US20160025737A1 - Biomarkers for assessment of preeclampsia - Google Patents

Biomarkers for assessment of preeclampsia Download PDF

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US20160025737A1
US20160025737A1 US14/341,024 US201414341024A US2016025737A1 US 20160025737 A1 US20160025737 A1 US 20160025737A1 US 201414341024 A US201414341024 A US 201414341024A US 2016025737 A1 US2016025737 A1 US 2016025737A1
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glyfn
subject
serum sample
preeclampsia
determined level
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Srinivasa R. Nagalla
Eric S. Bean
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Diabetomics Inc
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Assigned to DIABETOMICS, INC. reassignment DIABETOMICS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: DIABETOMICS, LLC
Priority to PCT/US2015/041796 priority patent/WO2016014832A1/en
Priority to EP15824634.8A priority patent/EP3172572A4/de
Priority to CN201580051757.6A priority patent/CN107076760A/zh
Publication of US20160025737A1 publication Critical patent/US20160025737A1/en
Priority to US15/833,656 priority patent/US10996228B2/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
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Embodiments herein relate to the field of screening tools for fetal/maternal wellness, and, more specifically, to biomarkers for the assessment of preeclampsia.
  • Preeclampsia is a potentially life-threatening complication unique to pregnancy, and it occurs in up to 7% of all pregnancies.
  • Hypertensive disorders, including preeclampsia are the second leading cause of maternal mortality worldwide, and are responsible for 10%-25% of all maternal deaths.
  • clinical manifestations of preeclampsia may occur late in the course of the disease, and may be associated with adverse maternal and neonatal outcomes.
  • Robust biomarkers for screening, diagnosis, and monitoring, particularly with respect to severe preeclampsia are necessary to appropriately manage preeclampsia and to mitigate adverse outcomes. This is particularly the case in developing countries, where the burden of disease is greatest, and where medical intervention is often ineffective due to late presentation.
  • the incidence of preeclampsia has been increasing since 1990, which may be directly related to the increase in obesity. Early and robust diagnostic tests are urgently needed in order to provide for appropriate triage to skilled medical facilities and management of preeclamptics.
  • FIGS. 1A-1D are tables showing GlyFn serum biomarker concentration within a longitudinal cohort by preeclampsia status and trimester ( FIG. 1A ), total fibronectin serum biomarker concentration within the longitudinal cohort by preeclampsia status and trimester ( FIG. 1B ), the average weekly change in GlyFn concentration by week and preeclampsia status across all cohorts ( FIG. 1C ), and the average weekly change in total serum fibronectin concentration by week and preeclampsia status across all cohorts ( FIG. 1D ), in accordance with various embodiments;
  • FIG. 2 is a table showing maternal characteristics by preeclampsia status and cohort, in accordance with various embodiments
  • FIG. 3 is a graph illustrating that GlyFn levels were significantly higher in patients with preeclampsia across 1st, 2nd and 3rd trimesters, where GlyFn levels were measured in serum from 45 normotensive control (circles and solid lines) and 62 preeclampsia (pluses and dotted lines) subjects across first, second, and third trimesters, in accordance with various embodiments;
  • FIGS. 4A and 4B are a table showing serum biomarker concentration within the longitudinal cohort by preeclampsia status and trimester ( FIG. 4A ), and a plot of GlyFn concentration across the span of pregnancy in the longitudinal cohort ( FIG. 4B ), in accordance with various embodiments;
  • FIGS. 5A and 5B are a table showing serum biomarker concentrations in the normotensive and clinical preeclampsia cohorts ( FIG. 5A ), and a plot of 2 nd and 3 rd trimester GlyFn concentration in normotensive controls and clinical preeclampsia patients ( FIG. 5B ), in accordance with various embodiments;
  • FIGS. 6A and 6B are a table showing average weekly change in GlyFn concentration by week and preeclampsia status ( FIG. 6A ), and a receiver operating characteristic curves showing third-trimester preeclampsia classification performance of biomarkers within all cohorts ( FIG. 6B ), in accordance with various embodiments;
  • FIG. 7 is a table showing third-trimester preeclampsia classification performance of biomarkers within all cohorts, in accordance with various embodiments.
  • FIG. 8 is a table showing GlyFn POC values for prediction of preeclampsia at varying prevalence estimates, in accordance with various embodiments
  • FIG. 9 is a table showing a relationship of third-trimester GlyFn levels to clinical characteristics and outcomes in normotensives and clinical preeclampsia, in accordance with various embodiments.
