slow dissociation rates will remain intact. Additionally, the methods include the use of modified nucleotides in the production of candidate nucleic acid mixtures to generate aptamers or SOMAmers with improved off-rate performance.
[00332] A variation of this assay employs aptamers that include photoreactive functional groups that enable the aptamers to covalently bind or “photocrosslink” their target molecules. See, e.g., U.S. Patent No. 6,544,776 entitled “Nucleic Acid Ligand Diagnostic Biochip". These photoreactive aptamers are also referred to as photoaptamers. See, e.g., U.S. Patent No. 5,763,177, U.S. Patent No. 6,001,577, and U.S. Patent No. 6,291,184, each of which is entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX"; see also, e.g., U.S. Patent No. 6,458,539, entitled “Photoselection of Nucleic Acid Ligands". After the microarray is contacted with the sample and the photoaptamers have had an opportunity to bind to their target molecules, the photoaptamers are photoactivated, and the solid support is washed to remove any non- specifically bound molecules. Harsh wash conditions may be used, since target molecules that are bound to the photoaptamers are generally not removed, due to the covalent bonds created by the photoactivated functional group(s) on the photoaptamers. In this manner, the assay enables the detection of a biomarker value corresponding to a biomarker in the test sample.
[00333] In both of these assay formats, the aptamers or SOMAmers are immobilized on the solid support prior to being contacted with the sample. Under certain circumstances, however, immobilization of the aptamers or SOMAmers prior to contact with the sample may not provide an optimal assay. For example, pre-immobilization of the aptamers or SOMAmers may result in inefficient mixing of the aptamers or SOMAmers with the target molecules on the surface of the solid support, perhaps leading to lengthy reaction times and, therefore, extended incubation periods to permit efficient binding of the aptamers or SOMAmers to their target molecules. Further, when photoaptamers or photoSOMAmers are employed in the assay and depending upon the material utilized as a solid support, the solid support may tend to scatter or absorb the light used to effect the formation of covalent bonds between the photoaptamers or photoSOMAmers and their target molecules. Moreover, depending upon the method employed, detection of target molecules bound to their aptamers or photoSOMAmers can be subject to imprecision, since the surface of the solid support may also be exposed to and affected by any labeling agents that are used. Finally, immobilization of the aptamers or SOMAmers on the solid support generally involves an aptamer or SOMAmer-preparation step (i.e., the immobilization) prior to exposure of the aptamers or SOMAmers to the sample, and this preparation step may affect the activity or functionality of the aptamers or SOMAmers.
[00334] SOMAmer assays that permit a SOMAmer to capture its target in solution and then employ separation steps that are designed to remove specific components of the SOMAmer-target mixture prior to detection have also been described (see U.S. Patent Application Publication 20090042206, entitled “Multiplexed Analyses of Test Samples"). The described SOMAmer assay methods enable the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., a SOMAmer). The described methods create a nucleic acid surrogate (i.e, the SOMAmer) for detecting and quantifying a non-nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, including protein targets.
[00335] SOMAmers can be constructed to facilitate the separation of the assay components from a SOMAmer biomarker complex (or photoSOMAmer biomarker covalent complex) and permit isolation of the SOMAmer for detection and/or quantification. In one embodiment, these constructs can include a cleavable or releasable element within the SOMAmer sequence. In other embodiments, additional functionality can be introduced into the SOMAmer, for example, a labeled or detectable component, a spacer component, or a specific binding tag or immobilization element. For example, the SOMAmer can include a tag connected to the SOMAmer via a cleavable moiety, a label, a spacer component separating the label, and the cleavable moiety. In one embodiment, a cleavable element is a photocleavable linker. The photocleavable linker can be attached to a biotin moiety and a spacer section, can include an NHS group for derivatization of amines, and can be used to introduce a biotin group to a SOMAmer, thereby allowing for the release of the SOMAmer later in an assay method.
[00336] Homogenous assays, done with all assay components in solution, do not require separation of sample and reagents prior to the detection of signal. These methods are rapid and easy to use. These methods generate signal based on a molecular capture or binding reagent that reacts with its specific target. For estimation or determination of the current state of kidney function, the molecular capture reagents can be an aptamer (e.g., modified aptamer or SOMAmer reagent) or an antibody or the like and the specific target would be a biomarker as in Table 5.
[00337] In one embodiment, a method for signal generation takes advantage of anisotropy signal change due to the interaction of a fluorophore-labeled capture reagent with its specific biomarker target. When the labeled capture reagent reacts with its target, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower changing the anisotropy value. By monitoring the anisotropy change, binding events may be used to quantitatively measure the biomarkers in solutions. Other methods include fluorescence polarization assays, molecular beacon methods, time resolved fluorescence quenching, chemiluminescence, fluorescence resonance energy transfer, and the like.
[00338] An exemplary solution-based SOMAmer assay that can be used to detect a biomarker value corresponding to a biomarker in a biological sample includes the following: (a) preparing a mixture by contacting the biological sample with a SOMAmer that includes a first tag and has a specific affinity for the biomarker, wherein a SOMAmer affinity complex is formed when the biomarker is present in the sample; (b) exposing the mixture to a first solid support including a first capture element, and allowing the first tag to associate with the first capture element; (c) removing any components of the mixture not associated with the first solid support; (d) attaching a second tag to the biomarker component of the SOMAmer affinity complex; (e) releasing the SOMAmer affinity complex from the first solid support; (f) exposing the released SOMAmer affinity complex to a second solid support that includes a second capture element and allowing the second tag to associate with the second capture element; (g) removing any non-complexed SOMAmer from the mixture by partitioning the non-complexed SOMAmer from the SOMAmer affinity complex; (h) eluting the SOMAmer from the solid support; and (i) detecting the biomarker by detecting the SOMAmer component of the SOMAmer affinity complex.
[00339] Any means known in the art can be used to detect a biomarker value by detecting the SOMAmer component of a SOMAmer affinity complex. A number of different detection methods can be used to detect the SOMAmer component of an affinity complex, such as, for example, hybridization assays, mass spectroscopy, or QPCR. In some embodiments, nucleic acid sequencing methods can be used to detect the SOMAmer component of a SOMAmer affinity complex and thereby detect a biomarker value. Briefly, a test sample can be subjected to any kind of nucleic acid sequencing method to identify and quantify the sequence or sequences of one or more SOMAmers present in the test sample. In some embodiments, the sequence includes the entire SOMAmer molecule or any portion of the molecule that may be used to uniquely identify the molecule. In other embodiments, the identifying sequencing is a specific sequence added to the SOMAmer; such sequences are often referred to as “tags,” “barcodes,” or “zipcodes.” In some embodiments, the sequencing method includes enzymatic steps to amplify the SOMAmer sequence or to convert any kind of nucleic acid, including RNA and DNA that contain chemical modifications to any position, to any other kind of nucleic acid appropriate for sequencing.
[00340] In some embodiments, the sequencing method includes one or more cloning steps. In other embodiments the sequencing method includes a direct sequencing method without cloning.
[00341] In some embodiments, the sequencing method includes a directed approach with specific primers that target one or more SOMAmers in the test sample. In other embodiments, the sequencing method includes a shotgun approach that targets all SOMAmers in the test sample.
[00342] In some embodiments, the sequencing method includes enzymatic steps to amplify the molecule targeted for sequencing. In other embodiments, the sequencing method directly sequences single molecules. An exemplary nucleic acid sequencing-based method that can be used to detect a biomarker value corresponding to a biomarker in a biological sample includes the following: (a) converting a mixture of SOMAmers that contain chemically modified nucleotides to unmodified nucleic acids with an enzymatic step; (b) shotgun sequencing the resulting unmodified nucleic acids with a massively parallel sequencing platform such as, for example, the 454 Sequencing System (454 Life Sciences/Roche), the Illumina Sequencing System (Illumina), the ABI SOLiD Sequencing System (Applied Biosystems), the Heli Scope Single Molecule Sequencer (Helicos Biosciences), or the Pacific Biosciences Real Time Single- Molecule Sequencing System (Pacific BioSciences) or the Polonator G Sequencing System (Dover Systems); and (c) identifying and quantifying the SOMAmers present in the mixture by specific sequence and sequence count.
Determination of Biomarker Values using Immunoassays
[00343] Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format. To improve specificity and sensitivity of an assay method based on immuno- reactivity, monoclonal antibodies are often used because of their specific epitope recognition. Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies. Immunoassays have been designed for use with a wide range of biological sample matrices. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.
[00344] Quantitative results are generated through the use of a standard curve created with known concentrations of the specific analyte to be detected. The response or signal from an unknown sample is plotted onto the standard curve, and a quantity or value corresponding to the target in the unknown sample is established.
[00345] Numerous immunoassay formats have been designed. ELISA or EIA can be quantitative for the detection of an analyte. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly or indirectly, an enzyme. ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (1125) or fluorescence. Additional techniques include, for example, agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assay, and others (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition). [00346] Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays. Examples of procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.
[00347] Methods of detecting and/or quantifying a detectable label or signal generating material depend on the nature of the label. The products of reactions catalyzed by appropriate enzymes (where the detectable label is an enzyme; see above) can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.
[00348] Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 384 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.
Determination of Biomarker Values using Gene Expression Profiling
[00349] Measuring mRNA in a biological sample may be used as a surrogate for detection of the level of the corresponding protein in the biological sample. Thus, any of the biomarkers or biomarker panels described herein can also be detected by detecting the appropriate RNA. [00350] mRNA expression levels are measured by reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR). RT-PCR is used to create a cDNA from the mRNA. The cDNA may be used in a qPCR assay to produce fluorescence as the DNA amplification process progresses. By comparison to a standard curve, qPCR can produce an absolute measurement such as number of copies of mRNA per cell. Northern blots, microarrays, Invader assays, and RT-PCR combined with capillary electrophoresis have all been used to measure expression levels of mRNA in a sample. See Gene Expression Profiling: Methods and Protocols, Richard A. Shimkets, editor, Humana Press, 2004.
[00351] miRNA molecules are small RNAs that are non-coding but may regulate gene expression. Any of the methods suited to the measurement of mRNA expression levels can also be used for the corresponding miRNA. Recently many laboratories have investigated the use of miRNAs as biomarkers for disease. Many diseases involve wide-spread transcriptional regulation, and it is not surprising that miRNAs might find a role as biomarkers. The connection between miRNA concentrations and disease is often even less clear than the connections between protein levels and disease, yet the value of miRNA biomarkers might be substantial. Of course, as with any RNA expressed differentially during disease, the problems facing the development of an in vitro diagnostic product will include the requirement that the miRNAs survive in the diseased cell and are easily extracted for analysis, or that the miRNAs are released into blood or other matrices where they must survive long enough to be measured. Protein biomarkers have similar requirements, although many potential protein biomarkers are secreted intentionally at the site of pathology and function, during disease, in a paracrine fashion. Many potential protein biomarkers are designed to function outside the cells within which those proteins are synthesized.
