WO2015164617A1 - Biomarqueurs de la tuberculose dans l'urine et leurs utilisations - Google Patents

Biomarqueurs de la tuberculose dans l'urine et leurs utilisations Download PDF

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WO2015164617A1
WO2015164617A1 PCT/US2015/027318 US2015027318W WO2015164617A1 WO 2015164617 A1 WO2015164617 A1 WO 2015164617A1 US 2015027318 W US2015027318 W US 2015027318W WO 2015164617 A1 WO2015164617 A1 WO 2015164617A1
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Prior art keywords
biomarker
biomarkers
mtb
composition
aptamer
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PCT/US2015/027318
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English (en)
Inventor
Urs Ochsner
David Sterling
Stephen Kraemer
Nebojsa Janjic
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Somalogic, Inc.
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Publication of WO2015164617A1 publication Critical patent/WO2015164617A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria

Definitions

  • the present application relates generally to biomarkers for tuberculosis infection and methods of detection thereof.
  • the invention relates to one or more biomarkers, biomarker panels, methods, devices, reagents, systems, and kits for detecting and/or characterizing tuberculosis infection in an individual from a urine sample.
  • Tuberculosis is caused by a bacterium called Mycobacterium tuberculosis.
  • the bacteria usually attack the lungs, but TB bacteria can attack any part of the body such as the kidney, spine, and brain. If not treated properly, TB disease can be fatal. Not everyone infected with TB bacteria becomes sick.
  • two TB-related conditions exist: latent TB infection and TB disease. Both latent TB infection and TB disease can be treated.
  • methods are provided for detecting the level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention.
  • biomarkers are selected from MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, A95C, or any sub- combinations thereof.
  • a method comprises detecting the level of one or more biomarkers in a sample from a subject.
  • a sample is a urine sample
  • a method of detecting or diagnosing TB infection in a subject comprises forming a biomarker panel having N biomarker proteins from Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., comprising MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51 , EsxB, EsxA, A85 A, A85B, A95C, or any sub-combinations thereof), and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • N is 1 to 5.
  • N is 2 to 10.
  • N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10. In some embodiments, N is 10 to 20. In some embodiments, N is 5 to 20. In some embodiments, N is 5 to 30. In some embodiments, N is 10 to 30. In some embodiments, N is 20 to 30. In some embodiments, at least one (e.g., 1-13) of the N biomarker proteins is selected from MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C.
  • at least one (e.g., 1-13) of the N biomarker proteins is selected from MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85
  • methods comprise panels of any combination of the Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, A95C, or any sub-combinations thereof), in addition to any other TB biomarkers.
  • Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention
  • biomarker panels are provided having reagents for the detection of N biomarker proteins from the Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., comprising MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, A95C, or any sub-combinations thereof).
  • N is 1 to 5.
  • N is 2 to 10.
  • N is 3 to 10.
  • N is 4 to 10.
  • N is 5 to 10.
  • N is 10 to 20.
  • N is 5 to 20. In some embodiments, N is 5 to 30. In some embodiments, N is 10 to 30. In some embodiments, N is 20 to 30. In some embodiments, at least one (e.g., 1-13) of the N biomarker proteins is selected from MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C.
  • panels of any combination of the Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, A95C, or any sub-combinations thereof, in addition to any other TB biomarkers.
  • the each biomarker may be a protein biomarker.
  • the method may comprise contacting biomarkers of the sample from the subject with a set of biomarker capture reagents, wherein each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a biomarker being detected.
  • each biomarker capture reagent of the set of biomarker capture reagents specifically binds to a different biomarker being detected.
  • each biomarker capture reagent may be an antibody or an aptamer.
  • each biomarker capture reagent may be an aptamer.
  • At least one aptamer may be a slow off-rate aptamer.
  • at least one slow off-rate aptamer may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with modifications.
  • the modifications are hydrophobic modifications.
  • the modifications are hydrophobic base modifications.
  • each slow off-rate aptamer binds to its target protein with an off rate (t1 ⁇ 2) of > 30 minutes, > 60 minutes, > 90 minutes, > 120 minutes, > 150 minutes, > 180 minutes, > 210 minutes, or > 240 minutes.
  • the sample may be a urine sample.
  • the urine sample is filtered, concentrated (e.g., 2-fold, 5-fold, 10 fold, 20-fold, 50-fold, 100-fold, or more), diluted, or un-manipulated.
  • a method may further comprise treating the subject for TB infection.
  • treating the subject for TB infection comprises a treatment regimen of administering one or more of: isoniazid (INH), rifampin (RIF), rifapentine (RPT), ethambutol (EMB), pyrazinamide (PZA), and/or another approved TB therapeutic to the subject.
  • IH isoniazid
  • RIND rifampin
  • RPT rifapentine
  • EMB ethambutol
  • PZA pyrazinamide
  • methods of monitoring progression or severity of TB infection and/or monitoring effectiveness of treatment in a subject are provided.
  • a method comprises detecting the level of one or more TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C) in a sample from the subject at a first time point.
  • the method further comprises measuring the level one or more of the biomarkers at a second time point.
  • TB infection severity is improving (e.g., declining) if the level of said biomarkers improved at the second time point than at the first time point.
  • kits are provided.
  • a kit comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine aptamers, at least ten aptamers, wherein each aptamer specifically binds to a different target protein selected from the Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • a kit comprises N aptamers.
  • N is 1 to 30. In some embodiments, N is 2 to 30. In some embodiments, N is 3 to 30. In some embodiments, N is 4 to 30. In some embodiments, N is 5 to 30. In some embodiments, N is 1 to 10. In some embodiments, N is 2 to 10. In some embodiments, N is 3 to 10. In some embodiments, N is 4 to 10. In some embodiments, N is 5 to 10.
  • At least one of the N biomarker proteins is selected from the Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • compositions comprising proteins of a sample from a subject and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine aptamers, or more (e.g., 10, 11, 12, 13, or more) wherein each aptamer specifically binds to a different target protein selected from the Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • Mtb biomarkers identified in experiments conducted during development of embodiments of the present invention
  • a composition comprises proteins of a sample from a subject and N aptamers.
  • N is 1 to 30.
  • N is 2 to 30.
  • N is 3 to 30.
  • N is 4 to 30.
  • N is 5 to 30.
  • N is 1 to 10.
  • N is 2 to 10.
  • N is 3 to 10.
  • N is 4 to 10.
  • N is 5 to 10.
  • the sample in a composition is a urine sample.
  • the urine sample is filtered, concentrated (e.g., 2-fold, 5-fold, 10 fold, 20-fold, 50-fold, 100-fold, or more), diluted, or un-manipulated.
  • a kit or composition may comprise at least one aptamer that is a slow off-rate aptamer.
  • each aptamer of a kit or composition may be a slow off-rate aptamer.
  • at least one slow off-rate aptamer comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with modifications.
  • at least one nucleotide with a modification is a nucleotide with a hydrophobic base modification.
  • each nucleotide with a modification is a nucleotide with a hydrophobic base modification.
  • each slow off-rate aptamer in a kit binds to its target protein with an off rate (t1 ⁇ 2) of > 30 minutes, > 60 minutes, > 90 minutes, > 120 minutes, > 150 minutes, > 180 minutes, > 210 minutes, or > 240 minutes.