  • FIGS. 10A and 10B illustrate a schematic diagram showing an example of a lateral flow immunoassay ( FIG. 10A ) and a lateral flow test device ( FIG. 10B ) that may be used in accordance with various embodiments disclosed herein.
  • Coupled may mean that two or more elements are in direct physical or electrical contact. However, “coupled” may also mean that two or more elements are not in direct contact with each other, but yet still cooperate or interact with each other.
  • a phrase in the form “NB” or in the form “A and/or B” means (A), (B), or (A and B).
  • a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).
  • a phrase in the form “(A)B” means (B) or (AB) that is, A is an optional element.
  • Disclosed herein in various embodiments are methods for assessing the risk of, predicting, diagnosing, and monitoring preeclampsia in a subject. Methods disclosed herein also may be used for distinguishing between mild preeclampsia and severe preeclampsia. Also disclosed are methods of predicting or diagnosing low birth weight and/or HELLP syndrome, a life-threatening complication of preeclampsia involving hemolysis, elevated liver enzymes, and low platelet count. In various embodiments, the methods may include measuring the level of glycosylated fibronectin (GlyFn) in a biological sample from a subject, such as a serum sample. Although serum samples are described herein, one of skill in the art will appreciate that the disclosed methods may be adapted for use with other biological samples, such as whole blood, plasma, urine, saliva, or other bodily fluids.
  • GlyFn glycosylated fibronectin
  • Fibronectin is an abundant protein with a wide spectrum of functions.
  • An exemplary GENBANK® Accession number for human fibronectin is Genbank Accession No. P02751.
  • the Fn gene encodes a collection of isoforms that differ in sequence and length.
  • the majority of the Fn present in serum or plasma is termed plasma Fn (pFn), which is produced and secreted in a soluble form by hepatocytes, while so-called cellular Fn (cFn) is produced by numerous cell types, including fibroblasts, endothelial cells, and smooth muscle cells.
  • pFn and cFn are the presence of alternatively spliced extra domains A and B (ECA/B) that are absent in pFn but variably present in cFn. It is increasingly clear that cFn is also found in the circulation, especially in various pathological conditions, including diabetes and inflammation. Both pFn and cFn exhibit complex patterns of glycosylation, and elevated levels of a specific glycosylated version of Fn (Fn-SNA) in maternal serum have been shown to predict gestational diabetes. However, prior to the present disclosure, elevated GlyFn was not known to be associated with preeclampsia.
  • FIGS. 1A-1D are tables showing GlyFn serum biomarker concentration within a longitudinal cohort by preeclampsia status and trimester ( FIG. 1A ), total fibronectin serum biomarker concentration within the longitudinal cohort by preeclampsia status and trimester ( FIG. 1B ), the average weekly change in GlyFn concentration by week and preeclampsia status across all cohorts ( FIG. 1C ), and the average weekly change in total serum fibronectin concentration by week and preeclampsia status across all cohorts ( FIG.
  • total fibronectin levels ( FIG. 1B ) and rate of change in total fibronecting levels ( FIG. 1D ) are not predictive of preeclampsia status.
  • the disclosed methods include obtaining a biological sample, such as a serum sample, whole blood sample, plasma sample, or saliva sample, from a pregnant subject.
  • a biological sample such as a serum sample, whole blood sample, plasma sample, or saliva sample
  • the level of glycosylated fibronectin (GlyFn) in the sample is then determined using any of several possible methods, and the level of GlyFn in the sample is compared to a control value, such as a reference value representative of a GlyFn level normally found in a subject who will not go on to develop preeclampsia.
  • the subject may be categorized as not having preeclampsia or being at low risk of developing preeclampsia.
  • the sample GlyFn value is determined to be elevated relative to the control value (e.g., elevated to a statistically significant degree with respect to the control value or outside a predetermined range of “normal” values) then the subject may be determined to have preeclampsia or be at elevated risk of developing preeclampsia.
  • control value may be a reference value (or a range of “normal” values) representative of a level of GlyFn in a sample from a first trimester subject who will not go on to develop preeclampsia.
  • an “elevation” in the sample GlyFn level relative to the first trimester control value may be at least a 15% elevation, such as a 20% elevation, a 30% elevation, a 40% elevation, a 50% elevation, a 60% elevation, a 70% elevation, an 80% elevation, a 90% elevation, a 100% elevation, or even more, such as a 125% elevation or a 150% elevation.