Detection of Biomarkers Using In Vivo Molecular Imaging Technologies
[00352] Any of the described biomarkers (see Table 5) may also be used in molecular imaging tests. For example, an imaging agent can be coupled to any of the described biomarkers, which can be used to aid in estimation of determination of the current state of kidney function, to monitor response to therapeutic interventions, to select a population for clinical trials among other uses.
[00353] In vivo imaging technologies provide non-invasive methods for determining the state of a particular disease or condition in the body of an individual. For example, entire portions of the body, or even the entire body, may be viewed as a three dimensional image, thereby providing valuable information concerning morphology and structures in the body. Such technologies may be combined with the detection of the biomarkers described herein to provide information concerning the renal health status of an individual.
[00354] The use of in vivo molecular imaging technologies is expanding due to various advances in technology. These advances include the development of new contrast agents or labels, such as radiolabels and/or fluorescent labels, which can provide strong signals within the body; and the development of powerful new imaging technology, which can detect and analyze these signals from outside the body, with sufficient sensitivity and accuracy to provide useful information. The contrast agent can be visualized in an appropriate imaging system, thereby providing an image of the portion or portions of the body in which the contrast agent is located. The contrast agent may be bound to or associated with a capture reagent, such as a SOMAmer or an antibody, for example, and/or with a peptide or protein, or an oligonucleotide (for example, for the detection of gene expression), or a complex containing any of these with one or more macromolecules and/or other particulate forms.
[00355] The contrast agent may also feature a radioactive atom that is useful in imaging. Suitable radioactive atoms include technetium-99m or iodine-123 for scintigraphic studies.
Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as, for example, iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Such labels are well known in the art and could easily be selected by one of ordinary skill in the art.
[00356] Standard imaging techniques include but are not limited to magnetic resonance imaging, computed tomography scanning (coronary calcium score), positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography angiography, and the like. For diagnostic in vivo imaging, the type of detection instrument available is a major factor in selecting a given contrast agent, such as a given radionuclide and the particular biomarker that it is used to target (protein, mRNA, and the like). The radionuclide chosen typically has a type of decay that is detectable by a given type of instrument. Also, when selecting a radionuclide for in vivo diagnosis, its half-life should be long enough to enable detection at the time of maximum uptake by the target tissue but short enough that deleterious radiation of the host is minimized.
[00357] Exemplary imaging techniques include but are not limited to PET and SPECT, which are imaging techniques in which a radionuclide is synthetically or locally administered to an individual. The subsequent uptake of the radiotracer is measured over time and used to obtain information about the targeted tissue and the biomarker. Because of the high-energy (gamma- ray) emissions of the specific isotopes employed and the sensitivity and sophistication of the instruments used to detect them, the two-dimensional distribution of radioactivity may be inferred from outside of the body.
[00358] Commonly used positron-emitting nuclides in PET include, for example, carbon- 11, nitrogen-13, oxygen-15, and fluorine-18. Isotopes that decay by electron capture and/or gamma-emission are used in SPECT and include, for example iodine-123 and technetium-99m. An exemplary method for labeling amino acids with technetium-99m is the reduction of pertechnetate ion in the presence of a chelating precursor to form the labile technetium-99m- precursor complex, which, in turn, reacts with the metal binding group of a bifunctionally modified chemotactic peptide to form a technetium-99m-chemotactic peptide conjugate.
[00359] Antibodies are frequently used for such in vivo imaging diagnostic methods. The preparation and use of antibodies for in vivo diagnosis is well known in the art. Labeled antibodies which specifically bind any of the biomarkers in Table 5 can be injected into an individual suspected of having renal insufficiency, detectable according to the particular biomarker used, for the purpose of diagnosing or evaluating the disease status or condition of the individual. The label used will be selected in accordance with the imaging modality to be used, as previously described. Localization of the label permits determination of the tissue damage or other indications related to renal insufficiency. The amount of label within an organ or tissue also allows determination of the involvement of the current state of kidney health biomarkers in that organ or tissue.
[00360] Similarly, SOMAmers may be used for such in vivo imaging diagnostic methods. For example, a SOMAmer that was used to identify a particular biomarker described in Table 5 (and therefore binds specifically to that particular biomarker) may be appropriately labeled and injected into an individual being evaluated for renal insufficiency, detectable according to the particular biomarker, for the purpose of diagnosing or evaluating the levels of tissue damage, components of inflammatory response and other factors associated with the renal insufficiency in the individual. The label used will be selected in accordance with the imaging modality to be used, as previously described. Localization of the label permits determination of the site of the processes leading to increased risk. The amount of label within an organ or tissue also allows determination of the infiltration of the pathological process in that organ or tissue. SOMAmer- directed imaging agents could have unique and advantageous characteristics relating to tissue penetration, tissue distribution, kinetics, elimination, potency, and selectivity as compared to other imaging agents.
[00361] Such techniques may also optionally be performed with labeled oligonucleotides, for example, for detection of gene expression through imaging with antisense oligonucleotides. These methods are used for in situ hybridization, for example, with fluorescent molecules or radionuclides as the label. Other methods for detection of gene expression include, for example, detection of the activity of a reporter gene.
[00362] Another general type of imaging technology is optical imaging, in which fluorescent signals within the subject are detected by an optical device that is external to the subject. These signals may be due to actual fluorescence and/or to bioluminescence. Improvements in the sensitivity of optical detection devices have increased the usefulness of optical imaging for in vivo diagnostic assays.
[00363] The use of in vivo molecular biomarker imaging is increasing, including for clinical trials, for example, to more rapidly measure clinical efficacy in trials for new disease or condition therapies and/or to avoid prolonged treatment with a placebo for those diseases, such as multiple sclerosis, in which such prolonged treatment may be considered to be ethically questionable.
[00364] For a review of other techniques, see N. Blow, Nature Methods, 6, 465-469,
2009.
Determination of Biomarker Values using Mass Spectrometry Methods
[00365] A variety of configurations of mass spectrometers can be used to detect biomarker values. Several types of mass spectrometers are available or can be produced with various configurations. In general, a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument- control system, and a data system. Difference in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities. For example, an inlet can be a capillary-column liquid chromatography source or can be a direct probe or stage such as used in matrix-assisted laser desorption. Common ion sources are, for example, electrospray, including nanospray and microspray or matrix-assisted laser desorption. Common mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time-of-flight mass analyzer. Additional mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, New York (2000)).
[00366] Protein biomarkers and biomarker values can be detected and measured by any of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI- MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflex III TOF/TOF, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)N, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)N, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), quantitative mass spectrometry, and ion trap mass spectrometry.
[00367] Sample preparation strategies are used to label and enrich samples before mass spectroscopic characterization of protein biomarkers and determination biomarker values. Labeling methods include but are not limited to isobaric tag for relative and absolute quantitation (iTRAQ) and stable isotope labeling with amino acids in cell culture (SILAC). Capture reagents used to selectively enrich samples for candidate biomarker proteins prior to mass spectroscopic analysis include but are not limited to SOMAmers, antibodies, nucleic acid probes, chimeras, small molecules, an F(ab’)2 fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand-binding receptor, affybodies, nanobodies, ankyrins, domain antibodies, alternative antibody scaffolds (e.g. diabodies etc) imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleic acids, threose nucleic acid, a hormone receptor, a cytokine receptor, and synthetic receptors, and modifications and fragments of these.
Determination of Biomarker Values using a Proximity Ligation Assay
[00368] A proximity ligation assay can be used to determine biomarker values. Briefly, a test sample is contacted with a pair of affinity probes that may be a pair of antibodies or a pair of SOMAmers, with each member of the pair extended with an oligonucleotide. The targets for the pair of affinity probes may be two distinct determinates on one protein or one determinate on each of two different proteins, which may exist as homo- or hetero-multimeric complexes. When probes bind to the target determinates, the free ends of the oligonucleotide extensions are brought into sufficiently close proximity to hybridize together. The hybridization of the oligonucleotide extensions is facilitated by a common connector oligonucleotide which serves to bridge together the oligonucleotide extensions when they are positioned in sufficient proximity. Once the oligonucleotide extensions of the probes are hybridized, the ends of the extensions are joined together by enzymatic DNA ligation.
[00369] Each oligonucleotide extension comprises a primer site for PCR amplification. Once the oligonucleotide extensions are ligated together, the oligonucleotides form a continuous DNA sequence which, through PCR amplification, reveals information regarding the identity and amount of the target protein, as well as, information regarding protein-protein interactions where the target determinates are on two different proteins. Proximity ligation can provide a highly sensitive and specific assay for real-time protein concentration and interaction information through use of real-time PCR. Probes that do not bind the determinates of interest do not have the corresponding oligonucleotide extensions brought into proximity and no ligation or PCR amplification can proceed, resulting in no signal being produced.
[00370] The foregoing assays enable the detection of biomarker values that are useful in methods for determining or estimating glomerular filtration rate (eGFR) and/or renal insufficiency, where the methods comprise detecting, in a biological sample from an individual, biomarker values that each correspond to a biomarker selected from the group consisting of the biomarkers provided in Table 5, wherein an assessment, as described in detail below, using the biomarker values indicates the current state of kidney health in the individual. While certain of the described renal biomarkers are useful alone for estimating or determining current state of kidney health, methods are also described herein for the grouping of multiple subsets of the renal health biomarkers that are each useful as a panel of three or more biomarkers.. In accordance with any of the methods described herein, biomarker values can be detected and evaluated individually or they can be detected and evaluated collectively, as for example in a multiplex assay format.
[00371] A biomarker “signature” for a given diagnostic or predictive test contains a set of markers, each marker having different levels in the populations of interest. Different levels, in this context, may refer to different means of the marker levels for the individuals in two or more groups, or different variances in the two or more groups, or a combination of both. For the simplest form of a diagnostic test, markers can be used to assign an unknown sample from an individual into one of two groups, either renal insufficiency or not. The assignment of a sample into one of two or more groups is known as classification, and the procedure used to accomplish this assignment is known as a classifier or a classification method. Classification methods may also be referred to as scoring methods. There are many classification methods that can be used to construct a diagnostic classifier from a set of biomarker values. In general, classification methods are most easily performed using supervised learning techniques where a data set is collected using samples obtained from individuals within two (or more, for multiple classification states) distinct groups one wishes to distinguish. Since the class (group or population) to which each sample belongs is known in advance for each sample, the classification method can be trained to give the desired classification response. It is also possible to use unsupervised learning techniques to produce a diagnostic classifier.