  • FIGURES Figure 1 shows SDS-PAGE analysis of Mtb proteins used for SELEX.
  • Figure 2 shows a schematic of the SELEX process, including the structures of certain modified bases used in the randomized libraries.
  • Figure 3A-B shows qualification of t£-specific SOMAmers for SOMAscan by slide hybridization assay using (A) protein titrations, and (B) pull-down assays.
  • Figure 4 shows examples of validation of Mtb SOMAmers with fractions from a human macrophage infection model (quadruplicate samples).
  • Figure 5 shows a volcano plot of TB markers in urine, from a small pilot study.
  • Figure 6 shows scatter plots of Mtb SOMAmer measurements in urine of TB versus non-TB patients, in HIV-negative and HIV-positive populations. Samples with elevated Mtb SOMAmer measurements are highlighted.
  • Figure 7 shows detection of Mtb proteins in urine, shown as the number of proteins with elevated cut-off 2.5-5 standard deviations above the non-TB medians.
  • Figure 8 shows correlation of protein size and detection in urine.
  • Figure 9 shows spike and recovery of EsxB in urine, serum, and plasma.
  • Figure 10 shows a volcano plot of TB markers in plasma, from a small pilot study.
  • Figure 11 shows a volcano plot of TB markers in urine, from a small pilot study.
  • Figure 12 shows background signals of Mtb SOMAmers compared to spuriomers observed in 40% serum (Non-TB serum from FIND and QC serum).
  • Figure 13 shows a schematic of post-SELEX modifications to optimize performance of lead candidate reagents, via truncations from both ends until activity is lost (red line), identification of nonessential positions (-) to be replaced by linker (HEG, hexaethylene glycol) and positions that can accommodate 2'0-methyl substitutions (m), and introduction of 5-position variants (iB, isobutyl-dU, Th, thiophene-dU).
  • linker HOG, hexaethylene glycol
  • m 2-methyl substitutions
  • iB isobutyl-dU, Th, thiophene-dU
  • Figure 14 shows an example of SOMAscan accuracy determined via spike and recovery assays and dilution linearity assessment.
  • the present application relates generally to biomarkers for tuberculosis infection and methods of detection thereof.
  • the invention relates to one or more biomarkers, biomarker panels, methods, devices, reagents, systems, and kits for detecting and/or characterizing tuberculosis infection in an individual from a urine sample.
  • 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 may include other elements not expressly listed.
  • a “capture agent' or “capture reagent” refers to a molecule that is capable of binding specifically to a biomarker.
  • a “target protein capture reagent” refers to a molecule that is capable of binding specifically to a target protein.
  • Nonlimiting exemplary capture reagents include aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, and modifications and fragments of any of the aforementioned capture reagents.
  • a capture reagent is selected from an aptamer and an antibody.
  • antibody refers to full-length antibodies of any species and fragments and derivatives of such antibodies, including Fab fragments, F(ab') 2 fragments, single chain antibodies, Fv fragments, and single chain Fv fragments.
  • antibody also refers to synthetically-derived antibodies, such as phage display-derived antibodies and fragments, affybodies, nanobodies, etc.
  • marker and “biomarker” 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 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. In some embodiments, a biomarker is a target protein.
  • biomarker level and “level” 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.
  • level depends on the specific design and components of the particular analytical method employed to detect the biomarker.
  • a “control level” of a target molecule refers to the level of the target molecule in the same sample type from an individual that does not have the disease or condition, or from an individual that is not suspected or at risk of having the disease or condition, or from an individual that has a non-progressive form of the disease or condition.
  • a “control level” of a target molecule need not be determined each time the present methods are carried out, and may be a previously determined level that is used as a reference or threshold to determine whether the level in a particular sample is higher or lower than a normal level.
  • a control level in a method described herein is the level that has been observed in one or more subjects without TB infection or active TB.
  • a control level in a method described herein is the average or mean level, optionally plus or minus a statistical variation, that has been observed in a plurality of subjects without TB infection or active TB.
  • individual and “subject” are used interchangeably to refer 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 (such as TB) 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.
  • 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 response after the administration of a treatment or therapy to the individual.
  • detecting or “determining” with respect to a biomarker level includes the use of both the instrument used to observe and record a signal corresponding to a biomarker level and the material/s required to generate that signal.
  • the level is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared
  • 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
  • polytetrafluoroethylene butyl rubber, styrenebutadiene rubber, natural rubber, polyethylene, polypropylene, (poly)tetrafluoroethylene, (poly)vinylidenefluoride,
  • Suitable solid support particles that can be used include, e.g., encoded particles, such as Luminex ® -type encoded particles, magnetic particles, and glass particles.
  • host biomarkers are biological molecules (e.g., proteins) that are endogenous to an individual, the expression or level of which is altered (e.g., increased or decreased) upon infection by a pathogenic agent (e.g., Mycobacterium tuberculosis).
  • a pathogenic agent e.g., Mycobacterium tuberculosis
  • Detection and/or quantification of host biomarkers allows for diagnosis of pathogen infection.
  • pathogen biomarkers are molecules (e.g., proteins) that are not endogenous to an individual, but produced by a pathogen (e.g., Mycobacterium tuberculosis) that has infected the individual. Detection and/or quantification of pathogen biomarkers (e.g., Mtb biomarkers) allows for diagnosis of pathogen infection.
  • pathogen biomarkers e.g., Mtb biomarkers
  • the present application includes biomarkers, methods, devices, reagents, systems, and kits for detecting, identifying, characterizing, and/or diagnosing infection of a subject (e.g., human subject) with Mycobacterium tuberculosis ⁇ Mtb) infection (e.g., TB infection) or tuberculosis (TB).
  • a subject e.g., human subject
  • Mycobacterium tuberculosis ⁇ Mtb infection e.g., TB infection
  • TB tuberculosis
  • one or more biomarkers are provided for use either alone or in various combinations to identify TB infection.
  • exemplary embodiments include the TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • biomarkers are useful alone or in combination for detecting TB infection, methods are also described herein for grouping the one or more of the biomarkers with additional biomarkers not described herein. In some embodiments, panels of at least two, at least three, at least four, at least five, or at least 6 biomarkers described herein are provided.
  • methods comprises contacting a sample (e.g., urine, concentrated urine, filtered urine, diluted urine, etc.) or a portion of the sample from a subject with at least one capture reagent, wherein each capture reagent specifically binds a biomarker whose presence or levels are being detected.
  • the method comprises contacting the sample, or proteins from the sample, with at least one aptamer, wherein each aptamer specifically binds a biomarker whose levels are being detected.
  • methods are provided for determining whether a subject is infected with Mycobacterium tuberculosis (TB infection) and/or is suffering from Tuberculosis (TB). Methods are also provided for assessing the effectiveness of TB treatment.
  • biomarkers are indicative of co-infection with TB and human immunodeficiency virus (HIV).
  • biomarkers are indicative of infection with TB but not HIV.
  • methods comprise detecting the presence of one or more biomarkers (e.g., Mtb biomarkers).
  • methods comprise measuring the level or concentrations of one or more biomarkers by any number of analytical methods, including any of the analytical methods described herein.