  • a normal (non-preeclamptic) range of GlyFn in a sample from a first trimester subject may range from 10-150 ⁇ g/ml, and an abnormal (e.g., preeclamptic) level of GlyFn may be greater than 150 ⁇ g/ml, such as about 175 ⁇ g/ml or higher.
  • the method may be a method of determining the risk of preeclampsia in a subject during the second trimester.
  • the control value may be a reference value (or range of “normal” reference values) representative of a level of GlyFn in a sample from a second trimester subject who will not go on to develop preeclampsia.
  • an “elevation” in the sample GlyFn level relative to the second trimester control value may be at least a 15% elevation, such as a 20% elevation, a 30% elevation, a 40% elevation, a 50% elevation, a 60% elevation, a 70% elevation, an 80% elevation, a 90% elevation, a 100% elevation, or even more, such as a 125% elevation or a 150% elevation.
  • a normal (non-preeclamptic) range of GlyFn in a sample from a second trimester subject may range from 10-150 ⁇ g/ml, and an abnormal (e.g., preeclamptic) level of GlyFn may be greater than 150 ⁇ g/ml, such as about 175 ⁇ g/ml or higher.
  • the method may be a method of determining the risk of preeclampsia in a subject during the third trimester.
  • the control value may be a reference value (or range of “normal” reference values) representative of a level of GlyFn in a sample from a third trimester subject who will not go on to develop preeclampsia.
  • an “elevation” in the sample GlyFn level relative to the third trimester control value may be at least a at least a 30% elevation, such as a 40% elevation, a 50% elevation, a 60% elevation, a 70% elevation, a 80% elevation, a 90% elevation, a 100% elevation, a 125% elevation, a 150% elevation, or even more, such as a 200% elevation or a 300% elevation.
  • a normal (non-preeclamptic) range of GlyFn in a sample from a first trimester subject may range from 10-150 ⁇ g/ml, and an abnormal (e.g., preeclamptic) level of GlyFn may be greater than 150 ⁇ g/ml, such as about 200 ⁇ g/ml or higher.
  • Still other embodiments may be methods of assessing the risk of low birth weight, methods of assessing the risk of HELLP syndrome, or methods of diagnosing preeclampsia in a subject.
  • the subject may be in the third trimester of pregnancy, and a level of GlyFn in the serum sample from the subject that is equal to or greater than about 100 ⁇ g/ml, such as about 110 ⁇ g/ml, about 120 ⁇ g/ml, about 130 ⁇ g/ml, about 140 ⁇ g/ml, about 150 ⁇ g/ml, about 160 ⁇ g/ml, about 170 ⁇ g/ml, about 180 ⁇ g/ml, about 200 ⁇ g/ml, or even more, may indicate that the subject has preeclampsia.
  • a level of GlyFn in the serum sample from the subject that is equal to or greater than about about 250 ⁇ g/ml, such as about 275 ⁇ g/ml, about 300 ⁇ g/ml, about 325 ⁇ g/ml, about 350 ⁇ g/ml, about 375 ⁇ g/ml, about 400 ⁇ g/ml, about 425 ⁇ g/ml, about 450 ⁇ g/ml, about 475 ⁇ g/ml, about 500 ⁇ g/ml, or even more, may indicate that the subject is at risk of having a low birth weight (small for gestational age or SGA) baby or of developing HELLP syndrome.
  • SGA small for gestational age
  • a level of GlyFn in the serum sample from the subject that is equal to or greater than about 500 ⁇ g/ml or even more may indicate that the subject has a high risk of having a low birth weight (SGA) baby or of developing HELLP syndrome.
  • SGA birth weight
  • the method may further include obtaining at least one additional serum sample from the subject at least one week after the first serum sample was obtained, such as a series of weekly samples between weeks 33-38 of pregnancy.
  • the method may include determining the level of glycosylated fibronectin (GlyFn) in the second serum sample and comparing the level of GlyFn in the second serum sample with the previously determined level of GlyFn in the first serum sample.
  • GlyFn glycosylated fibronectin
  • a weekly increase in the level of GlyFn in the second (or subsequent) serum sample compared to the level of GlyFn in the first (or previous) serum sample may indicate that a diagnosis of preeclampsia is warranted.
  • a weekly increase of between about 15 ⁇ g/ml and 125 ⁇ g/ml such as about 25 ⁇ g/ml, about 35 ⁇ g/ml, about 45 ⁇ g/ml, about 55 ⁇ g/ml, about 65 ⁇ g/ml, about 75 ⁇ g/ml, about 85 ⁇ g/ml, about 95 ⁇ g/ml, about 105 ⁇ g/ml, about 115 ⁇ g/ml, or about 125 ⁇ g/ml, may indicate that the subject has mild preeclampsia.
