Highly sensitive system and methods for analysis of troponin

Abstract

The invention provides methods, compositions, kits, and systems for the sensitive detection of cardiac troponin, Such methods, compositions, kits, and systems are useful in diagnosis, prognosis, and determination of methods of treatment in conditions that involve release of cardiac troponin.

1 . A method for determining the presence or absence of a single molecule of troponin or a fragment or complex thereof in a sample, comprising
 i) labeling said molecule, fragment, or complex, if present, with a label; and   ii) detecting the presence or absence of said label, wherein detection of the presence of said label indicates the presence of said single molecule, fragment, or complex of troponin in said sample.   
     
     
         2 . The method of  claim 1  wherein the troponin is a cardiac isoform of troponin. 
     
     
         3 . The method of  claim 2  wherein the troponin is selected from the group consisting of cardiac troponin I (cTnI) and cardiac troponin C (cTnC). 
     
     
         4 . The method of  claim 3  wherein the troponin is cTnI. 
     
     
         5 . The method of  claim 1 , wherein said detecting is capable of detecting said single molecule of troponin at a limit of detection of less than about 50 pg/ml. 
     
     
         6 . The method of  claim 1  wherein said method is capable of detecting said troponin at a level of detection of less than about 20 pg/ml. 
     
     
         7 . The method of  claim 1 , wherein said label comprises a fluorescent moiety. 
     
     
         8 . The method of  claim 1 , wherein said fluorescent moiety capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. 
     
     
         9 . The method of  claim 1  wherein said fluorescent moiety comprises a molecule that comprises 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 group. 
     
     
         10 . The method of  claim 1  wherein said fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. 
     
     
         11 . The method of  claim 1  wherein said fluorescent moiety comprises AlexaFluor 647. 
     
     
         12 . The method of  claim 1  wherein said label further comprises a binding partner for said troponin molecule, fragment, or complex. 
     
     
         13 . The method of  claim 12  wherein said binding partner comprises an antibody specific to said troponin molecule, fragment, or complex. 
     
     
         14 . The method of  claim 13  wherein said antibody is specific to a specific region of the troponin molecule. 
     
     
         15 . The method of  claim 14  wherein said antibody is specific to a region comprising amino acids 27-41 of cardiac troponin I. 
     
     
         16 . The method of  claim 13  wherein said antibody comprises a polyclonal antibody. 
     
     
         17 . The method of  claim 13  wherein said antibody is a monoclonal antibody. 
     
     
         18 . The method of  claim 1  further comprising capturing said troponin or troponin complex on a solid support. 
     
     
         19 . The method of  claim 18  wherein the solid support is selected from the group consisting of a microtiter plate and paramagnetic beads. 
     
     
         20 . The method of  claim 18  wherein said solid support comprises a capture partner specific for said troponin or troponin complex that is attached to said solid support. 
     
     
         21 . The method of  claim 20  wherein said attachment of said capture partner to said solid support is noncovalent. 
     
     
         22 . The method of  claim 20  wherein said attachment of said capture partner to said solid support is covalent. 
     
     
         23 . The method of  claim 22  wherein said covalent attachment of said capture partner is such that said capture partner is attached to said solid support in a specific orientation. 
     
     
         24 . The method of  claim 23  wherein said specific orientation serves to maximize specific binding of said troponin or troponin complex to said capture partner. 
     
     
         25 . The method of  claim 20  wherein said capture partner comprises an antibody. 
     
     
         26 . The method of  claim 25  wherein said antibody is a monoclonal antibody. 
     
     
         27 . The method of  claim 25  wherein said antibody is specific to amino acids 87-91 of cardiac troponin I. 
     
     
         28 . The method of  claim 25  wherein said antibody is specific to amino acids 41-49 of cardiac troponin I. 
     
     
         29 . The method of  claim 1  wherein said sample is a blood, serum, or plasma sample. 
     
     
         30 . The method of  claim 29  wherein said sample is a serum sample. 
     
     
         31 . The method of  claim 1  wherein said label comprises a fluorescent moiety, and wherein step ii) comprises passing said label through an single molecule detector. 
     
     
         32 . The method of  claim 31  wherein said single molecule detector comprises
 a) an electromagnetic radiation source for stimulating said fluorescent moiety;   b) a capillary flow cell for passing said fluorescent moiety;   c) a source of motive force for moving said fluorescent moiety in said capillary flow cell;   d) an interrogation space defined within said capillary flow cell for receiving electromagnetic radiation emitted from said electromagnetic source;   e) an electromagnetic radiation detector operably connected to said interrogation space for measuring an electromagnetic characteristic of said stimulated fluorescent moiety; and   f) a microscope objective lens situated between said interrogation space and said detector, wherein the lens has a numerical aperture of 0.6 or greater.   
     