  • biomarkers are, for example, present at different levels in TB-positive and TB-negative subjects (e.g., present in TB + subjects and absent in TB " subjects).
  • detection of the differential levels of a biomarker in an individual can be used, for example, to permit the determination of whether the individual has TB infection, active TB, etc. ).
  • detection of the presence of a biomarker in an individual can be used, for example, to permit the determination that the individual has TB infection and/or active TB, etc.
  • any of the biomarkers described herein may be used to monitor TB infection in an individual over time, and to permit the determination of treatment is effective.
  • biomarker levels and/or presence e.g., one or more of the TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C) as a stand-alone diagnostic test
  • biomarker levels are tested in conjunction with other markers or assays indicative of TB (e.g., skin test, sputum culture, blood test, tissue culture, body fluid culture, chest x-ray, etc.).
  • biomarkers can also be evaluated in conjunction with other types of data, particularly data that indicates an individual's risk for TB (e.g., lifestyle, location, age, 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.
  • biomarker presence and/or level is detected using a capture reagent.
  • 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 aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, F(ab') 2 fragments, single chain antibody fragments, Fv fragments, single chain Fv fragments, nucleic acids, lectins, ligand-binding receptors, affybodies, nanobodies, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, and synthetic receptors, and modifications and fragments of these.
  • a biomarker level is detected using a biomarker/capture reagent complex.
  • the biomarker presence and/or level 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 presence and/or level is detected directly from the biomarker in a biological sample (e.g., urine).
  • a biological sample e.g., urine
  • 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 analyze one or more of multiple biomarkers to be detected in a biological sample.
  • a fluorescent tag is used to label a component of the biomarker/capture reagent complex to enable the detection of the biomarker level.
  • 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 level.
  • 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 Bio luminescence & 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 level.
  • Suitable chemiluminescent materials include any of oxalyl chloride, Rodamin 6G,
  • Ru(bipy) 3 2+ 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
  • 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.
  • 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 presence and/or levels for the biomarkers described herein can be detected using any analytical methods including, singleplex aptamer assays, multiplexed aptamer assays, singleplex or multiplexed immunoassays, mRNA expression profiling, miRNA expression profiling, mass spectrometric analysis,
  • 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".
  • the aptamers bind to their respective target molecules present in the sample and thereby enable a determination of a biomarker level corresponding to a biomarker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SELEX can also be used to identify aptamers that have desirable off-rate
  • an aptamer comprises at least one nucleotide with a modification, such as a base modification.
  • an aptamer comprises at least one nucleotide with a hydrophobic modification, such as a hydrophobic base modification, allowing for hydrophobic contacts with a target protein. Such hydrophobic contacts, in some
  • an aptamer contribute to greater affinity and/or slower off-rate binding by the aptamer.
  • an aptamer comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with hydrophobic modifications, where each hydrophobic modification may be the same or different from the others.
  • an aptamer comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 hydrophobic modifications.
  • a slow off-rate aptamer (including an aptamers comprising at least one nucleotide with a hydrophobic modification) has an off-rate (t1 ⁇ 2) of > 30 minutes, > 60 minutes, > 90 minutes, > 120 minutes, > 150 minutes, > 180 minutes, > 210 minutes, or > 240 minutes.
  • an 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.
  • 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.
  • the assay enables the detection of a biomarker level corresponding to a biomarker in the test sample.
  • the aptamers are immobilized on the solid support prior to being contacted with the sample.
  • immobilization of the aptamers prior to contact with the sample may not provide an optimal assay.
  • pre-immobilization of the aptamers may result in inefficient mixing of the aptamers 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 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 and their target molecules.
  • immobilization of the aptamers on the solid support generally involves an aptamer- preparation step (i.e., the immobilization) prior to exposure of the aptamers to the sample, and this preparation step may affect the activity or functionality of the aptamers.
  • the described aptamer 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., an aptamer).
  • a nucleic acid i.e., an aptamer
  • the described methods create a nucleic acid surrogate (i.e, the aptamer) 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.
  • Aptamers can be constructed to facilitate the separation of the assay components from an aptamer biomarker complex (or photoaptamer biomarker covalent complex) and permit isolation of the aptamer for detection and/or quantification.
  • these constructs can include a cleavable or releasable element within the aptamer sequence.
  • additional functionality can be introduced into the aptamer, for example, a labeled or detectable component, a spacer component, or a specific binding tag or immobilization element.
  • the aptamer can include a tag connected to the aptamer 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 an aptamer, thereby allowing for the release of the aptamer later in an assay method.
  • 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.
  • the molecular capture reagents comprise an aptamer or an antibody or the like and the specific target may be a TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • a TB biomarkers identified in experiments conducted during development of embodiments of the present invention e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • a method for signal generation takes advantage of anisotropy signal change due to the interaction of a fiuorophore-labeled capture reagent with its specific biomarker target.
  • the labeled capture reacts with its target, the increased molecular weight causes the rotational motion of the fiuorophore 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 aptamer assay that can be used to detect a biomarker level in a biological sample includes the following: (a) preparing a mixture by contacting the biological sample with an aptamer that includes a first tag and has a specific affinity for the biomarker, wherein an aptamer 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 aptamer affinity complex; (e) releasing the aptamer affinity complex from the first solid support; (f) exposing the released aptamer 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 aptamer from the mixture by partitioning the non-complex
  • a nonlimiting exemplary method of detecting biomarkers in a biological sample using aptamers is described, for example, in Kraemer et al, PLoS One 6(10): e26332 (2011); herein incorporated by reference in its entirety.
  • 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 and fragments thereof 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 level 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 (I 125 ) or fluorescence. Additional techniques include, for example,
  • 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.
  • ELISA enzyme-linked immunosorbent assay
  • FRET fluorescence resonance energy transfer
  • TR-FRET time resolved-FRET
  • 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.
  • detectable label 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 386 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, in some embodiments, be used as a surrogate for detection of the level of the corresponding protein in the biological sample.
  • a biomarker or biomarker panel described herein can 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.
  • 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.
  • a biomarker described herein may be used in molecular imaging tests.
  • an imaging agent can be coupled to a capture reagent, which can be used to detect the biomarker in vivo.
  • In vivo imaging technologies provide non-invasive methods for determining the state of a particular disease 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 biomarker in vivo.
  • 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 an aptamer 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 an aptamer 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.
  • Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as, for example, iodine-123, iodine-131, indium-111, fiuorine-19, carbon-13, nitrogen-15, oxygen- 17, gadolinium, manganese or iron.
  • MRI magnetic resonance imaging
  • Standard imaging techniques include but are not limited to magnetic resonance imaging, computed tomography scanning, positron emission tomography (PET), single photon emission computed tomography (SPECT), and the like.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • 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.
  • 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
  • the two-dimensional distribution of radioactivity may be inferred from outside of the body.
  • 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
  • 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.
  • aptamers may be used for such in vivo imaging diagnostic methods.
  • an aptamer that was used to identify a particular biomarker described herein may be appropriately labeled and injected into an individual to detect the biomarker in vivo.
  • the label used will be selected in accordance with the imaging modality to be used, as previously described.
  • Aptamer-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.
  • the biomarkers described herein may be detected in a variety of tissue samples using histological or cytological methods.