  • a weekly increase of more than about 150 ⁇ g/ml such as about 175 ⁇ g/ml, about 200 ⁇ g/ml, about 225 ⁇ g/ml, about 300 ⁇ g/ml, about 325 ⁇ g/ml, about 350 ⁇ g/ml, about 375 ⁇ g/ml, about 400 ⁇ g/ml, or about 425 ⁇ g/ml or more, may indicate that the subject has severe preeclampsia.
  • Statistical methods for determining if the abundance of a protein of interest is increased or decreased relative to a reference sample are well known in the art, and are described below.
  • determination of the level of GlyFn in a biological fluid such as whole blood, plasma, serum, saliva, or urine, may be performed using a variety of methods known to those of skill in the art.
  • the reference sample and test sample may be treated exactly the same way, in order to correctly represent the relative abundance of GlyFn and obtain accurate results.
  • the proteins present in the biological samples may be separated by 2D-gel electrophoresis according to their charge and molecular weight.
  • the proteins may first be separated by their charge using isoelectric focusing (one-dimensional gel electrophoresis), for example using immobilized pH-gradient (IPG) strips, which are commercially available.
  • the second dimension may be an SDS-PAGE analysis, where the focused IPG strip may be used as the sample.
  • proteins may then be visualized with conventional dyes, such as Coomassie Blue or silver staining, and imaged using known techniques and equipment, such as, for example Bio-Rad GS800 densitometer and PDQUESTTM software.
  • individual spots may then be cut from the gel, de-stained, and subjected to tryptic digestion, allowing the peptide mixtures to be analyzed by mass spectrometry (MS).
  • MS mass spectrometry
  • the peptides may be separated, for example by capillary high pressure liquid chromatography (HPLC) and may be analyzed by MS either individually, or in pools.
  • HPLC capillary high pressure liquid chromatography
  • the amino acid sequences of the peptide fragments and the proteins from which they derived may be determined. Although it is possible to identify and sequence all or some of the proteins present in a proteomic profile, this typically is not necessary for the diagnostic use of the methods disclosed herein.
  • a diagnosis of or risk of preeclampsia may be based on characteristic similarities or differences between a reference sample and a test sample.
  • the expression signature may be a peak representing GlyFn that differs, qualitatively or quantitatively, from the mass spectrum of a corresponding normal sample.
  • any statistically significant change in the amplitude or shape of an existing peak may reflect a change in a leve of GlyFn relative to a control.
  • Protein arrays may utilize protein arrays to monitor GlyFn levels, enabling high-throughput analysis.
  • Protein arrays are known to those of skill in the art, and generally are formed by immobilizing proteins, such as antibodies specific for proteins of interest, like GlyFn, on a solid surface, such as glass, silicon, nitrocellulose, or PVDF using any of a variety of covalent and non-covalent attachment chemistries well known in the art.
  • the arrays may be probed with fluorescently labeled proteins from two different sources, such as normal and test samples, and fluorescence intensity may reflect the expression level of a target protein, such as GlyFn.
  • immunoassays may be homogeneous or heterogeneous.
  • an enzyme-linked immunosorbant assay ELISA
  • a solid surface may be coated with a solid phase antibody, and the test sample may be allowed to react with the bound antibody. Any unbound antigen may then be washed away, and a known amount of enzyme-labeled antibody may then be reacted. The label may then be quantified as a direct measurement of the amount of protein of interest present in the sample.
  • ELISA may also be used as a competitive assay.
  • the test sample containing the protein of interest may be mixed with a precise amount of enzyme-labeled protein of interest, and both may compete for binding to an antibody attached to a solid surface.
  • excess free enzyme-labeled protein may be washed off before the substrate for the enzyme is added, and the color intensity resulting from the enzyme-substrate interaction may be used as a measure of the amount of protein of interest in the test sample.
  • Various other embodiments may quantify the proteins of interest using an Enzyme Multiplied Immunoassay Technique (EMIT), which may include a test sample, enzyme-labeled molecules of the proteins of interest, antibodies specific to the proteins of interest, and a specific enzyme chromogenic substrate.
  • EMIT Enzyme Multiplied Immunoassay Technique
  • an excess of the specific antibodies may be added to the test sample, and the proteins of interest may then bind to the antibodies.