[00372] Common approaches for developing diagnostic classifiers include decision trees; bagging, boosting, forests and random forests; rule inference based learning; Parzen Windows; linear models; logistic; neural network methods; unsupervised clustering; K-means; hierarchical ascending/ descending; semi-supervised learning; prototype methods; nearest neighbor; kernel density estimation; support vector machines; hidden Markov models; Boltzmann Learning; and classifiers may be combined either simply or in ways which minimize particular objective functions. For a review, see, e.g., Pattern Classification, R.O. Duda, et ah, editors, John Wiley & Sons, 2nd edition, 2001; see also, The Elements of Statistical Learning - Data Mining, Inference, and Prediction, T. Hastie, et ah, editors, Springer Science+Business Media, LLC, 2nd edition, 2009; each of which is incorporated by reference in its entirety.
[00373] To produce a classifier using supervised learning techniques, a set of samples called training data are obtained. In the context of diagnostic tests, training data includes samples from the distinct groups (classes) to which unknown samples will later be assigned. For example, samples collected from individuals in a control population and individuals in a particular disease, condition or event population can constitute training data to develop a classifier that can classify unknown samples (or, more particularly, the individuals from whom the samples were obtained) as either having the disease, condition or elevated risk of an event or being free from the disease, condition or elevated risk of an event. The development of the classifier from the training data is known as training the classifier. Specific details on classifier training depend on the nature of the supervised learning technique (see, e.g., Pattern Classification, R.O. Duda, et ah, editors, John Wiley & Sons, 2nd edition, 2001; see also, The Elements of Statistical Learning - Data Mining, Inference, and Prediction, T. Hastie, et ah, editors, Springer Science+Business Media, LLC, 2nd edition, 2009).
[00374] Since typically there are many more potential biomarker values than samples in a training set, care must be used to avoid over-fitting. Over-fitting occurs when a statistical model describes random error or noise instead of the underlying relationship. Over-fitting can be avoided in a variety of ways, including, for example, by limiting the number of markers used in developing the classifier, by assuming that the marker responses are independent of one another, by limiting the complexity of the underlying statistical model employed, and by ensuring that the underlying statistical model conforms to the data.
[00375] In order to identify a set of biomarkers associated with occurrence of events, the combined set of control and early event samples were analyzed using Principal Component Analysis (PCA). PCA displays the samples with respect to the axes defined by the strongest variations between all the samples, without regard to the case or control outcome, thus mitigating the risk of overfitting the distinction between case and control. Since the occurrence of serious thrombotic events has a strong component of chance involved, requiring unstable plaque to rupture in vital vessels to be reported, one would not expect to see a clear separation between the control and event sample sets. While the observed separation between case and control is not large, it occurs on the second principal component, corresponding to around 10% of the total variation in this set of samples, which indicates that the underlying biological variation is relatively simple to quantify.
[00376] In the next set of analyses, biomarkers can be analyzed for those components of difference between samples which were specific to the separation between the control samples and early event samples. One method that may be employed is the use of DSGA (Bair,E. and Tibshirani,R. (2004) Semi-supervised methods to predict patient survival from gene expression data. PLOS Biol., 2, 511-522) to remove (deflate) the first three principal component directions of variation between the samples in the control set. Although the dimensionality reduction is performed on the control set to discover, both the samples in the control and the samples from the early event samples are run through the PCA. Separation of cases from early events can be observed along the horizontal axis.
Cross validated selection of proteins relevant to renal function estimation or determination [00377] In order to avoid over-fitting of protein predictive power to idiosyncratic features of a particular selection of samples, a cross-validation and dimensional reduction approach can be taken. Cross-validation involves the multiple selection of sets of samples to determine the association of risk by protein combined with the use of the unselected samples to monitor the ability of the method to apply to samples which were not used in producing the model of risk (The Elements of Statistical Learning - Data Mining, Inference, and Prediction, T. Hastie, et ah, editors, Springer Science+Business Media, LLC, 2nd edition, 2009). We applied the supervised PCA method of Tibshirani et al (Bair,E. and Tibshirani,R. (2004) Semi-supervised methods to predict patient survival from gene expression data. PLOS Biol., 2, 511-522.) which is applicable to high dimensional datasets in the modeling of risk of renal insufficiency. The supervised PCA (SPCA) method inolves the univariate selection of a set of proteins statistically associated with the observed event hazard in the data and the determination of the correlated component which combines information from all of these proteins. This determination of the correlated component is a dimensionality reduction step which not only combines information across proteins, but also mitigates the likelihood of overfitting by reducing the number of independent variables from the full protein menu of over 1000 proteins down to a few principal components (in this work, we only examined the first principal component).
Univariate analysis and multivariate analysis of the relationship of individual proteins to time to event
[00378] The Cox proportional hazard model (Cox, David R (1972). "Regression Models and Life-Tables". Journal of the Royal Statistical Society. Series B (Methodological) 34 (2): 187-220.)) is widely used in medical statistics. Cox regression avoids fitting a specific function of time to the cumulative survival, and instead employs a model of relative risk referred to a baseline hazard function (which may vary with time). The baseline hazard function describes the common shape of the survival time distribution for all individuals, while the relative risk gives the level of the hazard for a set of covariate values (such as a single individual or group), as a multiple of the baseline hazard. The relative risk is constant with time in the Cox model.
Kits
[00379] Any combination of the biomarkers of Table 5 can be detected using a suitable kit, such as for use in performing the methods disclosed herein. Furthermore, any kit can contain one or more detectable labels as described herein, such as a fluorescent moiety, etc.
[00380] In one embodiment, a kit includes (a) one or more capture reagents (such as, for example, at least one SOMAmer or antibody) for detecting one or more biomarkers in a biological sample, wherein the biomarkers include any of the biomarkers set forth in Table 5 and optionally (b) one or more software or computer program products for computing current state of kidney health. Alternatively, rather than one or more computer program products, one or more instructions for manually performing the above steps by a human can be provided.
[00381] The combination of a solid support with a corresponding capture reagent having a signal generating material is referred to herein as a “detection device” or “kit". The kit can also include instructions for using the devices and reagents, handling the sample, and analyzing the data. Further the kit may be used with a computer system or software to analyze and report the result of the analysis of the biological sample.
[00382] The kits can also contain one or more reagents (e.g., solubilization buffers, detergents, washes, or buffers) for processing a biological sample. Any of the kits described herein can also include, e.g., buffers, blocking agents, mass spectrometry matrix materials, antibody capture agents, positive control samples, negative control samples, software and information such as protocols, guidance and reference data.
[00383] In one aspect, the invention provides kits for the analysis of current state of kidney health. The kits include PCR primers for one or more SOMAmers specific to biomarkers selected from Table 5. The kit may further include instructions for use and correlation of the biomarkers with an estimation or determination of current state of kidney health. The kit may also include a DNA array containing the complement of one or more of the aptamers or SOMAmer reagents specific for the biomarkers selected from Table 5, reagents, and/or enzymes for amplifying or isolating sample DNA. The kits may include reagents for real-time PCR, for example, TaqMan probes and/or primers, and enzymes.
[00384] For example, a kit can comprise (a) reagents comprising at least capture reagent for quantifying one or more biomarkers in a test sample, wherein said biomarkers comprise the biomarkers set forth in Table 5, or any other biomarkers or biomarkers panels described herein, and optionally (b) one or more algorithms or computer programs for performing the steps of comparing the amount of each biomarker quantified in the test sample to one or more predetermined cutoffs and assigning a score for each biomarker quantified based on said comparison, combining the assigned scores for each biomarker quantified to obtain a total score, comparing the total score with a predetermined score, and using said comparison to determine whether an individual has renal insufficiency. Alternatively, rather than one or more algorithms or computer programs, one or more instructions for manually performing the above steps by a human can be provided.
Computer Methods and Software
[00385] Once a biomarker or biomarker panel is selected, a method for diagnosing an individual can comprise the following: 1) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; 3) perform any data normalization or standardization required for the method used to collect biomarker values; 4) calculate the marker score; 5) combine the marker scores to obtain a total diagnostic or predictive score; and 6) report the individual’s diagnostic or predictive score. In this approach, the diagnostic or predictive score may be a single number determined from the sum of all the marker calculations that is compared to a preset threshold value that is an indication of the presence or absence of disease. Or the diagnostic or predictive score may be a series of bars that each represent a biomarker value and the pattern of the responses may be compared to a pre-set pattern for determination of the presence or absence of disease, condition or the increased risk (or not) of an event.
[00386] At least some embodiments of the methods described herein can be implemented with the use of a computer. An example of a computer system 100 is shown in Figure 2. With reference to Figure 2, system 100 is shown comprised of hardware elements that are electrically coupled via bus 108, including a processor 101, input device 102, output device 103, storage device 104, computer-readable storage media reader 105a, communications system 106, processing acceleration (e.g., DSP or special-purpose processors) 107 and memory 109. Computer-readable storage media reader 105a is further coupled to computer-readable storage media 105b, the combination comprehensively representing remote, local, fixed and/or removable storage devices plus storage media, memory, etc. for temporarily and/or more permanently containing computer-readable information, which can include storage device 104, memory 109 and/or any other such accessible system 100 resource. System 100 also comprises software elements (shown as being currently located within working memory 191) including an operating system 192 and other code 193, such as programs, data and the like.
[00387] With respect to Figure 2, system 100 has extensive flexibility and configurability. Thus, for example, a single architecture might be utilized to implement one or more servers that can be further configured in accordance with currently desirable protocols, protocol variations, extensions, etc. However, it will be apparent to those skilled in the art that embodiments may well be utilized in accordance with more specific application requirements. For example, one or more system elements might be implemented as sub-elements within a system 100 component (e.g., within communications system 106). Customized hardware might also be utilized and/or particular elements might be implemented in hardware, software or both. Further, while connection to other computing devices such as network input/output devices (not shown) may be employed, it is to be understood that wired, wireless, modem, and/or other connection or connections to other computing devices might also be utilized.
[00388] In one aspect, the system can comprise a database containing features of biomarkers characteristic of estimating or determining renal health. The biomarker data (or biomarker information) can be utilized as an input to the computer for use as part of a computer implemented method. The biomarker data can include the data as described herein.