  • endo- and trans- bronchial biopsies, fine needle aspirates, cutting needles, and core biopsies can be used for histology.
  • Bronchial washing and brushing, pleural aspiration, and sputum, can be used for cyotology.
  • Any of the biomarkers identified herein can be used to stain a specimen as an indication of disease.
  • one or more capture reagent/s specific to the corresponding biomarker/s are used in a cytological evaluation of a sample and may include one or more of the following: collecting a cell sample, fixing the cell sample, dehydrating, clearing, immobilizing the cell sample on a microscope slide, permeabilizing the cell sample, treating for analyte retrieval, staining, destaining, washing, blocking, and reacting with one or more capture reagent/s in a buffered solution.
  • the cell sample is produced from a cell block.
  • one or more capture reagent/s specific to the corresponding biomarkers are used in a histological evaluation of a tissue sample and may include one or more of the following: collecting a tissue specimen, fixing the tissue sample, dehydrating, clearing, immobilizing the tissue sample on a microscope slide, permeabilizing the tissue sample, treating for analyte retrieval, staining, destaining, washing, blocking, rehydrating, and reacting with capture reagent/s in a buffered solution.
  • fixing and dehydrating are replaced with freezing.
  • the one or more aptamer/s specific to the corresponding biomarker/s are reacted with the histological or cytological sample and can serve as the nucleic acid target in a nucleic acid amplification method.
  • Suitable nucleic acid amplification methods include, for example, PCR, q-beta replicase, rolling circle amplification, strand displacement, helicase dependent amplification, loop mediated isothermal amplification, ligase chain reaction, and restriction and circularization aided rolling circle amplification.
  • the one or more capture reagent/s specific to the corresponding biomarkers for use in the histological or cytological evaluation are mixed in a buffered solution that can include any of the following: blocking materials, competitors, detergents, stabilizers, carrier nucleic acid, polyanionic materials, etc.
  • a “cytology protocol” generally includes sample collection, sample fixation, sample immobilization, and staining.
  • Cell preparation can include several processing steps after sample collection, including the use of one or more aptamers for the staining of the prepared cells.
  • mass spectrometers can be used to detect biomarker levels.
  • 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.
  • 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 levels 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
  • Labeling methods include but are not limited to isobaric tag for relative and absolute quantitation
  • Capture reagents used to selectively enrich samples for candidate biomarker proteins prior to mass spectroscopic analysis include but are not limited to aptamers, 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.
  • the foregoing assays enable the detection of biomarker levels that are useful in the methods described herein, where the methods comprise detecting, in a biological sample from an individual, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine biomarkers selected from the TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C).
  • biomarkers selected from the TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CHIO, RL7, TPX, CF30, KAD, MPT51, Esx
  • a biomarker "signature" for a given diagnostic test contains a set of markers, each marker having characteristic levels in the populations of interest.
  • Characteristic levels may refer to the mean or average of the biomarker levels for the individuals in a particular group.
  • a diagnostic method described herein can be used to assign an unknown sample from an individual into one of two groups, either TB positive or TB negative.
  • 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 levels.
  • classification methods are performed using supervised learning techniques in which 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.
  • diagnostic classifiers include decision trees; bagging + boosting + 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.
  • Pattern Classification R.O. Duda, et al, editors, John Wiley & Sons, 2nd edition, 2001
  • 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 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 or being free from the disease.
  • 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. Training a naive Bayesian classifier is an example of such a supervised learning technique (see, e.g., Pattern Classification, R.O. Duda, et al, editors, John Wiley & Sons, 2nd edition, 2001; see also, The Elements of Statistical Learning - Data Mining, Inference, and Prediction, T.
  • 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 way, 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.
  • An illustrative example of the development of a diagnostic test using a set of biomarkers includes the application of a naive Bayes classifier, a simple probabilistic classifier based on Bayes theorem with strict independent treatment of the biomarkers.
  • Each biomarker is described by a class-dependent probability density function (pdf) for the measured RFU values or log RFU (relative fluorescence units) values in each class.
  • PDF probability density function
  • the joint pdfs for the set of markers in one class is assumed to be the product of the individual class- dependent pdfs for each biomarker.
  • Training a naive Bayes classifier in this context amounts to assigning parameters ("parameterization") to characterize the class dependent pdfs. Any underlying model for the class-dependent pdfs may be used, but the model should generally conform to the data observed in the training set.
  • the performance of the naive Bayes classifier is dependent upon the number and quality of the biomarkers used to construct and train the classifier.
  • a single biomarker will perform in accordance with its KS-distance (Kolmogorov-Smirnov).
  • the addition of subsequent markers with good KS distances (>0.3, for example) will, in general, improve the classification performance if the subsequently added markers are independent of the first marker.
  • KS-distance Kolmogorov-Smirnov
  • KS distances >0.3, for example
  • many high scoring classifiers can be generated with a variation of a greedy algorithm. (A greedy algorithm is any algorithm that follows the problem solving metaheuristic of making the locally optimal choice at each stage with the hope of finding the global optimum.)
  • ROC receiver operating characteristic
  • TPR true positive rate
  • FPR false positive rate
  • the area under the ROC curve (AUC) is commonly used as a summary measure of diagnostic accuracy. It can take values from 0.0 to 1.0.
  • the AUC has an important statistical property: the AUC of a classifier is equivalent to the probability that the classifier will rank a randomly chosen positive instance higher than a randomly chosen negative instance (Fawcett T, 2006. An introduction to ROC analysis. Pattern Recognition Letters .27: 861-874). This is equivalent to the Wilcoxon test of ranks (Hanley, J.A., McNeil, B.J., 1982. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 143, 29- 36.).
  • Exemplary embodiments use any number of the TB biomarkers identified in experiments conducted during development of embodiments of the present invention (e.g., MP64, ACR, CH602, PstSl, DnaK, MASZ, CH10, RL7, TPX, CF30, KAD, MPT51, EsxB, EsxA, A85A, A85B, and A95C) in various combinations to produce diagnostic tests for identifying individuals with TB.
  • the markers can be combined in many ways to produce classifiers. For example, certain combinations of biomarkers may produce tests that are more sensitive (or more specific) than other combinations.
  • a biological sample is run in one or more assays to produce the relevant quantitative biomarker levels used for classification.
  • the measured biomarker levels are used as input for the classification method that outputs a classification and an optional score for the sample that reflects the confidence of the class assignment.
  • a biological sample is optionally diluted and run in a multiplexed aptamer assay, and data is assessed as follows.
  • the data from the assay are optionally normalized and calibrated, and the resulting biomarker levels are used as input to a Bayes classification scheme.
  • the log-likelihood ratio is computed for each measured biomarker individually and then summed to produce a final classification score, which is also referred to as a diagnostic score. The resulting assignment as well as the overall
  • classification score can be reported.
  • the individual log-likelihood risk factors computed for each biomarker level can be reported as well.
  • kits 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 aptamer or antibody) for detecting one or more biomarkers in a biological sample, and optionally (b) one or more software or computer program products for predicting whether the individual from whom the biological sample was obtained suffers from or is infected with TB.
  • capture reagents such as, for example, at least one aptamer or antibody
  • software or computer program products for predicting whether the individual from whom the biological sample was obtained suffers from or is infected with TB.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • kits comprises a solid support, a capture reagent, and a signal generating material.