  • a measured amount of the corresponding enzyme-labeled proteins may then be added to the mixture, and antibody binding sites not occupied by proteins of interest from the test sample may be occupied with molecules of the enzyme-labeled protein.
  • enzyme activity may be reduced because only free enzyme-labeled protein can act on the substrate, and the amount of converted substrate may reflect the amount of free enzyme left in the mixture.
  • a high concentration of the protein of interest in the sample may result in higher absorbance readings.
  • kits for the quantification of the proteins of interest in a test sample.
  • these kits may include, in separate containers, one or more monoclonal or polyclonal antibodies having binding specificity for GlyFn, and, optionally, anti-antibody immunoglobulins, particularly labeled anti-antibody immunoglobulins.
  • a sample capture device such as a lateral flow device (for example a lateral flow test strip) that may allow quantification of GlyFn.
  • Lateral flow devices are available in numerous different configurations, but in one example, a test strip may include a flow path from an upstream sample application area to a test site, such as from a sample application area through a mobilization zone to a capture zone.
  • the mobilization zone may contain a mobilizable marker that may interact with the protein of interest
  • the capture zone may contain a reagent that binds the protein of interest for detection and/or quantification.
  • exemplary sample collection kits may include an absorbent medium, such as filter paper, that may include indicia for the placement of the test sample on the medium.
  • Such kits also may include a lancing device for obtaining a blood sample from a subject, and optionally, a mailer for sending the test sample to a physician or laboratory for analysis.
  • sample collection kits may be used, for example, during standard prenatal exams, such as the eight week, twelve week, sixteen week, twenty week, twenty-four week, twenty-eight week, thirty week, or subsequent-week visit, and/or sample collection may be performed when blood is obtained for other standard prenatal tests.
  • a longitudinal cohort consisted of 60 women who were sampled serially throughout pregnancy, with the first sample taken between 6 and 14 weeks of gestation and an additional sample obtained in each trimester. Forty-five women remained normotensive and 15 developed preeclampsia at various gestational ages.
  • a clinical preeclampsia cohort of 47 patients who were diagnosed with preeclampsia at various gestational ages was analyzed to measure the rate of change in GlyFn levels during the course of their preeclampsia.
  • Preeclampsia status was defined having a systolic blood pressure ⁇ 140 mmHg or a diastolic blood pressure ⁇ 90 mmHg with proteinuria ⁇ 300 mg/day.
  • the study participants were recruited from the Department of Obstetrics and Gynecology, Oulu University Hospital, Oulu, Finland, and the Finnish maternity cohort serum bank at the National Institute for Health and Welfare between 2004 and 2006.
  • the research protocol was approved by the Oulu University Hospital Ethics Committee, and all participants provided informed consent.
  • Maternal blood was spun, aliquoted, and stored at ⁇ 80° C. until subjected to the assays described below.
  • GlyFn plate assay Reacti-Bind plates (Thermo Scientific, Rockford, Ill.) were coated with an Fc fragment-specific goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.; cat #115-005-071) in carbonate buffer, pH 9.6, and incubated at 4° C. overnight, followed by washing with PBS-0.05% Tween 20. Plates were blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS), pH 7.2, for one hour at room temperature. Plates were then washed with PBS-0.05% Tween 20 buffer and an anti-GlyFn monoclonal antibody was added and incubated for 45 minutes at room temperature.
  • PBS phosphate-buffered saline
  • antibodies for use in the methods and devices of this disclosure may be monoclonal or polyclonal.
  • monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein ( Nature 256:495-497, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane ( Antibodies, A Laboratory Manual , CSHL, New York, 1988).
  • GlyFn POC assay A fluorescence immunoassay, comprising an automated cassette reader (LRE Medical cPoC Reader, LRE Medical, Oceanside, Calif.) and a disposable, single-use, plastic assay cartridge was developed that employs standard immunoassay techniques to specifically and quantitatively detect GlyFn in serum specimens.
  • the polyclonal anti-Fn antibody employed in the plate assay described above was conjugated to a Tide FluorTM 5WS succinimidyl ester fluorescent tag (AAT Bioquest, Sunnyvale, Calif.; cat #2281) and served as the detection antibody.
  • the monoclonal anti-GlyFn antibody employed in the plate assay described above served as the capture antibody and was immobilized on a solid phase (test zone).
  • Goat polyclonal anti-rabbit IgG, Fc antibody (Jackson ImmunoResearch Laboratories, Inc.; cat #111-045-046) was immobilized in a separate capture zone to act as a reference for the test zone and to provide assurance that the device performed properly.