     
         33 . A method for determining a diagnosis, prognosis, or method of treatment in an individual comprising
 i) determining a concentration of cardiac troponin in a sample or determining the concentrations of cardiac troponin in a series of samples from said individual, wherein said concentration is determined by a cardiac troponin assay with a limit of detection for said cardiac troponin in said sample of less than about 50 pg/ml; and   ii) determining a diagnosis, prognosis, or method of treatment in said individual, based on said concentration in said sample, or on said concentrations in said series of samples.   
     
     
         34 . The method of  claim 33  wherein step ii) comprises an analysis selected from the group consisting of comparing said concentration or series of concentrations to a normal value for said concentration, comparing said concentration or series of concentrations to a predetermined threshold level, comparing said concentration or series of concentrations to a baseline value, and determining a rate of change of concentration for said series of concentrations. 
     
     
         35 . The method of  claim 34  wherein step ii) comprises comparing said concentration of troponin in said sample with a predetermined threshold concentration, and determining a diagnosis, prognosis, or method of treatment if the sample concentration is greater than the threshold level. 
     
     
         36 . The method of  claim 35  wherein said threshold concentration is determined by determining the 99 th  percentile concentration of troponin in a group of normal individuals, and setting said threshold concentration at said 99 th  percentile concentration. 
     
     
         37 . The method of  claim 33  wherein at least one sample is taken during or after a cardiac stress test. 
     
     
         38 . The method of  claim 33  wherein said cardiac troponin is selected from the group consisting of cardiac troponin I and cardiac troponin T. 
     
     
         39 . The method of  claim 33  wherein the cardiac troponin is cardiac troponin I. 
     
     
         40 . The method of  claim 38  wherein said concentration of cardiac troponin is a concentration of total cardiac troponin. 
     
     
         41 . The method of  claim 40  wherein said concentration of cardiac troponin is a concentration of a cardiac troponin complex, cardiac troponin fragment, phosphorylated cardiac troponin, oxidized cardiac troponin, or a combination thereof. 
     
     
         42 . The method of  claim 41  wherein said concentration of cardiac troponin is compared to total cardiac troponin. 
     
     
         43 . The method of  claim 33  wherein said diagnosis, prognosis, or method of treatment is a diagnosis, prognosis, or method of treatment of myocardial infarct. 
     
     
         44 . The method of  claim 43  wherein said diagnosis, prognosis, or method of treatment comprises risk stratification for level of risk of myocardial infarct. 
     
     
         45 . The method of  claim 43 , wherein said concentration or series of concentrations is determined at or near the time the individual presents to a health professional with one or more symptoms indicative of myocardial ischemia or infarct. 
     
     
         46 . The method of  claim 45  wherein said one or more symptoms is selected from the group consisting of chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath. 
     
     
         47 . The method of  claim 33  wherein said concentration is determined by a method that comprises detecting single molecules of troponin, or complexes or fragments thereof. 
     
     
         48 . The method of  claim 47  comprising labeling said troponin or troponin complex with a label that comprises a fluorescent moiety. 
     
     
         49 . The method of  claim 48  wherein said fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. 
     
     
         50 . The method of  claim 48  wherein said fluorescent moiety comprises a molecule that comprises 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. 
     
     
         51 . The method of  claim 48  wherein said fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. 
     
     
         52 . The method of  claim 51  wherein said fluorescent moiety comprises AlexaFluor 647. 
     
     
         53 . The method of  claim 48  wherein said label further comprises a binding partner for said troponin. 
     
     
         54 . The method of  claim 53  wherein said binding partner comprises an antibody specific to said troponin. 
     
     
         55 . The method of  claim 54  wherein said antibody comprises a polyclonal antibody. 
     
     
         56 . The method of  claim 33  further comprising capturing said troponin or troponin complex on a solid support. 
     
     
         57 . The method of  claim 56  wherein the solid support is selected from the group consisting of a microtiter plate and paramagnetic beads. 
     
     
         58 . The method of  claim 56  wherein said solid support comprises a capture partner specific for said troponin or troponin complex that is attached to said solid support. 
     
     
         59 . The method of  claim 33  wherein step i) further comprises assessing another indicator for said individual, and step ii) comprises determining a diagnosis, prognosis, or method of treatment in said individual, based on said concentration of troponin and said assessment of said other indicator of said non-troponin marker in said sample, or on said concentrations in said series of samples. 
     