  • 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.
  • 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.
  • kits are provided for the analysis of TB, wherein the kits comprise PCR primers for one or more biomarkers described herein.
  • a kit may further include instructions for use and correlation of the biomarkers with TB diagnosis.
  • a kit may include a DNA array containing the complement of one or more of the biomarkers described herein, 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 one capture reagent for determining the level of one or more biomarkers in a test sample, 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.
  • an algorithm or computer program assigns a score for each biomarker quantified based on said comparison and, in some embodiments, combines the assigned scores for each biomarker quantified to obtain a total score.
  • an algorithm or computer program compares the total score with a predetermined score, and uses the comparison to determine whether a subject is infected with TB.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • a method for assessing TB in an individual may 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; and 3) report the results of the biomarker levels.
  • the results of the biomarker levels are reported qualitatively rather than quantitatively, such as, for example, a proposed diagnosis (e.g., "TB infection", "X% risk of TB infection,” etc.) or simply a positive TB / negative TB result.
  • a method for assessing TB in an individual may 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; 4) calculate each biomarker level; and 5) report the results of the biomarker levels.
  • the biomarker levels are combined in some way and a single value for the combined biomarker levels is reported.
  • the reported value may be a single number determined from the sum of all the marker calculations that is compared to a pre - set threshold value that is an indication of the presence or absence of disease.
  • the diagnostic 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.
  • the subject is treated for TB infection.
  • medications used to treat latent TB infection include: isoniazid (INH), rifampin (RIF), and rifapentine (RPT).
  • TB disease is treated by taking several drugs for 6 to 9 months.
  • drugs currently approved by the U.S. Food and Drug Administration (FDA) for treating TB include: isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA).
  • Regimens for treating TB disease have an initial phase of 2 months, followed by a choice of several options for the continuation phase of either 4 or 7 months (total of 6 to 9 months for treatment).
  • M. tuberculosis (Mtb) proteins have been over-expressed in E. coli and purified in recombinant, tagged form (Table 1, Figure 1). These proteins include
  • Mtb genes encoding antigen-85 (FpvA, FpvB, FpvC), ESAT-6, CFP10, and PstSl were cloned into the pET-51b vector that features an amino-terminal Strep-tag and a carboxy-terminal His-tag. Additional plasmids for over- expression of His-tagged MPT64, Acr, GroEL2, OnaK, GlcB, GroES, RpIL, Tpx, Cfp30, Adk, and MPT51 were from BEI Resources.
  • SELEX was performed on the Mtb proteins, using multiple ssDNA libraries containing different modified nucleotides (Figure 2).
  • the starting library contained of 1 nmol (10 14 -10 15 molecules) of sequences with a 40mer random region flanked by 20mer fixed regions for PCR amplification between the rounds of SELEX (Gold, 2010; herein incorporated by reference in its entirety).
  • 48 individual clones from each active pool were sequenced and tested in 32 P-radiolabel affinity binding assays. The best candidates were produced synthetically by solid support phosphoramidite chemistry as 50mer truncated versions with a 5'PBOC (photocleavable biotin D-spacer-cy3).
  • the esxB gene is located in the RD1 region of the chromosome that is deleted in the BCG vaccine strain (Harboe, 1996; Teutschbein, 2007; herein incorporated by reference in their entireties). EsxB levels increased over time in lysates and media supernatants from Mtb H37Rv-infected macrophages, but EsxB was not detected in exosome preparations.
  • RL7 ribosomal protein RpIL was detected in exosomes, lysates and supernatants from t3 ⁇ 4-infected macrophages, and the RL7 levels increased over time.
  • the antigen-85 proteins also known as fibronectin-binding proteins FnbA, FnbB, and FnbC, were not detected by the corresponding SOMAmers.
  • fibronectin which was present at lower levels in exosomes and in supernatant from t3 ⁇ 4-infected macrophages compared to uninfected macrophages. The data are consistent with sequestration of antigen- 85 and fibronectin upon complex formation between these bacterial and host proteins
  • the Mtb SOMAmers proved useful for the detection of Mtb proteins in various sample matrices, including plasma and urine from some TB patients, but showed high background in serum when added to the SOMAmer mix used for detection of low-abundance proteins in 40% serum.
  • Normalization and calibration of the SOMAscan urine measurements was done using the standard hybridization and median normalization procedures, with measurement of creatinine levels and total protein in urine as basis for alternate means of normalization.
  • concentrating urine will, in some embodiments, increase assay sensitivity. Such a step will not only concentrate the proteins in urine (e.g., at least 10-fold), but will also eliminate small molecules such as urea that could have
  • Protocols have been established to generate SOMAmer pairs for sandwich-type assays and have isolated such reagent pairs for a panel of cardiovascular risk biomarkers, and the utility of filter-, plate-, or bead-based sandwich assays using combinations of antibodies and SOMAmers or SOMAmers alone has been demonstrated (Ochsner, 2013; Ochsner, 2014; herein incorporated by reference in their entireties).
  • Such assays not only allow the use of larger sample sizes (1 mL or more), but also the concentration of analytes via capture prior to detection with quantitative colorimetric and fluorescent readouts.
  • SOMAmer pairs for the Mtb proteins can be developed. Reagent pairs can be identified among existing SOMAmers or selected via sandwich SELEX using protein-SOMAmer complexes.
  • SOMAmer pairs would be particularly useful to detect Mtb proteins in urine, where the low-abundant antigens can be concentrated from a large sample volume via a capture SOMAmer, but also as reagents generally applicable to many possible platforms for the development of a TB test.
  • the levels of detection in bead- and plate -based assays may be determined, and algorithms for calling a positive or negative result can be developed. Potential loss of sensitivity in a sandwich assay compared to a hybridization assay will be off-set by a larger sample volume and concentration effect in this assay.
  • Urine was clearly a less complex matrix compared to either serum or plasma. While signals were generally lower, background was extremely low (typical medians ⁇ 50 RFU for irrelevant spuriomers).
  • Mtb SOMAmers exhibited high background due to nonspecific binding to other serum components, which led to poor detection limits under nonoptimized conditions. In contrast, many of the Mtb SOMAmers performed well in a less complex sample matrix such as urine, where low background was observed.
  • the strategy for generation of "low background" Mtb SOMAmers involves passive counter-selection with human serum during the SELEX process to remove sequences that are potential serum protein binders: Selection in round 1 is performed in the presence of 40% serum, subsequent rounds with 8% serum, using serum competitor buffer prepared as a concentrated stock (80% human serum, 2 ⁇ prothrombin, and 2 ⁇ casein). Alternating rounds of SELEX (R5, R7, R9, Rl 1) will not use serum counter-selection to avoid carry-over of sequences that may be bound to certain serum proteins that form complexes with the Mtb protein targets.
  • a total of 82 selections (2 SELEX plates) will be performed. Active pools obtained after 7-11 rounds will be sent for Ion Torrent ePCR and Sequencing (estimated 50 pools). Clone picks (6 per pool, estimated 300 total) will be synthesized as 5' PBDC 50-mers for screening in standard filter binding assays and then moved into the slide screening assay. The best performing SOMAmers (1-3 per target, estimated 40-80 total) that also show low background in the serum titrations as part of the slide screening assays will be synthesized at larger (1 ⁇ ) scale, HPLC-purified, and incorporated into SOMAscan.