  • Serum was diluted in assay buffer and applied to the test strip.
  • the serum flows down the diagnostic lane via capillary action, taking the fluorescent detection antibody into suspension.
  • GlyFn in the specimen binds to the fluorescent antibody to form a multivalent complex that is captured by the antibody immobilized in the test zone.
  • the cartridge is inserted into the cassette reader and quantitative measurements of glyFn concentration in the range from 10 to 2000 ⁇ g/mL are displayed on the meter screen and/or printout after 10 minutes.
  • sFlt1 levels were determined by ELISA. Due to the large amount of serum needed for this assay, 13 participants were unable to be assayed for this analyte.
  • PIGF levels were determined using a commercial kit (R&D Systems Human PIGF Quantikine ELISA Kit; cat # DPG00). Due to inadequate serum sample, this analysis was subset to 57 subjects. Plates were read using an Epoch plate reader (BioTek, Winooski, Vt.) at 450 nm, and data were processed using Gen5 software version 1.10.8 and analyzed as described below.
  • samples were subset to the third trimester and the normotensive group was compared to the clinical preeclampsia cohort via non-parametric Wilcoxon t-tests.
  • a subset of patients with two repeated measures between 33 and 38 weeks were used to calculate the average weekly change.
  • Receiver-operating characteristic (ROC) curves were generated using predicted probabilities from logistic regression models using a single age-matched measure from 14 to 40 weeks of gestation for each subject from all cohorts.
  • the area under the ROC curve (AUROC) and corresponding 95% confidence limits were calculated using simple logistic regression. Sensitivity and specificity were reported based on thresholds chosen, and 95% confidence limits calculated by the score method with a continuity correction are reported.
  • Statistical tests of differences in ROC curves were calculated using contrast matrices of differences. Hypothetical predictive values and 95% confidence intervals (CI) were calculated using the standard logit method using a population prevalence of 3%, 5%, or 7%.
  • GlyFn was compared to gestational age at delivery, birth weight, systolic blood pressure, and diastolic blood pressure.
  • comparative analyses were performed with GlyFn with respect to gestational age of preeclampsia start, uric acid, ALAT, proteinuria, HELLP syndrome, small-for-gestational age, and placental insufficiency.
  • Pearson correlation coefficients and linear regression slopes were calculated.
  • linear regression slopes were calculated to reflect a change in GlyFn of 100 ⁇ g.
  • Fisher's exact tests were performed by categorizing participants as those with or without GlyFn levels ⁇ 500 ⁇ g.
  • FIG. 2 is a table showing maternal characteristics by preeclampsia status and cohort, in accordance with various embodiments.
  • Patients in the clinical preeclampsia group were more likely to give birth earlier (p ⁇ 0.01) and have lower neonatal birth weights (p ⁇ 0.01; FIG. 2 ).
  • There was no difference in maternal age (p 0.14) and nulliparity (0.31) between the cohorts.
  • Median gestational age at diagnosis of preeclampsia was significantly later in the longitudinal preeclampsia group than in the clinical preeclampsia cohort.
  • FIG. 3 is a graph illustrating a comparison of the longitudinal normotensive and preeclampsia groups, showing that, within each trimester, GlyFn levels were significantly higher in patients with preeclampsia than in controls
  • FIGS. 4A and 4B are a table showing serum biomarker concentration within the longitudinal cohort by preeclampsia status and trimester ( FIG. 4A ), and a plot of GlyFn concentration across the span of pregnancy in the longitudinal cohort ( FIG. 4B ), in accordance with various embodiments.
  • FIGS. 5A and 5B are a table showing serum biomarker concentrations in the normotensive and clinical preeclampsia cohorts ( FIG. 5A ), and a plot of 2 nd and 3 rd trimester GlyFn concentration in normotensive controls and clinical preeclampsia patients ( FIG. 5B ), in accordance with various embodiments.
  • levels of GlyFn, sFLt1, PIGF, and the sFLT1/PIGF ratio were compared between age-matched samples from the normotensive control and clinical preeclampsia cohorts. There was a significant difference in all serum biomarkers between participants with and without preeclampsia (p ⁇ 0.01).
  • FIGS. 6A and 6B are a table showing average weekly change in GlyFn concentration by week and preeclampsia status ( FIG. 6A ), and a receiver operating characteristic curves showing third-trimester preeclampsia classification performance of biomarkers within all cohorts ( FIG. 6B ), in accordance with various embodiments
  • FIG. 7 is a table showing third-trimester preeclampsia classification performance of biomarkers within all cohorts
  • FIG. 8 is a table showing GlyFn POC values for prediction of preeclampsia at varying prevalence estimates, in accordance with various embodiments.