[00389] In one aspect, the system further comprises one or more devices for providing input data to the one or more processors. [00390] The system further comprises a memory for storing a data set of ranked data elements.
[00391] In another aspect, the device for providing input data comprises a detector for detecting the characteristic of the data element, e.g., such as a mass spectrometer or gene chip reader.
[00392] The system additionally may comprise a database management system. User requests or queries can be formatted in an appropriate language understood by the database management system that processes the query to extract the relevant information from the database of training sets.
[00393] The system may be connectable to a network to which a network server and one or more clients are connected. The network may be a local area network (LAN) or a wide area network (WAN), as is known in the art. Preferably, the server includes the hardware necessary for running computer program products (e.g., software) to access database data for processing user requests.
[00394] The system may include an operating system (e.g., UNIX or Linux) for executing instructions from a database management system. In one aspect, the operating system can operate on a global communications network, such as the internet, and utilize a global communications network server to connect to such a network.
[00395] The system may include one or more devices that comprise a graphical display interface comprising interface elements such as buttons, pull down menus, scroll bars, fields for entering text, and the like as are routinely found in graphical user interfaces known in the art. Requests entered on a user interface can be transmitted to an application program in the system for formatting to search for relevant information in one or more of the system databases. Requests or queries entered by a user may be constructed in any suitable database language. [00396] The graphical user interface may be generated by a graphical user interface code as part of the operating system and can be used to input data and/or to display inputted data. The result of processed data can be displayed in the interface, printed on a printer in communication with the system, saved in a memory device, and/or transmitted over the network or can be provided in the form of the computer readable medium.
[00397] The system can be in communication with an input device for providing data regarding data elements to the system (e.g., expression values). In one aspect, the input device can include a gene expression profiling system including, e.g., a mass spectrometer, gene chip or array reader, and the like.
[00398] The methods and apparatus for analyzing current state of kidney health via an estimation or determination of GFR with biomarker information according to various embodiments may be implemented in any suitable manner, for example, using a computer program operating on a computer system. A conventional computer system comprising a processor and a random access memory, such as a remotely-accessible application server, network server, personal computer or workstation may be used. Additional computer system components may include memory devices or information storage systems, such as a mass storage system and a user interface, for example a conventional monitor, keyboard and tracking device. The computer system may be a stand-alone system or part of a network of computers including a server and one or more databases.
[00399] The current state of kidney health via the estimation or determination of GFR with the biomarker analysis system can provide functions and operations to complete data analysis, such as data gathering, processing, analysis, reporting and/or diagnosis. For example, in one embodiment, the computer system can execute the computer program that may receive, store, search, analyze, and report information relating to the current state of kidney health biomarkers. The computer program may comprise multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate a current state of kidney health status. Calculation of current state of kidney health may optionally comprise generating or collecting any other information, including additional biomedical information, regarding the condition of the individual relative to the disease, condition or event, identifying whether further tests may be desirable, or otherwise evaluating the health status of the individual.
[00400] Referring now to Figure 3, an example of a method of utilizing a computer in accordance with principles of a disclosed embodiment can be seen. In Figure 3, a flowchart 3000 is shown. In block 3004, biomarker information can be retrieved for an individual. The biomarker information can be retrieved from a computer database, for example, after testing of the individual’s biological sample is performed. The biomarker information can comprise biomarker values that each correspond to one or more of the biomarkers of Table 5. In block 3008, a computer can be utilized to perform a computation with each of the biomarker values. And, in block 3012, an estimation or determination can be made of glomerular filtration rate.
The indication can be output to a display or other indicating device so that it is viewable by a person. Thus, for example, it can be displayed on a display screen of a computer or other output device.
[00401] Some embodiments described herein can be implemented so as to include a computer program product. A computer program product may include a computer readable medium having computer readable program code embodied in the medium for causing an application program to execute on a computer with a database.
[00402] As used herein, a “computer program product” refers to an organized set of instructions in the form of natural or programming language statements that are contained on a physical media of any nature (e.g., written, electronic, magnetic, optical or otherwise) and that may be used with a computer or other automated data processing system. Such programming language statements, when executed by a computer or data processing system, cause the computer or data processing system to act in accordance with the particular content of the statements. Computer program products include without limitation: programs in source and object code and/or test or data libraries embedded in a computer readable medium. Furthermore, the computer program product that enables a computer system or data processing equipment device to act in pre-selected ways may be provided in a number of forms, including, but not limited to, original source code, assembly code, object code, machine language, encrypted or compressed versions of the foregoing and any and all equivalents.
[00403] In one aspect, a computer program product is provided for the estimation or determination of glomerular filtration rate. The computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises biomarker values that each correspond to one or more of the biomarkers of Table 5; and code that executes a computational method that indicates current state of kidney health of the individual as a function of the biomarker values.
[00404] In still another aspect, a computer program product is provided for indicating a likelihood of renal insufficiency. The computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises a biomarker value corresponding to one or more of the biomarkers of Table 5; and code that executes a computational method that indicates current state of kidney health as a function of the biomarker value.
[00405] While various embodiments have been described as methods or apparatuses, it should be understood that embodiments can be implemented through code coupled with a computer, e.g., code resident on a computer or accessible by the computer. For example, software and databases could be utilized to implement many of the methods discussed above. Thus, in addition to embodiments accomplished by hardware, it is also noted that these embodiments can be accomplished through the use of an article of manufacture comprised of a computer usable medium having a computer readable program code embodied therein, which causes the enablement of the functions disclosed in this description. Therefore, it is desired that embodiments also be considered protected by this patent in their program code means as well. Furthermore, the embodiments may be embodied as code stored in a computer-readable memory of virtually any kind including, without limitation, RAM, ROM, magnetic media, optical media, or magneto-optical media. Even more generally, the embodiments could be implemented in software, or in hardware, or any combination thereof including, but not limited to, software running on a general purpose processor, microcode, PLAs, or ASICs.
[00406] It is also envisioned that embodiments could be accomplished as computer signals embodied in a carrier wave, as well as signals (e.g., electrical and optical) propagated through a transmission medium. Thus, the various types of information discussed above could be formatted in a structure, such as a data structure, and transmitted as an electrical signal through a transmission medium or stored on a computer readable medium.
[00407] It is also noted that many of the structures, materials, and acts recited herein can be recited as means for performing a function or step for performing a function. Therefore, it should be understood that such language is entitled to cover all such structures, materials, or acts disclosed within this specification and their equivalents, including the matter incorporated by reference.
[00408] The biomarker identification process, the utilization of the biomarkers disclosed herein, and the various methods for determining biomarker values are described in detail above with respect to evaluating an individual’s eGFR and whether that individual may have renal insufficiency. However, the application of the process, the use of identified biomarkers, and the methods for determining biomarker values are fully applicable to other specific types of diseases or medical conditions, or to the identification of individuals who may or may not be benefited by an ancillary medical treatment.
EXAMPLES
[00409] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the application as defined by the appended claims. All examples described herein were carried out using standard techniques, which are well known and routine to those of skill in the art. Routine molecular biology techniques described in the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et ak, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001).
Example 1. Model Specification [00410] 1.1 Model result description. Estimated glomerular filtration rate (eGFR) reported in ml/min/1.73m2 with values from 5 to 100 ml/min/1.73 m2.
[00411] 1.2 Final model information. In one aspect, the final model is a linear model with 39 features with non-zero coefficients. The model was trained on a loglO transformed eGFR endpoint. This transformation means that final model estimation or determinations must be back-transformed (i.e., ioestimatlon) to deliver eGFR determinations in the scale familiar to clinicians. The final model is a linear model for the log of the eGFR. The equation for the linear model is: loglO(eGFR) = Intercept + sum(coefficient*feature level)
For this model, the intercept = 1.677504887 and the feature names and coefficients are shown in Table 5. The feature level is the RFU (relative fluorescence units) as measured in a sample by a proteomic assay, for example, an aptamer-based assay.
Table 4: Final Model Information
Figure imgf000071_0001
Table 5: Model Features
Figure imgf000071_0002
Figure imgf000072_0001
Further R2 values are provided in Tables 6a- 6e for selected Table 5 features and combinations of Table 5 features.
Table 6a
Figure imgf000072_0002
Table 6b
Figure imgf000072_0003
Table 6c
Figure imgf000073_0001
Figure imgf000074_0001
Table 6d
Figure imgf000074_0002
Table 6e
Figure imgf000074_0003
Example 2. Datasets for Test Development and Validation
[00412] 2.1 Development and validation cohort(s). CRIC is a multi-site observational study initiated to explore the relationship between chronic renal insufficiency and cardiovascular disease and has since expanded to measure many outcomes that are thought to be associated with renal insufficiency such as cognitive decline and frailty. CRIC enrolled patients ages 21 to 74 years of age, half of whom have diabetes mellitus. Participants had annual in-person follow up visits (where urine and plasma were collected and stored) and telephone interviews every 6 months, where study outcomes and general health status were ascertained. Study recruitment began in 2003 and recruitment lasted for about 2.5 years at 13 clinical sites in the United States; investigators continue to monitor this cohort. The SomaLogic CRIC dataset includes clinical data and second annual visit samples (collected July 2003 through December 2009) for 3413 participants with kidney disease who were not yet experiencing end stage renal disease by the second annual visit.
[00413] The Covance study is a cross-sectional study designed to measure baseline information from normal individuals based on clinical labs, lifestyle factors, and health history.
It was conducted in 2008 by a Contract Research Organization, Covance, under SomaLogic sponsorship. Participants provided 8 study blood samples. Height, weight, and vital signs were obtained, along with an extensive self-reported health history and clinical laboratory measures, including serum creatinine to calculate eGFR. This population-based cohort recruited from 6 U.S. based clinical sites (Honolulu, HI; Portland, OR; Boise, ID; San Diego, CA; Austin, TX; and Dallas, TX) and is comprised of 1029 participants stratified by age, sex, and ethnicity. [00414] These datasets were combined for model training and verification with the goal of offering a test which can be utilized for those with both healthy and abnormal kidney function. The hold-out datasets from Covance and CRIC were combined for the validation phase.
[00415] 2.2 Dataset Stratification. For this test, each cohort (CRIC and Covance) was split independently into training (70%), verification (15%), and validation (15%) sets, allowing identification of a robust model while mitigating overfitting issues. The respective splits were then combined (i.e., CRIC training with Covance training and CRIC verification with Covance verification) for refinement analyses. The validation data for both cohorts was not used in the POC or refinement stages.