  • the Mtb SOMAmer reagents Prior to testing any clinical samples, the Mtb SOMAmer reagents will be validated for binding to native Mtb proteins in SOMAscan using fractions from a human macrophage infection model, specifically, using lysates, supernatants, and exosomes collected at 72 h post-infection from quadruplicate cultures of infected vs. uninfected cells (estimated 24 samples). The Mtb SOMAmers will also be evaluated in competition immunoassays, which will allow the direct comparison with available antibodies.
  • performance of SOMAmers is improved substantially by post- SELEX modifications (Davies, 2012; Gelinas, 2014; herein incorporated by reference in their entireties), which involves both systematic and targeted changes of the original modified 50mer DNA SOMAmer (Figure 13).
  • All SOMAmers are prepared at small scale by high through-put synthesis in house. First, the sequence is truncated from both the 5' and 3' end to a minimal length. Second, a linker scan identifies nonessential positions via replacement of individual nucleotides by a C3 spacer (lacking the sugar ring and the base but maintaining the three carbons between phosphates), and positions are identified that can accommodate 2 ⁇ - methyl substitutions.
  • SOMAmer 5557-2 was validated as a specific reagent to detect CFP10 in fractions from the macrophage infection model, where it distinguished Mtb from M. bovis BCG. Furthermore, this SOMAmer showed low background in SOMAscan of serum and was capable to identify some of the TB patients using urine samples. A sequential series of truncations, linker scan, and substitutions, requiring the synthesis of 100 analogs at 50 nmol scale is contemplated. The best candidate(s) are identified in radiolabel filter binding assays and will be synthesized at larger (1 ⁇ ) scale, HPLC -purified, and incorporated into SOMAscan.
  • Circulating antigens in serum are likely complexed with antibodies which hinder detection. Immune complex dissociation under mild acidic conditions prior to antigen or antibody detection has been described for HIV (p24), hepatitis C (antibody), leishmaniasis, dengue (NS-1 antigen).
  • serum is mixed with one volume of 1.5 M glycine pH 2.8 and incubated for 1 h at 37°C to dissociate Ab-Ag immune complexes, followed by neutralization with one volume of 1.5 M Tris-HCI pH 9.7.
  • this treatment increased the rate of NS-1 antigen detection in serum from infected patients from 18% to 78% (Koraka, 2003; herein incorporated by reference in its entirety).
  • the assay refinement strategy will focus on the optimization of serum dilutions to establish performance criteria for the Mtb SOMAmers.
  • Mtb SOMAmers suffered from high background signals when used in the SOMAmer mix for 40% serum to detect low-abundant proteins.
  • Matrix titrations will be performed to define the optimal serum concentrations for each analyte. Since Mtb proteins are not present in normal serum, spike and recovery and dilution linearity assessments will be used to characterize the performance of SOMAscan with respect to accuracy, signal-to-noise ratio and lower/upper limits of detection for Mtb- specific SOMAmers, as shown for an example ( Figure 14).
  • the dilution of serum from 40% to 1% can increase the signal-to-noise ratios, which ultimately defines the overall assay performance.
  • optimized serum dilutions can lead to improved detection of Mtb proteins and accuracy of the proteomic measurements.
  • four of them were measured in 1% serum and five of them in 0.005% (so these are medium to high abundance proteins) based on previous optimization of the conditions for each individual SOMAmer.
  • the top TB biomarkers identified as the classifier (host and microbial) are assembled and measured in a TB panel, where measurements can be reported in concentration space and can be optimized for performance and validated to the level acceptable for clinical research applications. Panels can also be moved to alternate read-out formats for better turn-around times and reduced cost.
  • SOMApanel streamlined multiplexed SOMAmer- based assay
  • the bio- analytical work plan for panel assay development is based on guidance for the validation of immunoassays for protein biomarkers to support pre-clinical and clinical studies (Valentin, 2011; herein incorporated by reference in its entirety).
  • a SOMAmer-based multiplex panel assay for the quantitative determination of the Mtb proteins, for the 9 host response (HR9) markers, and for other markers identified will be developed. The performance will be verified in the standard slide hybridization assay, except that only panel SOMAmers will be present during the incubation and that only probes for the panel SOMAmers will be required on the slides.
  • Panel assay performance is assessed on a suspension microarray (Luminex) platform using a different bead type (color) for each SOMAmer-specific anti-sense probe.
  • Hybridization to the bead-immobilized probes and fluorescent measurements using the Luminex platform will replace the backend part of the SOMAscan assay (hybridization to a slide array and slide reading) and will reduce cost several-fold.
  • Minimum required dilution and linear dilution range in matrix for each marker in the panel will be determined by using protein calibrator standard curves run in parallel in the same experiments.
  • Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages.
  • Gold L Ayers 0, Bertino J. Bock C, Bock A, Brody EN, et al. Aptamer-based multiplexed proteomic technology for biomarker discovery.

Abstract

La présente invention porte d'une manière générale sur des biomarqueurs d'une infection tuberculeuse et sur des procédés de détection correspondants. Dans divers modes de réalisation, l'invention concerne un ou plusieurs biomarqueurs, des panels de biomarqueurs, des méthodes, des dispositifs, des réactifs, des systèmes et des kits de détection et/ou de caractérisation d'une infection par la tuberculose chez un individu à partir d'un échantillon d'urine.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105572352A (zh) * 2016-02-17 2016-05-11 遵义医学院附属医院 一组结核潜伏感染诊断标志物及其用途
CN106047882A (zh) * 2016-06-01 2016-10-26 湖南大学 一组特异性结合结核分枝杆菌的核酸适配体及其应用
RU2636490C1 (ru) * 2017-01-24 2017-11-23 Общество с ограниченной ответственностью "Передовые диагностические платформы" Аллерген, содержащий комбинацию последовательностей белков, кодируемых генами EsxA, EsxB, EspJ, EspK M.tuberculosis complex, и способ его применения для диагностики инфекции M.tuberculosis complex
WO2019244163A1 (fr) * 2018-06-22 2019-12-26 Translational Health Science And Technology Institute Aptamère contre m.tb mpt51 et utilisations associées
US10526665B2 (en) 2016-03-04 2020-01-07 University Of Notre Dame Du Lac Exosomal biomarkers diagnostic of tuberculosis

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US5580737A (en) 1990-06-11 1996-12-03 Nexstar Pharmaceuticals, Inc. High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
US5660985A (en) 1990-06-11 1997-08-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
US5705337A (en) 1990-06-11 1998-01-06 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chemi-SELEX