  • the clinical utility of these biomarkers for detection of preeclampsia was tested via ROC curves.
  • the AUROCs for GlyFn, sFlt1, PIGF, and the sFlt1/PIGF ratio are show in FIG. 7 . Since the sFlt1 assay requires significant serum quantities, this analysis was restricted to 15 control and 39 preeclampsia participants between 20 and 39 weeks of gestation with sufficient serum for sFlt1 analysis.
  • GlyFn demonstrated a sensitivity of 0.97 (0.85 to 1.00) and a specificity of 0.93 (0.66 to 1.00).
  • the positive predictive and negative predictive values were 47% (95% CI: 23-72%) and 89% (95% CI: 80-98%), respectively ( FIG. 8 ).
  • FIG. 9 is a table showing the relationship of third-trimester GlyFn levels to clinical characteristics and outcomes in normotensives and clinical preeclampsia, in accordance with various embodiments.
  • GlyFn values had a significant linear relationship with gestational age at delivery, birth weight, blood pressure, uric acid, and ALAT.
  • GlyFn is shown herein to be a powerful biomarker for preeclampsia, and as such, GlyFn may be used in various assessment and diagnostic methods, such as assessing the progression of preeclampsia over time based on elevated levels in first-trimester maternal serum, and monitoring the progressive increase throughout pregnancy in order to predict or diagnose preeclampsia in a subject.
  • increasing GlyFn levels were correlated with important clinical characteristics and outcomes, including earlier delivery, decreases in birth weight, and increases in blood pressure, uric acid, and ALAT.
  • GlyFn is a uniquely useful analyte to monitor preeclampsia, since GlyFn levels remain constant in controls throughout pregnancy. The best sensitivity and specificity for prediction of preeclampsia were found to be at a cutoff for GlyFn of 176.4 ⁇ g/ml.
  • measurement of serum GlyFn levels may be used in methods for the management of preeclampsia, methods of prediction of poor clinical outcomes (low birth weight, HELLP, etc.), and methods of distinguishing between mild and severe preeclampsia.
  • GlyFn fraction presumably reflects a strong involvement of GlyFn with the pathological processes that initiate preeclampsia. Without being bound by theory, this, in turn, may reflect the particular involvement in preeclampsia development of Fn splice variants or proteolytic fragments that exhibit unique glycosylation patterns. It is of interest that oxygen levels have recently been reported to regulate expression of the core-1 O-glycan Gal ⁇ 1-3GalNac epitope in human placenta; thus, without being bound by theory, placental insufficiency may contribute to altered glycoprotein level in preeclampsia.
  • GlyFn with gestational diabetes as well as preeclampsia may be a consequence of the fact that both conditions are associated with inflammation and endothelial dysfunction.
  • first-trimester inflammation and endothelial dysfunction related to disrupted spiral artery remodeling may be linked to increased levels of a specific form of glycosylated Fn.
  • the distinct patterns of GlyFn abundance in in these two related conditions remains of interest (e.g., consistently elevated in all trimesters in gestational diabetes but a progressive increase during the course of preeclampsia), but it may indicate that the factors that trigger gestational diabetes are established early in pregnancy and remain at a constant level, while initiation and development of preeclampsia involves a continuous increase in the conditions that produce GlyFn.
  • the methods disclosed herein enable the use of GlyFn as a biomarker for monitoring the severity of preeclampsia, as well as the use of GlyFn for predicting the development of preeclampsia, particularly in a first or second trimester patient.
  • sFlt1 and PIGF are currently used in investigational studies for the diagnosis of preeclampsia, but not for early prediction or monitoring of disease progression.
  • the correlation found between GlyFn and clinical outcomes is important and unique, in that it establishes a method for predicting which patients will have poor maternal and/or fetal outcomes.
  • GlyFn is significantly different between patients with and without preeclampsia across the span of pregnancy (including before the clinical presentation of preeclampsia), which has not been shown for other preeclampsia analytes and supports the idea of an early pathogenesis of the disease.
  • elevated GlyFn levels may serve as an early indicator of risk for preeclampsia.
  • FN LFIA Glycosylated Fibronectin Lateral Flow Immunoassay
  • GlyFn levels may be assessed using a lateral flow device.
  • Various lateral flow assay methods may be utilized to test for the presence or absence or quantity of an analyte, such as GlyFn, in a biological sample.