[00416] 2.2.1 Model development data
Table 7: Summary statistics of clinical covariates in model training and verification data splits
Figure imgf000075_0001
Figure imgf000076_0001
*These ethnicities were reported as either African American or Black in the native data sets.
[00417] 2.2.2 Model validation data
Table 8: Summary statistics for relevant covariates in model validation dataset
Figure imgf000076_0002
Figure imgf000077_0001
Example 3. Results from Development
[00418] 3.1 Data QC and Pre- Analytics Results. Previous data QC and a feasibility POC has been conducted on the CRIC cohort for this endpoint. Before combining with the CRIC dataset, Covance was analyzed with standard data QC and pre-analytics POC tools, after which 5 (0.4%) samples were removed due to row check failure. After combining the respective data splits from each cohort, data quality control and pre-analytics checks were run on the combined data. Particular attention was given to normalization scale factors, to ensure that the normalization process does not confound signal associated with kidney function. No scale factors were noted to be outside the typical expected ranges. The normalization scale factors were checked for association with the endpoint. An R2 of 0.425 was observed between the endpoint and the 0.5 dilution group scale factor. The final model selected in refinement contained no features from this dilution group. Only features that passed target confirmation specificity testing were used for any analyses. The samples for this test were run on assay version 4.0 from January 21, 2019 until September 30, 2019.
[00419] 3.2 POC Approach and Results. The support vector machines (SVM) exceeded the feasibility criterion of 0.600 (R2 = 0.889) and so model development moved into refinement. POCs were re-run on the combined dataset. POC reports and all model development work can be found in the bitbucket repo and the POC tech report.
[00420] 3.3 Refinement Approach and Results. Models developed in refinement used the training and verification dataset(s), as combined from the respective CRIC and Covance splits. The final model was developed using a loglO transformation on the endpoint. This was found to reduce consistent underprediction of eGFR for those with most severe chronic kidney disease (stage 4 and stage 5), as well as retain homoscedasticity in the model residuals. Only features with greater than 0.75 correlation between assay versions 4.0 and 4.1 were used in model development. Initial feature selection using stability selection yielded 73 features, which was further pared down to a final feature list of 39 features by repeated training of elastic net models. Five repeats of 10-fold cross validation were used in model training. After each iteration of elastic net linear model training, the features with the smallest coefficients were eliminated and the model retrained. This process was repeated until both training and verification metrics began to suffer. The final feature list was used to train a linear model, which had more favorable performance metrics on both training and verification than an elastic net model given the same reduced feature list. Final metrics are shown in Table 9. R2 is reported in loglO space, while other metrics are calculated using the estimations, back-transformed into the native unit used for eGFR measurement. The root mean squared error (RMSE) was calculated on the model estimations as well. In order to better characterize the error across different stages of CKD, which have different ranges of eGFR, RMSE was normalized (NRMSE) to the average eGFR in each respective disease stage. NRMSE as a relative error demonstrates that the model performs sufficiently well across the meaningful range of eGFR.
Table 9: Training and verification metrics for the final model
Figure imgf000078_0001
[00421] In addition, the refinement process also included assessments for robustness. The proposed model passes all checks for robust performance. Given some observed bias in the residuals for those with eGFR > 100 ml/min/1.73m2, it is recommended that values over 100 be reported as ‘greater than 100’ for LDT use. This will avoid reporting otherwise healthy values that may be unusually high in the clinical setting, while preserving the ability to distinguish values in the unhealthy range. Threshold values other than 100 with greater clinical significance could be used for mapping as well.
Example 4. Model Validation Plan and Results
[00422] 4.1 Clinical Validation Plan. Validation was assessed on the 15% holdout set, which is a combined 15% representation from each cohort (Covance, CRIC). These data were stored in a separate file and not used in model development. The final model was used to generate estimations or determinations on these data. These estimations or determinations are in log 10 space, and the validation metric was assessed on these transformed data. A model R2 = 0.8 in estimation or determination of GFR as calculated by CKD epi (2009) is the minimum validation threshold, assessed directly on model estimations or determinations of the log 10 transformed endpoint.
[00423] Clinical Results on Validation Data. The model performs similarly in validation as in the training and verification datasets. The validation R2 metric exceeds the validation criteria of R2 > 0.8 (Table 10).
Table 10: Model validation results
Figure imgf000079_0001
Example 5. Impacts of Imputation
[00424] Results from simulations show that the best approach for handling out-of-range aptamers is to set analyte values to zero (the average value) in Table 11.
Table 11: Imputation for out-of-range RFU values.
Figure imgf000079_0002
Example 6. Variation due to assay noise
[00425] Ninety-five percent and 99% upper and lower bounds for variation in the model estimation or determination due to the assay are listed in Table 12.
Table 12: Error bounds for variation in assay noise
Figure imgf000079_0003
REFERENCES
All references listed below, or anywhere else throughout this description, are hereby incorporated by reference herein in their entireties:
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“doi: 10.1371/journal. pone.0136994” LaBleu et al. (2013) “Identification of human epididymis protein-4 as a fibroblast- derived mediator of fibrosis” Nature Medicine 19: pp. 227-231. Zhang et al. (2019) “Hypoxia-induced HE4 in tubular epithelial cells promotes extracellular matrix accumulation and renal fibrosis viaNF-kB" The FASEB Journal 34(2): 2554-2567. Li et al. (Mar 3 2020) “Analysis of Spatiotemporal Urine Protein Dynamics to Identify New Biomarkers for Sepsis-Induced Acute Kidney Injury” Front Physiol. 11: 139. Nakagawa et al. (2013) “Myofibroblasts in Fiboritc Kidneys” Curr Pathobiol Rep. 1(3): doi: 10.1007/s40139-013-0025-8. Rayego-Mateos (2018) “Role of Epidermal Growth Factor Receptor (EGFR) and Its Ligands in Kidney Inflammation and Damage” Mediators Inflamm. doi: 10.1155/2018/8739473. Ngo et al. (Oct 62020) “Circulating testican-2 is a podocyte-derived marker of kidney health” Proc Natl Acad Sci USA 117(40): pp. 25026-25035. Ravindan et al. (Jan 302020) “Proteomic Analysis of Complement Proteins in Membranous Nephropathy” Kidney IntRep. 5(5): pp. 618-626. de Jorge (2018) “Factor H Competitor Generated by Gene Conversion Events Associates with Atypical Hemolytic Uremic Syndrome” J Am Soc Nephrol 29(1): pp. 240-249. Rouillard et al. (2016) “Kidney Disease Gene Set: GWASdb SNP -Disease Associations” The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database (Oxford). Rouillard et al. (2016) “Kidney Gene Set: BioGPS Human Cell Type and Tissue Gene Expression Profiles” The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database (Oxford).

Claims

What is claimed is:
1. A method comprising: a) measuring the level of TMEDA protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
2. A method comprising: a) measuring the level of ARMEL protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
3. A method compri sing : a) measuring the level of CAC02 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of CAC02 and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
4. A method comprising: a) measuring the level of NAGPA protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of NAGPA and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
5. The method of claim 1 , wherein the method comprises measuring Cystatin C and TMEDA, DSC2 and TMEDA, HPRT and TMEDA, FABP and TMEDA, PPIC and TMEDA, GBRAP and TMEDA, ISK7 and TMEDA, ARMEL and TMEDA, FSH and TMEDA, RBP and TMEDA, HE4 and TMEDA, C01A1 and TMEDA, PGRP-L and TMEDA, ERBB1 and TMEDA, Testican- 2 and TMEDA, FHR1 and TMEDA, SRCA and TMEDA, CAC02 and TMEDA, NAGPA and TMEDA, DLK1 and TMEDA, SLIT2 and TMEDA, HCC-1 and TMEDA, CDON and TMEDA, COFA1 and TMEDA, PEARl and TMEDA, Ribonuclease UK114 and TMEDA, Activin A and TMEDA, Heparin cofactor II and TMEDA, SUMF1 and TMEDA, MP2K4 and TMEDA, SELW and TMEDA, HYALl and TMEDA, al -Antitrypsin and TMEDA, MMP-7 and TMEDA, QSOX2 and TMEDA, IGDC4 and TMEDA, S100A6 and TMEDA, ATS 13 and TMEDA.
6. The method of claim 2, wherein the method comprises measuring Cystatin C and ARMEL, TMEDA and ARMEL, DSC2 and ARMEL, HPRT and ARMEL, FABP and ARMEL, PPIC and ARMEL, GBRAP and ARMEL, ISK7 and ARMEL, F SH and ARMEL, RBP and ARMEL, HE4 and ARMEL, C01A1 and ARMEL, PGRP-L and ARMEL, ERBBl and ARMEL, Testican-2 and ARMEL, FHRl and ARMEL, SRCA and ARMEL, CAC02 and ARMEL, NAGPA and ARMEL, DLK1 and ARMEL, SLIT2 and ARMEL, HCC-1 and ARMEL, CDON and ARMEL, COFA1 and ARMEL, PEARl and ARMEL, Ribonuclease UK114 and ARMEL, Activin A and ARMEL, Heparin cofactor II and ARMEL, SUMF1 and ARMEL, MP2K4 and ARMEL, SELW and ARMEL, HYALl and ARMEL, al -Antitrypsin and ARMEL, MMP-7 and ARMEL, QSOX2 and ARMEL, IGDC4 and ARMEL, S100A6 and ARMEL, ATS 13 and ARMEL.
7. The method of claim 3, wherein the method comprises measuring Cystatin C and CAC02, TMEDA and CAC02, DSC2 and CAC02, HPRT and CAC02, FABP and CAC02, PPIC and CAC02, GBRAP and CAC02, ISK7 and CAC02, ARMEL and CAC02, FSH and CAC02, RBP and CAC02, HE4 and CAC02, COl Al and CAC02, PGRP-L and CAC02, ERBBl and CAC02, Testican-2 and CAC02, FHRl and CAC02, SRCA and CAC02, NAGPA and CAC02, DLK1 and CAC02, SLIT2 and CAC02, HCC-1 and CAC02, CDON and CAC02, COFA1 and CAC02, PEARl and CAC02, Ribonuclease UK114 and CAC02, Activin A and CAC02, Heparin cofactor II and CAC02, SUMF1 and CAC02, MP2K4 and CAC02, SELW and CAC02, HYALl and CAC02, a 1 -Antitrypsin and CAC02, MMP-7 and CAC02, QSOX2 and CAC02, IGDC4 and CAC02, S100A6 and CAC02, ATS 13 and CAC02.