US5763177A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US6001577A (en) 1998-06-08 1999-12-14 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US6242246B1 (en) 1997-12-15 2001-06-05 Somalogic, Inc. Nucleic acid ligand diagnostic Biochip
US6458539B1 (en) 1993-09-17 2002-10-01 Somalogic, Inc. Photoselection of nucleic acid ligands
WO2007005627A2 (fr) * 2005-07-01 2007-01-11 Forsyth Dental Infirmary For Children Essais de detection d'antigenes de la tuberculose et vaccins
US20090004667A1 (en) 2007-01-16 2009-01-01 Somalogic, Inc. Method for generating aptamers with improved off-rates
US20090042206A1 (en) 2007-01-16 2009-02-12 Somalogic, Inc. Multiplexed Analyses of Test Samples
US20090098549A1 (en) 2007-07-17 2009-04-16 Somalogic, Inc. Selex and photoselex
US20120077695A1 (en) 2010-09-27 2012-03-29 Somalogic, Inc. Mesothelioma Biomarkers and Uses Thereof
US20120101002A1 (en) 2008-09-09 2012-04-26 Somalogic, Inc. Lung Cancer Biomarkers and Uses Thereof
WO2013151122A1 (fr) * 2012-04-05 2013-10-10 株式会社ビーエル Procédé et kit pour la détection immunologique de complexe mycobacterium tuberculosis
WO2014059336A1 (fr) * 2012-10-12 2014-04-17 University Of Notre Dame Du Lac Exosomes et biomarqueurs diagnostiques

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5580737A (en) 1990-06-11 1996-12-03 Nexstar Pharmaceuticals, Inc. High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
US5660985A (en) 1990-06-11 1997-08-26 Nexstar Pharmaceuticals, Inc. High affinity nucleic acid ligands containing modified nucleotides
US5705337A (en) 1990-06-11 1998-01-06 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: chemi-SELEX
US5763177A (en) 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
US6291184B1 (en) 1990-06-11 2001-09-18 Somalogic, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
US6458539B1 (en) 1993-09-17 2002-10-01 Somalogic, Inc. Photoselection of nucleic acid ligands
US6544776B1 (en) 1997-12-15 2003-04-08 Somalogic, Inc. Nucleic acid ligand diagnostic biochip
US6242246B1 (en) 1997-12-15 2001-06-05 Somalogic, Inc. Nucleic acid ligand diagnostic Biochip
US6458543B1 (en) 1997-12-15 2002-10-01 Somalogic, Incorporated Nucleic acid ligand diagnostic biochip
US6503715B1 (en) 1997-12-15 2003-01-07 Somalogic, Inc. Nucleic acid ligand diagnostic biochip
US6001577A (en) 1998-06-08 1999-12-14 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
WO2007005627A2 (fr) * 2005-07-01 2007-01-11 Forsyth Dental Infirmary For Children Essais de detection d'antigenes de la tuberculose et vaccins
US20090004667A1 (en) 2007-01-16 2009-01-01 Somalogic, Inc. Method for generating aptamers with improved off-rates
US20090042206A1 (en) 2007-01-16 2009-02-12 Somalogic, Inc. Multiplexed Analyses of Test Samples
US20090098549A1 (en) 2007-07-17 2009-04-16 Somalogic, Inc. Selex and photoselex
US20120101002A1 (en) 2008-09-09 2012-04-26 Somalogic, Inc. Lung Cancer Biomarkers and Uses Thereof
US20120077695A1 (en) 2010-09-27 2012-03-29 Somalogic, Inc. Mesothelioma Biomarkers and Uses Thereof
WO2013151122A1 (fr) * 2012-04-05 2013-10-10 株式会社ビーエル Procédé et kit pour la détection immunologique de complexe mycobacterium tuberculosis
WO2014059336A1 (fr) * 2012-10-12 2014-04-17 University Of Notre Dame Du Lac Exosomes et biomarqueurs diagnostiques

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
"Bioluminescence & Chemiluminescence: Progress & Current Applications", January 2002, WORLD SCIENTIFIC PUBLISHING COMPANY
"Gene Expression Profiling: Methods and Protocols", 2004, HUMANA PRESS
"ImmunoAssay: A Practical Guide", 2005, TAYLOR & FRANCIS, LTD.
"Pattern Classification", 2001, JOHN WILEY & SONS
"The Elements of Statistical Learning - Data Mining, Inference, and Prediction", 2009, SPRINGER SCIENCE+BUSINESS MEDIA, LLC
BEKMURZAYEVA A; SYPABEKOVA M; KANAYEVA D: "Tuberculosis diagnosis using immunodominant, secreted antigens of Mycobacterium tuberculosis", TUBERCULOSIS (EDINB, vol. 93, no. 4, 2013, pages 381 - 8
BILL & MELINDA GATES FOUNDATION PROPOSAL NARRATIVE SUPPLEMENTAL AMENDMENT 1114/13, 2013, pages 14 - 19
BOUMAZOS S; GRINFELD J; ALEXANDER KM; MURCHISON JT; WALLACE WA; MCFARLANE P ET AL.: "Association of FcgammaRlla R131H polymorphism with idiopathic pulmonary fibrosis severity and progression", BMC PULM MED, vol. 10, 2010, pages 51
BURLINGAME ET AL., ANAL. CHEM., vol. 70, 1998, pages 647 R - 716R
CHANGTAI ZHU ET AL: "Evaluation of the clinical value of ELISA based on MPT64 antibody aptamer for serological diagnosis of pulmonary tuberculosis", BMC INFECTIOUS DISEASES, BIOMED CENTRAL, LONDON, GB, vol. 12, no. 1, 20 April 2012 (2012-04-20), pages 96, XP021132604, ISSN: 1471-2334, DOI: 10.1186/1471-2334-12-96 *
CHOU SH; LO EH; NING M: "Plasma-type gelsolin in subarachnoid hemorrhage: novel biomarker today, therapeutic target tomorrow?", CRIT CARE, vol. 18, no. 1, 2014, pages 1 01
D. R. NAPOLITANO ET AL: "Identification of Mycobacterium tuberculosis Ornithine Carboamyltransferase in Urine as a Possible Molecular Marker of Active Pulmonary Tuberculosis", CLINICAL AND VACCINE IMMUNOLOGY, vol. 15, no. 4, 1 April 2008 (2008-04-01), pages 638 - 643, XP055194982, ISSN: 1556-6811, DOI: 10.1128/CVI.00010-08 *
DAVIES DR; GELINAS AD; ZHANG C; ROHLOFF JC; CARTER JD; O'CONNELL 0 ET AL.: "Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets", PROC NAIL ACAD SCI USA, vol. 109, no. 49, 2012, pages 19971 - 6
DEGROOTE MA; NAHID P; JARLSBERG L; JOHNSON JL; WEINER M; MUZANYI G ET AL.: "Elucidating novel serum biomarkers associated with pulmonary tuberculosis treatment", PLOS ONE, vol. 8, no. 4, 2013, pages E61 002
DIEPLINGER, B. ET AL.: "Analytical characterization and clinical evaluation of an enzyme-linked immunosorbent assay for measurement of afamin in human plasma", CLIN CHIM ACTA, vol. 425, 2013, pages 236 - 41
FAWCETT T: "An introduction to ROC analysis", PATTERN RECOGNITION LETTERS, vol. 27, 2006, pages 861 - 874
FLORES LL; STEINGART KR; DENDUKURI N; SCHILLER I; MINION J; PAI M ET AL.: "Systematic review and meta-analysis of antigen detection tests for the diagnosis of tuberculosis", CLIN VACCINE IMMUNOL, vol. 18, no. 10, 2011, pages 1616 - 27
GELINAS AD; DAVIES DR; EDWARDS TE; ROHLOFF JC; CARTER JD; ZHANG C; GUPTA S; ISHIKAWA Y; HIROTA M; NAKAISHI Y: "Crystal structure ofinterleuki n-6 in complex with modified nucleic acid ligand", J. BIOI. CHEM., vol. 289, 2014
GIRI PK; KRUH NA; DOBOS KM; SCHOREY JS: "Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages", PROTEOMICS, vol. 10, no. 17, 2010, pages 3190 - 202
GOLD L; AYERS 0; BERTINO J; BOCK C; BOCK A; BRODY EN ET AL.: "Aptamer-based multiplexed proteomic technology for biomarker discovery", PLOS ONE, vol. 5, no. 12, 2010, pages E15004
GUPTA A; KAUL A; TSOLAKI AG; KISHORE U; BHAKTA S: "Mycobacterium tuberculosis: immune evasion, latency and reactivation", IMMUNOBIOLOGY, vol. 217, no. 3, 2012, pages 36374
HANLEY, J.A.; MCNEIL, B.J.: "The meaning and use of the area under a receiver operating characteristic (ROC) curve", RADIOLOGY, vol. 143, 1982, pages 29 - 36
HARBOE, M. ET AL.: "Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG", INFECT IMMUN, vol. 64, no. 1, 1996, pages 16 - 22
J.R. LAKOWICZ: "Principles of Fluorescence Spectroscopy", 2004, SPRINGER SCIENCE + BUSINESS MEDIA, INC.