  • a “sandwich” assay method uses an antibody immobilized on a solid support, which forms part of a complex with a labeled antibody, to determine the presence of a target analyte by observing the presence and amount of bound analyte-labeled antibody complex.
  • the label may be an enzyme, colored microspheres, fluorescently-labeled microspheres, or may use other similar detection methods that provide for detection and/or quantification of analyte bound to the test line.
  • Conventional lateral flow test strips feature a solid support on which the sample receiving area and the target capture zones are supported.
  • the solid support material is one which is capable of supporting the sample receiving area and target capture zones and providing for the capillary flow of sample out from the sample receiving area to the target capture zones when the lateral flow test strip is exposed to an appropriate solvent or buffer which acts as a carrier liquid for the sample.
  • Specific classes of materials that may be used as support include organic or inorganic polymers, and natural and synthetic polymers. More specific examples of suitable solid supports include, without limitation, glass fiber, cellulose, nylon, crosslinked dextran, various chromatographic papers and nitrocellulose. One particularly useful material is nitrocellulose.
  • FIGS. 10A and 10B illustrate a schematic diagram showing an example of a lateral flow immunoassay ( FIG. 10A ) and a lateral flow test device ( FIG. 10B ) that may be used in accordance with various embodiments disclosed herein.
  • a lateral flow immunoassay FIG. 10A
  • a lateral flow test device FIG. 10B
  • 200 ⁇ g/mL Rabbit anti-GlyFn is immobilized on the membrane as a test line (0.5 ⁇ L/strip) and 300 ⁇ g/mL goat anti-mouse IgG is immobilized as the procedural control line (0.5 ⁇ L/strip).
  • Mouse anti-Fn-conjugated microspheres (10 ⁇ L of 150 ⁇ g/mL mouse anti-fibronectin, 1 mg/mL solids) are dried onto a conjugate pad that has been treated with a solution containing (per liter): 3.81 g sodium borate, 2.0 g dextran, 5.0 g BSA, 1.0 g Tween-20, and 0.5 g sodium azide, pH 8.0, followed by drying for 1 hour at 50° C.
  • the sample is then diluted 1:500 in HEPES Running Buffer (10 mM HEPES, 0.1 mM CaCl 2 , 155 mM NaCl, 0.1% NaN 3 , 0.75% Tween-20, and 0.01% polyvinyl alcohol).
  • HEPES Running Buffer 10 mM HEPES, 0.1 mM CaCl 2 , 155 mM NaCl, 0.1% NaN 3 , 0.75% Tween-20, and 0.01% polyvinyl alcohol.
  • the device is scanned, and the amount of GlyFn in the sample is determined by quantitative densitometry relative to a standard curve using purified GlyFn as a standard.

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Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5523229A (en) * 1994-03-22 1996-06-04 Trustees Of The University Of Pennsylvania Antibodies specific for oncofetal fibronectin
US20060020043A1 (en) * 2004-07-26 2006-01-26 Roger Berlin Methods and compositions for reducing C-reactive protein
US20060024725A1 (en) * 2004-07-30 2006-02-02 Robert Hussa Oncofetal fibronectin as a marker for pregnancy-related indications
US20070092911A1 (en) 2005-10-03 2007-04-26 Buechler Kenneth F Methods and compositions for diagnosis and /or prognosis in systemic inflammatory response syndromes
WO2009097584A1 (en) * 2008-01-30 2009-08-06 Proteogenix, Inc. Maternal serum biomarkers for detection of pre-eclampsia
US20120252015A1 (en) * 2011-02-18 2012-10-04 Bio-Rad Laboratories Methods and compositions for detecting genetic material
CN110208540B (zh) * 2008-10-31 2024-02-09 耶鲁大学 子痫前期检测和治疗的方法和组合物
CA2743570A1 (en) * 2008-11-20 2010-05-27 Perkinelmer Health Sciences, Inc. Method for determining the risk of pre-eclampsia using pigf-2 and pigf-3 markers
EP2836845B1 (de) * 2012-04-13 2018-05-09 DiabetOmics, Inc. Biomarker für schwangerschaftsdiabetes
US20160025737A1 (en) 2014-07-25 2016-01-28 Diabetomics, Llc Biomarkers for assessment of preeclampsia

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
De Vivo., Endoglin, PlGF and sFLt-1 as markers for predicting pre-eclampsia, AOEG, 2008, 837-842 *

Cited By (1)

* Cited by examiner, † Cited by third party
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