8. The method of claim 4, wherein the method comprises measuring Cystatin C and NAGPA, TMEDA and NAGPA, DSC2 and NAGPA, HPRT and NAGPA, FABP and NAGPA, PPIC and NAGPA, GBRAP and NAGPA, ISK7 and NAGPA, ARMEL and NAGPA, FSH and NAGPA, RBP and NAGPA, HE4 and NAGPA, C01A1 and NAGPA, PGRP-L and NAGPA, ERBB1 and NAGPA, Testican-2 and NAGPA, FHR1 and NAGPA, SRCA and NAGPA, CAC02 and NAGPA, DLK1 and NAGPA, SLIT2 and NAGPA, HCC-1 and NAGPA, CDON and NAGPA, COFA1 and NAGPA, PEARl and NAGPA, Ribonuclease UK114 and NAGPA, Activin A and NAGPA, Heparin cofactor II and NAGPA, SUMF1 and NAGPA, MP2K4 and NAGPA, SELW and NAGPA, HYALl and NAGPA, a 1 -Antitrypsin and NAGPA, MMP-7 and NAGPA, QSOX2 and NAGPA, IGDC4 and NAGPA, S100A6 and NAGPA, ATS 13 and NAGPA.
9. The method of claim 1, wherein the method comprises measuring TMEDA and ARMEL, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
10. The method of claim 1, wherein the method comprises measuring TMEDA and CAC02, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
11. The method of claim 1, wherein the method comprises measuring TMEDA and NAGPA, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
12. The method of claim 2, wherein the method comprises measuring ARMEL and CAC02, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK 114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
13. The method of claim 2, wherein the method comprises measuring ARMEL and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al-Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
14. The method of claim 3, wherein the method comprises measuring CAC02 and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al-Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
15. The method of any one of claims 1 to 14, wherein the estimated or determined GFR is between 5 and 100 ml/min/1.73m2.
16. The method of any one of claims 1 to 15, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
17. The method of any one of claims 1 to 16, wherein the sample is selected from blood, plasma, serum or urine.
18. The method of any one of claims 1 to 17, wherein the human subject is determined to have renal insufficiency.
19. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising TMEDA protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
20. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising ARMEL protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
21. A method compri sing : a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising CAC02 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
22. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising NAGPA protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
23. The method of claim 19, wherein the method comprises measuring Cystatin C and TMEDA, DSC2 and TMEDA, HPRT and TMEDA, FABP and TMEDA, PPIC and TMEDA, GBRAP and TMEDA, ISK7 and TMEDA, ARMEL and TMEDA, FSH and TMEDA, RBP and TMEDA, HE4 and TMEDA, COl Al and TMEDA, PGRP-L and TMEDA, ERBBl and TMEDA, Testican-2 and TMEDA, FHRl and TMEDA, SRCA and TMEDA, CAC02 and TMEDA, NAGPA and TMEDA, DLK1 and TMEDA, SLIT2 and TMEDA, HCC-1 and TMEDA, CDON and TMEDA, COFA1 and TMEDA, PEARl and TMEDA, Ribonuclease UK114 and TMEDA, Activin A and TMEDA, Heparin cofactor II and TMEDA, SUMF1 and TMEDA, MP2K4 and TMEDA, SELW and TMEDA, HYALl and TMEDA, al -Antitrypsin and TMEDA, MMP-7 and TMEDA, QSOX2 and TMEDA, IGDC4 and TMEDA, S100A6 and TMEDA, ATS 13 and TMEDA.
24. The method of claim 20, wherein the method comprises measuring Cystatin C and ARMEL, TMEDA and ARMEL, DSC2 and ARMEL, HPRT and ARMEL, FABP and ARMEL, PPIC and ARMEL, GBRAP and ARMEL, ISK7 and ARMEL, FSH and ARMEL, RBP and ARMEL, HE4 and ARMEL, C01A1 and ARMEL, PGRP-L and ARMEL, ERBBl and ARMEL, Testican-2 and ARMEL, FHR1 and ARMEL, SRCA and ARMEL, CAC02 and ARMEL, NAGPA and ARMEL, DLK1 and ARMEL, SLIT2 and ARMEL, HCC-1 and ARMEL, CDON and ARMEL, COFA1 and ARMEL, PEARl and ARMEL, Ribonuclease UK114 and ARMEL, Activin A and ARMEL, Heparin cofactor II and ARMEL, SUMFl and ARMEL, MP2K4 and ARMEL, SELW and ARMEL, HYALl and ARMEL, a 1 -Antitrypsin and ARMEL, MMP-7 and ARMEL, QSOX2 and ARMEL, IGDC4 and ARMEL, S100A6 and ARMEL, ATS 13 and ARMEL.
25. The method of claim 21, wherein the method comprises measuring Cystatin C and CAC02, TMEDA and CAC02, DSC2 and CAC02, HPRT and CAC02, FABP and CAC02, PPIC and CAC02, GBRAP and CAC02, ISK7 and CAC02, ARMEL and CAC02, FSH and CAC02, RBP and CAC02, HE4 and CAC02, C01A1 and CAC02, PGRP-L and CAC02, ERBBl and CAC02, Testican-2 and CAC02, FHR1 and CAC02, SRCA and CAC02, NAGPA and CAC02, DLK1 and CAC02, SLIT2 and CAC02, HCC-1 and CAC02, CDON and CAC02, COFA1 and CAC02, PEARl and CAC02, Ribonuclease UK114 and CAC02, Activin A and CAC02, Heparin cofactor II and CAC02, SUMFl and CAC02, MP2K4 and CAC02, SELW and CAC02, HYALl and CAC02, al -Antitrypsin and CAC02, MMP-7 and CAC02, QSOX2 and CAC02, IGDC4 and CAC02, S100A6 and CAC02, ATS 13 and CAC02.
26. The method of claim 22, wherein the method comprises measuring Cystatin C and NAGPA, TMEDA and NAGPA, DSC2 and NAGPA, HPRT and NAGPA, FABP and NAGPA, PPIC and NAGPA, GBRAP and NAGPA, ISK7 and NAGPA, ARMEL and NAGPA, FSH and NAGPA, RBP and NAGPA, HE4 and NAGPA, C01A1 and NAGPA, PGRP-L and NAGPA, ERBBl and NAGPA, Testican-2 and NAGPA, FHRl and NAGPA, SRCA and NAGPA, CAC02 and NAGPA, DLK1 and NAGPA, SLIT2 and NAGPA, HCC-1 and NAGPA, CDON and NAGPA, COFA1 and NAGPA, PEARl and NAGPA, Ribonuclease UK114 and NAGPA, Activin A and NAGPA, Heparin cofactor II and NAGPA, SUMFl and NAGPA, MP2K4 and NAGPA, SELW and NAGPA, HYALl and NAGPA, a 1 -Antitrypsin and NAGPA, MMP-7 and NAGPA, QSOX2 and NAGPA, IGDC4 and NAGPA, S100A6 and NAGPA, ATS 13 and NAGPA.
27. The method of claim 19, wherein the method comprises measuring TMEDA and ARMEL, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYAL1, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
28. The method of claim 19, wherein the method comprises measuring TMEDA and CAC02, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
29. The method of claim 19, wherein the method comprises measuring TMEDA and NAGPA, and at least one of the following proteins selected from Cystatin C, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
30. The method of claim 20, wherein the method comprises measuring ARMEL and CAC02, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK 114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
31. The method of claim 20, wherein the method comprises measuring ARMEL and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al-Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
32. The method of claim 21, wherein the method comprises measuring CAC02 and NAGPA, and at least one of the following proteins selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYAL1, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13.
33. The method of any one of claims 19 to 32, wherein the protein levels are used to estimate or determine glomerular filtration rate (GFR) for the human subject.
34. The method of any one of claims 19 to 33, wherein the estimated or determined GFR is between 5 and 100 ml/min/1.73m2.
35. The method of any one of claims 19 to 34, wherein the set of capture reagents is selected from aptamers, antibodies and a combinations of aptamers and antibodies.
36. The method of any one of claims 19 to 35, wherein the sample is selected from blood, plasma, serum or urine.
37. The method of any one of claims 19 to 36, wherein the human subject is determined to have renal insufficiency.
38. A method compri sing : a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a ARMEL protein; and b) measuring the level of each protein with the two capture reagents.
39. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a CAC02 protein; and b) measuring the level of each protein with the two capture reagents.
40. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a TMEDA protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
41. A method compri sing : a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a ARMEL protein and the second capture reagent has affinity for a CAC02 protein; and b) measuring the level of each protein with the two capture reagents.
42. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a ARMEL protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
43. A method comprising: a) contacting a sample from a human subject with two capture reagents, wherein one capture reagent has affinity for a CAC02 protein and the second capture reagent has affinity for a NAGPA protein; and b) measuring the level of each protein with the two capture reagents.
44. The method of any one of claims 38 to 43, further comprising measuring the level of a Cystatin C protein with a capture reagent having affinity for the Cystatin C protein.
45. The method of any one of claims 38 to 44, further comprising measuring the level of a DSC2 protein with a capture reagent having affinity for the DSC2 protein.
46. The method of any one of claims 38 to 45, further comprising measuring the level of a HPRT protein with a capture reagent having affinity for the HPRT protein.
47. The method of any one of claims 38 to 46, further comprising measuring the level of a FABP protein with a capture reagent having affinity for the FABP protein.
48. The method of any one of claims 38 to 47, further comprising measuring the level of a PPIC protein with a capture reagent having affinity for the PPIC protein.
49. The method of any one of claims 38 to 48, further comprising measuring the level of a GBRAP protein with a capture reagent having affinity for the GBRAP protein.
50. The method of any one of claims 38 to 49, further comprising measuring the level of a ISK7 protein with a capture reagent having affinity for the ISK7 protein.
51. The method of any one of claims 38 to 50, further comprising measuring the level of a FSH protein with a capture reagent having affinity for the FSH protein.
52. The method of any one of claims 38 to 51, further comprising measuring the level of a RBP protein with a capture reagent having affinity for the RBP protein.
53. The method of any one of claims 38 to 52, further comprising measuring the level of a HE4 protein with a capture reagent having affinity for the HE4 protein.
54. The method of any one of claims 38 to 53, further comprising measuring the level of a COl A1 protein with a capture reagent having affinity for the COl A1 protein.
55. The method of any one of claims 38 to 54, further comprising measuring the level of a PGRP-L protein with a capture reagent having affinity for the PGRP-L protein.