KASHINO SS; POLLOCK N; NAPOLITANO DR; RODRIGUES V, JR.; CAMPOS-NETO A.: "Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules", CLIN EXP IMMUNOL, vol. 153, no. 1 1, 2008, pages 56 - 62
KORAKA, P. ET AL.: "Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infection", J CLIN MICROBIOL, vol. 41, no. 9, 2013, pages 4154 - 4159
KRAEMER ET AL., PLOS ONE, vol. 6, no. 10, 2011, pages E26332
KRAEMER S; VAUGHT JD; BOCK C; GOLD L; KATILIUS E; KEENEY TR ET AL.: "From SOMAmer-based biomarker discovery to diagnostic and clinical applications: a SOMAmer-based, streamlined multiplex proteomic assay", PLOS ONE, vol. 6, no. 10, 2011, pages E26332
KUMAR, S.: "Identification of novel adhesins ofM. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis", PLOS ONE, vol. 8, no. 7, 2013, pages E69790
LIN WC; LU SL; LIN CF; CHEN CW; CHAO L; CHAO J: "Plasma kallistatin levels in patients with severe community-acquired pneumonia", CRIT CARE, vol. 17, no. 1, 2013, pages R27
MCNERNEY R; DALEY P: "Towards a point-of-eare test for active tuberculosis: obstacles and opportunities", NAT REV MICROBIOL, vol. 9, no. 3, 2011, pages 204 - 13
N. BLOW, NATURE METHODS, vol. 6, 2009, pages 465 - 469
NAPOLITANO DR; POLLOCK N; KASHINO SS; RODRIGUES V, JR.; CAMPOS-NETO A: "Identification of Mycobacterium tuberculosis ornithine carboamyltransferase in urine as a possible molecular marker of active pulmonary tuberculosis", CLIN VACCINE IMMUNOL, vol. 15, no. 4, 2008, pages 638 - 43
OCHSNER UA; GREEN LS; GOLD L; JANJIC N: "Systematic selection of modified aptamer pairs for diagnostic sandwich assays", BIOTECHNIQUES, vol. 56, no. 3, 2014, pages 125 - 133
OCHSNER UA; KATILIUS E; JANJIC N: "Detection of Clostridium difficile toxins A, B and binary toxin with slow off-rate modified aptamers", DIAGN MICROBIOLLNFECT DIS, vol. 76, no. 3, 2013, pages 278 - 85
PAI M: "Diagnostics for tuberculosis: what test developers want to know", EXPERT REV MOL DIAGN, vol. 13, no. 4, 2013, pages 311 - 4
PAI M; DENKINGER CM; KIK SV; RANGAKA MX; ZWERLING A; OXLADE 0 ET AL.: "Gamma interferon release assays for detection of Mycobacterium tuberculosis", INFECTION. CLIN MICROBIOL REV, vol. 27, no. 1, 2013, pages 3 - 20
PAI M; DOWDY, OW: "Tuberculosis: progress and challenges", THE LANCET RESPIRATORY MEDICINE, vol. 2, no. 1, 2014, pages 25 - 27
PAI NP; PAI M: "Point-of-eare diagnostics for HIV and tuberculosis: landscape, pipeline, and unmet needs", DISCOV MED, vol. 13, no. 68, 2012, pages 35 - 45
PARSONS LM; SOMOSKOVI A; GUTIERREZ C; LEE E; PARAMASIVAN CN; ABIMIKU A ET AL.: "Laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities", CLIN MICROBIOL REV, vol. 24, no. 2, 2011, pages 314 - 50
POLLOCK NR ET AL.: "Validation of Mycobacterium tuberculosis Rv1681 protein as a diagnostic marker of active pulmonary tuberculosis", J CLIN MICROBIOL, vol. 51, no. 5, 2013, pages 1367 - 1373
QIN LIANHUA ET AL: "The selection and application of ssDNA aptamers against MPT64 protein in Mycobacterium tuberculosis", CLINICAL CHEMISTRY AND LABORATORY MEDICINE, DE GRUYTER, DE, vol. 47, no. 4, 1 April 2009 (2009-04-01), pages 405 - 411, XP009184728, ISSN: 1434-6621, DOI: 10.1515/CCLM.2009.097 *
STEINGART KR ET AL.: "Performance of purified antigens for serodiagnosis of pulmonary tuberculosis: a meta-analysis", CLIN VACCINE IMMUNOL., vol. 16, no. 2, 2009, pages 260 - 76
TEUTSCHBEIN J.; SCHUMANN G; MOLLMANN U; GRABLEY S; COLE ST; MUNDER T: "A protein linkage map of the ESAT-6 secretion system 1 (ESX-1) of Mycobacterium tuberculosis", MICROBIOL RES, vol. 164, no. 3, 2007, pages 253 - 259
VALENTIN MA; MA S; ZHAO A; LEGAY F; AVRAMEAS A: "Validation of immunoassay for protein biomarkers: bioanalylical study plan implementation to support preclinical and clinical studies", J PHARM BIOMED ANAL, vol. 55, no. 5, 2011, pages 869 - 77
WALLIS. RS ET AL.: "Biomarkers for tuberculosis disease activity, cure, and relapse", LANCET INFECT DIS, vol. 9, no. 3, 2009, pages 162 - 72
WALZL G; RONACHER K; HANEKOM W; SCRIBA TJ; ZUMLA A: "Immunological biomarkers of tuberculosis", NAT REV IMMUNOL, vol. 11, no. 5, 2011, pages 343 - 54
WHO. GLOBAL TUBERCULOSIS REPORT, 2013
ZWERLING A; DOWDY D: "Economic evaluations of point of care testing strategies for active tuberculosis", EXPERT REV PHARMACOECON OUTCOMES RES, vol. 13, no. 3, 2013, pages 313 - 25

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