56. The method of any one of claims 38 to 55, further comprising measuring the level of a ERBB 1 protein with a capture reagent having affinity for the ERBB 1 protein.
57. The method of any one of claims 38 to 56, further comprising measuring the level of a Testican-2 protein with a capture reagent having affinity for the Testican-2 protein.
58. The method of any one of claims 38 to 57, further comprising measuring the level of a FHR1 protein with a capture reagent having affinity for the FHR1 protein.
59. The method of any one of claims 38 to 58, further comprising measuring the level of a SRCA protein with a capture reagent having affinity for the SRCA protein.
60. The method of any one of claims 38 to 59, further comprising measuring the level of a DLK1 protein with a capture reagent having affinity for the DLK1 protein.
61. The method of any one of claims 38 to 60, further comprising measuring the level of a SLIT2 protein with a capture reagent having affinity for the SLIT2 protein.
62. The method of any one of claims 38 to 61, further comprising measuring the level of a HCC-1 protein with a capture reagent having affinity for the HCC-1 protein.
63. The method of any one of claims 38 to 62, further comprising measuring the level of a CDON protein with a capture reagent having affinity for the CDON protein.
64. The method of any one of claims 38 to 63, further comprising measuring the level of a COFA1 protein with a capture reagent having affinity for the COFA1 protein.
65. The method of any one of claims 38 to 64, further comprising measuring the level of a PEARl protein with a capture reagent having affinity for the PEARl protein.
66. The method of any one of claims 38 to 65, further comprising measuring the level of a Ribonuclease UK114 protein with a capture reagent having affinity for the Ribonuclease UK114 protein.
67. The method of any one of claims 38 to 66, further comprising measuring the level of a Activin A protein with a capture reagent having affinity for the Activin A protein.
68. The method of any one of claims 38 to 67, further comprising measuring the level of a Heparin cofactor II protein with a capture reagent having affinity for the Heparin cofactor II protein.
69. The method of any one of claims 38 to 68, further comprising measuring the level of a SUMF1 protein with a capture reagent having affinity for the SUMF1 protein.
70. The method of any one of claims 38 to 69, further comprising measuring the level of a MP2K4 protein with a capture reagent having affinity for the MP2K4 protein.
71. The method of any one of claims 38 to 70, further comprising measuring the level of a SELW protein with a capture reagent having affinity for the SELW protein.
72. The method of any one of claims 38 to 71, further comprising measuring the level of a HYAL1 protein with a capture reagent having affinity for the HYAL1 protein.
73. The method of any one of claims 38 to 72, further comprising measuring the level of a al- Antitrypsin protein with a capture reagent having affinity for the al -Antitrypsin protein.
74. The method of any one of claims 38 to 73, further comprising measuring the level of a MMP-7 protein with a capture reagent having affinity for the MMP-7 protein.
75. The method of any one of claims 38 to 74, further comprising measuring the level of a QSOX2 protein with a capture reagent having affinity for the QSOX2 protein.
76. The method of any one of claims 38 to 75, further comprising measuring the level of a IGDC4 protein with a capture reagent having affinity for the IGDC4 protein.
77. The method of any one of claims 38 to 76, further comprising measuring the level of a S100A6 protein with a capture reagent having affinity for the S100A6 protein.
78. The method of any one of claims 38 to 77, further comprising measuring the level of a ATS 13 protein with a capture reagent having affinity for the ATS 13 protein.
79. A method comprising: a) measuring the level of TMEDA and ARMEL in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and ARMEL.
80. A method comprising: a) measuring the level of TMEDA and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and CAC02.
81. A method compri sing : a) measuring the level of TMEDA and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA and NAGPA.
82. A method comprising: a) measuring the level of ARMEL and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and CAC02.
83. A method comprising: a) measuring the level of ARMEL and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL and NAGPA.
84. A method comprising: a) measuring the level of CAC02 and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of CAC02 and NAGPA.
85. The method of any one of claims 79 to 84, further comprising measuring the level of a Cystatin C protein.
86. The method of any one of claims 79 to 85, further comprising measuring the level of a DSC2 protein.
87. The method of any one of claims 79 to 86, further comprising measuring the level of a HPRT protein.
88. The method of any one of claims 79 to 87, further comprising measuring the level of a FABP protein.
89. The method of any one of claims 79 to 88, further comprising measuring the level of a PPIC protein.
90. The method of any one of claims 79 to 89, further comprising measuring the level of a GBRAP protein.
91. The method of any one of claims 79 to 90, further comprising measuring the level of a ISK7 protein.
92. The method of any one of claims 79 to 91, further comprising measuring the level of a FSH protein.
93. The method of any one of claims 79 to 92, further comprising measuring the level of a RBP protein.
94. The method of any one of claims 79 to 93, further comprising measuring the level of a HE4 protein.
95. The method of any one of claims 79 to 94, further comprising measuring the level of a C01A1 protein.
96. The method of any one of claims 79 to 95, further comprising measuring the level of a PGRP-L protein.
97. The method of any one of claims 79 to 96, further comprising measuring the level of a ERBB1 protein.
98. The method of any one of claims 79 to 97, further comprising measuring the level of a Testican-2 protein.
99. The method of any one of claims 79 to 98, further comprising measuring the level of a FHR1 protein.
100. The method of any one of claims 79 to 99, further comprising measuring the level of a SRCA protein.
101. The method of any one of claims 79 to 100, further comprising measuring the level of a DLK1 protein.
102. The method of any one of claims 79 to 101, further comprising measuring the level of a SLIT2 protein.
103. The method of any one of claims 79 to 102, further comprising measuring the level of a HCC-1 protein.
104. The method of any one of claims 79 to 103, further comprising measuring the level of a CDON protein.
105. The method of any one of claims 79 to 104, further comprising measuring the level of a COFA1 protein.
106. The method of any one of claims 79 to 105, further comprising measuring the level of a PEAR1 protein.
107. The method of any one of claims 79 to 106, further comprising measuring the level of a Ribonuclease UK114 protein.
108. The method of any one of claims 79 to 107, further comprising measuring the level of a Activin A protein.
109. The method of any one of claims 79 to 108, further comprising measuring the level of a Heparin cofactor II protein.
110. The method of any one of claims 79 to 109, further comprising measuring the level of a SUMF1 protein.
111. The method of any one of claims 79 to 110, further comprising measuring the level of a MP2K4 protein.
112. The method of any one of claims 79 to 111, further comprising measuring the level of a SELW protein.
113. The method of any one of claims 79 to 112, further comprising measuring the level of a HYAL1 protein.
114. The method of any one of claims 79 to 113, further comprising measuring the level of a al -Antitrypsin protein.
115. The method of any one of claims 79 to 114, further comprising measuring the level of a MMP-7 protein.
116. The method of any one of claims 79 to 115, further comprising measuring the level of a QSOX2 protein.
117. The method of any one of claims 79 to 116, further comprising measuring the level of a IGDC4 protein.
118. The method of any one of claims 79 to 117, further comprising measuring the level of a S100A6 protein.
119. The method of any one of claims 79 to 118, further comprising measuring the level of a ATS 13 protein.
120. The method of any one of claims 79 to 119, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
121. A method compri sing : a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from TMEDA, ARMEL and CAC02; and b) measuring the level of each protein with the three capture reagents.
122. A method comprising: a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from TMEDA, ARMEL and NAGPA; and b) measuring the level of each protein with the three capture reagents.
123. A method compri sing : a) contacting a sample from a human subject with three capture reagents, wherein each of the three capture reagents has affinity for a protein selected from ARMEL, CAC02 and NAGPA; and b) measuring the level of each protein with the three capture reagents.
124. The method of any one of claims 121 to 123, further comprising measuring the level of a Cystatin C protein with a capture reagent having affinity for the Cystatin C protein.
125. The method of any one of claims 121 to 124, further comprising measuring the level of a DSC2 protein with a capture reagent having affinity for the DSC2 protein.
126. The method of any one of claims 121 to 125, further comprising measuring the level of a HPRT protein with a capture reagent having affinity for the HPRT protein.
127. The method of any one of claims 121 to 126, further comprising measuring the level of a FABP protein with a capture reagent having affinity for the FABP protein.
128. The method of any one of claims 121 to 127, further comprising measuring the level of a PPIC protein with a capture reagent having affinity for the PPIC protein.
129. The method of any one of claims 121 to 128, further comprising measuring the level of a GBRAP protein with a capture reagent having affinity for the GBRAP protein.
130. The method of any one of claims 121 to 129, further comprising measuring the level of a ISK7 protein with a capture reagent having affinity for the ISK7 protein.
131. The method of any one of claims 121 to 130, further comprising measuring the level of a FSH protein with a capture reagent having affinity for the FSH protein.
132. The method of any one of claims 121 to 131, further comprising measuring the level of a RBP protein with a capture reagent having affinity for the RBP protein.
133. The method of any one of claims 121 to 132, further comprising measuring the level of a HE4 protein with a capture reagent having affinity for the HE4 protein.
134. The method of any one of claims 121 to 133, further comprising measuring the level of a COl A1 protein with a capture reagent having affinity for the COl A1 protein.
135. The method of any one of claims 121 to 134, further comprising measuring the level of a PGRP-L protein with a capture reagent having affinity for the PGRP-L protein.
136. The method of any one of claims 121 to 135, further comprising measuring the level of a ERBB 1 protein with a capture reagent having affinity for the ERBB 1 protein.
137. The method of any one of claims 121 to 136, further comprising measuring the level of a Testican-2 protein with a capture reagent having affinity for the Testican-2 protein.
138. The method of any one of claims 121 to 137, further comprising measuring the level of a FHR1 protein with a capture reagent having affinity for the FHR1 protein.
139. The method of any one of claims 121 to 138, further comprising measuring the level of a SRCA protein with a capture reagent having affinity for the SRCA protein.
140. The method of any one of claims 121 to 139, further comprising measuring the level of a DLK1 protein with a capture reagent having affinity for the DLK1 protein.
141. The method of any one of claims 121 to 140, further comprising measuring the level of a SLIT2 protein with a capture reagent having affinity for the SLIT2 protein.
142. The method of any one of claims 121 to 141, further comprising measuring the level of a HCC-1 protein with a capture reagent having affinity for the HCC-1 protein.
143. The method of any one of claims 121 to 142, further comprising measuring the level of a CDON protein with a capture reagent having affinity for the CDON protein.
144. The method of any one of claims 121 to 143, further comprising measuring the level of a COFA1 protein with a capture reagent having affinity for the COFA1 protein.
145. The method of any one of claims 121 to 144, further comprising measuring the level of a PEARl protein with a capture reagent having affinity for the PEARl protein.
146. The method of any one of claims 121 to 145, further comprising measuring the level of a Ribonuclease UK114 protein with a capture reagent having affinity for the Ribonuclease UK114 protein.
147. The method of any one of claims 121 to 146, further comprising measuring the level of a Activin A protein with a capture reagent having affinity for the Activin A protein.
148. The method of any one of claims 121 to 147, further comprising measuring the level of a Heparin cofactor II protein with a capture reagent having affinity for the Heparin cofactor II protein.
149. The method of any one of claims 121 to 148, further comprising measuring the level of a SUMF1 protein with a capture reagent having affinity for the SUMF1 protein.
150. The method of any one of claims 121 to 149, further comprising measuring the level of a MP2K4 protein with a capture reagent having affinity for the MP2K4 protein.
151. The method of any one of claims 121 to 150, further comprising measuring the level of a SELW protein with a capture reagent having affinity for the SELW protein.
152. The method of any one of claims 121 to 151, further comprising measuring the level of a HYALl protein with a capture reagent having affinity for the HYAL1 protein.
153. The method of any one of claims 121 to 152, further comprising measuring the level of a al -Antitrypsin protein with a capture reagent having affinity for the al -Antitrypsin protein.
154. The method of any one of claims 121 to 153, further comprising measuring the level of a MMP-7 protein with a capture reagent having affinity for the MMP-7 protein.
155. The method of any one of claims 121 to 154, further comprising measuring the level of a QSOX2 protein with a capture reagent having affinity for the QSOX2 protein.
156. The method of any one of claims 121 to 155, further comprising measuring the level of a IGDC4 protein with a capture reagent having affinity for the IGDC4 protein.
157. The method of any one of claims 121 to 156, further comprising measuring the level of a S100A6 protein with a capture reagent having affinity for the S100A6 protein.
158. The method of any one of claims 121 to 157, further comprising measuring the level of a ATS 13 protein with a capture reagent having affinity for the ATS 13 protein.
159. A method compri sing : a) measuring the level of TMEDA, ARMEL and CAC02 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA, ARMEL and CAC02.
160. A method comprising: a) measuring the level of TMEDA, ARMEL and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of TMEDA, ARMEL and NAGPA.
161. A method compri sing : a) measuring the level of ARMEL, CAC02 and NAGPA in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ARMEL, CAC02 and NAGPA.
162. The method of any one of claims 159 to 161, further comprising measuring the level of a Cystatin C protein.
163. The method of any one of claims 159 to 162, further comprising measuring the level of a DSC2 protein.
164. The method of any one of claims 159 to 163, further comprising measuring the level of a HPRT protein.
165. The method of any one of claims 159 to 164, further comprising measuring the level of a FABP protein.
166. The method of any one of claims 159 to 165, further comprising measuring the level of a PPIC protein.
167. The method of any one of claims 159 to 166, further comprising measuring the level of a GBRAP protein.
168. The method of any one of claims 159 to 167, further comprising measuring the level of a ISK7 protein.
169. The method of any one of claims 159 to 168, further comprising measuring the level of a FSH protein.
170. The method of any one of claims 159 to 169, further comprising measuring the level of a RBP protein.
171. The method of any one of claims 159 to 170, further comprising measuring the level of a HE4 protein.
172. The method of any one of claims 159 to 171, further comprising measuring the level of a COIAI protein.
173. The method of any one of claims 159 to 172, further comprising measuring the level of a PGRP-L protein.
174. The method of any one of claims 159 to 173, further comprising measuring the level of a ERBB1 protein.
175. The method of any one of claims 159 to 174, further comprising measuring the level of a Testican-2 protein.
176. The method of any one of claims 159 to 175, further comprising measuring the level of a FHR1 protein.
177. The method of any one of claims 159 to 176, further comprising measuring the level of a SRCA protein.
178. The method of any one of claims 159 to 177, further comprising measuring the level of a DLK1 protein.
179. The method of any one of claims 159 to 178, further comprising measuring the level of a SLIT2 protein.
180. The method of any one of claims 159 to 179, further comprising measuring the level of a HCC-1 protein.
181. The method of any one of claims 159 to 180, further comprising measuring the level of a CDON protein.
182. The method of any one of claims 159 to 181, further comprising measuring the level of a COFA1 protein.
183. The method of any one of claims 159 to 182, further comprising measuring the level of a PEARl protein.
184. The method of any one of claims 159 to 183, further comprising measuring the level of a Ribonuclease UK114 protein.
185. The method of any one of claims 159 to 184, further comprising measuring the level of a Activin A protein.
186. The method of any one of claims 159 to 185, further comprising measuring the level of a Heparin cofactor II protein.
187. The method of any one of claims 159 to 186, further comprising measuring the level of a SUMF1 protein.
188. The method of any one of claims 159 to 187, further comprising measuring the level of a MP2K4 protein.
189. The method of any one of claims 159 to 188, further comprising measuring the level of a SELW protein.
190. The method of any one of claims 159 to 189, further comprising measuring the level of a HYAL1 protein.
191. The method of any one of claims 159 to 190, further comprising measuring the level of a al -Antitrypsin protein.
192. The method of any one of claims 159 to 191, further comprising measuring the level of a MMP-7 protein.
193. The method of any one of claims 159 to 192, further comprising measuring the level of a QSOX2 protein.
194. The method of any one of claims 159 to 193 further comprising measuring the level of a IGDC4 protein.
195. The method of any one of claims 159 to 194, further comprising measuring the level of a S100A6 protein.
196. The method of any one of claims 159 to 195, further comprising measuring the level of a ATS 13 protein.
197. The method of any one of claims 159 to 196, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
198. A method comprising: a) measuring the level of at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty- eight proteins, or thirty-nine proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of the at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight or thirty-nine proteins.
199. The method of claim 198, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
200. The method of claim 198 or 199, wherein the sample is selected from blood, plasma, serum or urine.
201. The method of any one of claims 198 to 200, wherein the human subject is determined to have renal insufficiency.
202. The method of any one of claims 198 to 201, further comprising measuring one or more of TMEDA, ARMEL, CAC02 and NAGPA.
203. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight proteins, or thirty-nine proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
204. The method of claim 203, wherein the set of capture reagents are selected from aptamers, antibodies and a combinations of aptamers and antibodies.
205. The method of claim 203 or 204, wherein the sample is selected from blood, plasma, serum or urine.
206. The method of any one of claims 203 to 205, wherein the human subject is determined to have renal insufficiency.
207. The method of any one of claims 203 to 206, further comprising measuring one or more of TMEDA, ARMEL, CAC02 and NAGPA.
208. A method comprising: a) measuring the level of Cystatin C protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS 13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of Cystatin C and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
209. A method comprising: a) measuring the level of DSC2 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C , TMEDA, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of DSC2 and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
210. A method compri sing : a) measuring the level of HPRT protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBBl, Testican-2, FHRl, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYAL1, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of HPRT and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
211. A method compri sing : a) measuring the level of FABP protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of FABP and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
212. A method compri sing : a) measuring the level of PPIC protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of PPIC and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty- one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty- seven, or thirty-eight proteins.
213. A method compri sing : a) measuring the level of GBRAP protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of GBRAP and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty -two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, or thirty-eight proteins.
214. A method compri sing : a) measuring the level of ISK7 protein, and the level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEAR1, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13 in a sample from a human subject; and b) estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of ISK7 and the level of the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty- one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty- seven, or thirty-eight proteins.
215. The method of any one of claims 208 to 214, wherein the measuring is performed using mass spectrometry, an aptamer based assay and/or an antibody based assay.
216. The method of any one of claims 208 to 215, wherein the sample is selected from blood, plasma, serum or urine.
217. The method of any one of claims 208 to 216, wherein the human subject is determined to have renal insufficiency.
218. A method compri sing : a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising Cystatin C protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, COIAI, PGRP-L, ERBB1, Testican-2, FHR1, SRC A, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
219. A method compri sing : a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising DSC2 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C , TMEDA, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
220. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising HPRT protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
221. A method compri sing : a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising FABP protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
222. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising PPIC protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
223. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising GBRAP protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty -two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBBl, Testican-2, FHRl, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents.
224. A method comprising: a) contacting a sample from a human subject with a set of capture reagents, wherein each capture reagent has affinity for a different protein of the set of proteins comprising ISK7 protein, and at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, or thirty-eight proteins selected from the group consisting of Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13; and b) measuring the level of each protein of the set of proteins with the set of capture reagents
225. The method of any one of claims 218 to 224, wherein the set of capture reagents is selected from aptamers, antibodies and a combinations of aptamers and antibodies.
226. The method of any one of claims 218 to 225, wherein the sample is selected from blood, plasma, serum or urine.
227. The method of any one of claims 218 to 226, wherein the human subject is determined to have renal insufficiency.
228. The method of any one of claims 19 to 32, 38 to 78, 121 to 197, 203 to 207, and 218 to 227, further comprising estimating or determining glomerular filtration rate (GFR) for the human subject based on the level of each protein measured.
229. The method of any one of claims 1 to 18, 3 to 37, 79 to 120, 198 to 202, 208 to 217, and 228, wherein estimating or determining GFR for the human subject is based on input of the level of each protein measured in a statistical model.
230. The method of claim 229, wherein the model is a linear model for the log of estimated GFR.
231. The method of claim 229 or 230, wherein the R2 value is > 0.65, 0.7, 0.75, or 0.8.
232. The method of any one of claims 229 to 231, wherein model output is an estimation or determination of GFR as a continuous integer value from 5 to 100 ml/min/1.73 m2, inclusive.
233. The method of any one of claims 229 to 232, wherein the model estimates or determines glomerular filtration rate (GFR) for the human subject based on the level of each protein measured selected from Cystatin C, TMEDA, DSC2, HPRT, FABP, PPIC, GBRAP, ISK7, ARMEL, FSH, RBP, HE4, C01A1, PGRP-L, ERBB1, Testican-2, FHR1, SRCA, CAC02, NAGPA, DLK1, SLIT2, HCC-1, CDON, COFA1, PEARl, Ribonuclease UK114, Activin A, Heparin cofactor II, SUMF1, MP2K4, SELW, HYALl, al -Antitrypsin, MMP-7, QSOX2, IGDC4, S100A6, and ATS13.
234. The method of any one of claims 228-233, wherein the level of each protein measured is determined from a relative florescence unit (RFU) or a protein concentration.
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