     
         60 . The method of  claim 59  wherein said other indicator is a clinical indicator of myocardial ischemia or infarct. 
     
     
         61 . The method of  claim 60  wherein said other indicator is the concentration of one or more non-troponin markers in said sample or said series of samples. 
     
     
         62 . The method of  claim 59 , wherein said one or more markers are markers of cardiac ischemia, or markers of inflammation and of plaque instability. 
     
     
         63 . The method of  claim 62 , wherein said one or more markers of cardiac ischemia are selected from the group consisting of creatine kinase (CK) and its myocardial fraction CK myocardial band (MB), aspartate aminotransferase, lactate dehydrogenase (LDH), α-hydroxybutyrate dehaydrogenase, myoglobin, glutamate oxaloacetate transaminase, glycogen phosphorylase BB, unbound free fatty acids, heart fatty acid binding protein (H-FABP), ischemia-modified albumin, myosin light chain 1, myosin light chain 2. 
     
     
         64 . The method of  claim 63 , wherein said one or more markers comprise one or more specific markers of myocardial injury. 
     
     
         65 . The method of  claim 33  wherein said diagnosis, prognosis, or method of treatment is a diagnosis, prognosis, or method of treatment of a condition that is not myocardial infarct. 
     
     
         66 . The method of  claim 65  wherein said condition is cardiac toxicity. 
     
     
         67 . The method of  claim 66  wherein said cardiac toxicity is associated with the administration of a drug to the individual. 
     
     
         68 . The method of  claim 65  wherein the condition is selected from the group consisting of acute rheumatic fever, amyloidosis, cardiac trauma (including contusion, ablation, pacing, firing, cardioversion, catheterization and cardiac surgery), reperfusion injury, congestive heart failure, end-stage renal failure, glycogen storage disease type II (Pompe's disease), heart transplantation, haeomoglobinopathy with transfusion haemosiderosis, hypertension, including gestational hypertension, hypotension, often with arrhythmias, hypothyroidism, myocarditis, pericarditis, post-operative non-cardiac surgery, pulmonary embolism, and sepsis. 
     
     
         69 . A composition for the detection of a troponin isoform comprising a binding partner to the troponin isoform attached to a fluorescent moiety, wherein said fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. 
     
     
         70 . The composition of  claim 69  wherein said binding partner comprises an antibody to said troponin isoform. 
     
     
         71 . The composition of  claim 70  wherein said antibody is a polyclonal antibody. 
     
     
         72 . The composition of  claim 70  wherein said antibody is a monoclonal antibody. 
     
     
         73 . The composition of  claim 69  wherein said troponin isoform is a cardiac isoform. 
     
     
         74 . The composition of  claim 73  wherein said cardiac isoform is selected from the group consisting of cTnI and cTnT. 
     
     
         75 . The composition of  claim 74  wherein said cardiac isoform is cTnI. 
     
     
         76 . The composition of  claim 70  wherein said antibody is specific to a specific region of the troponin molecule. 
     
     
         77 . The composition of  claim 76  wherein said antibody is specific to a region comprising amino acids 27-41 of cardiac troponin I. 
     
     
         78 . The composition of  claim 69  wherein said fluorescent moiety comprises a molecule that comprises 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. 
     
     
         79 . The composition of  claim 69  wherein said fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. 
     
     
         80 . The composition of  claim 79  wherein said fluorescent moiety comprises AlexaFluor 647. 
     
     
         81 . A composition comprising a set of standards for the determination of a concentration of a cardiac troponin, wherein at least one of the standards is at a concentration of cardiac troponin less than about 10 pg/ml. 
     
     
         82 . A kit comprising a composition comprising an antibody to cardiac troponin attached to a fluorescent dye moiety, wherein said moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules, wherein said composition is packaged in suitable packaging. 
     
     
         83 . The kit of  claim 82  wherein the cardiac troponin is cardiac troponin I or cardiac troponin T. 
     
     
         84 . The kit of  claim 82  wherein the cardiac troponin is cardiac troponin I. 
     
     
         85 . The kit of  claim 82  further comprising instructions. 
     
     
         86 . The kit of  claim 82  further comprising a composition comprising a capture antibody for said cardiac troponin I attached to a solid support. 
     
     
         87 . The kit of  claim 86  wherein said solid support comprises a microtiter plate or paramagnetic microparticles. 
     
     
         88 . The kit of  claim 82  further comprising a component selected from the group consisting of wash buffer, assay buffer, elution buffer, and calibrator diluent. 
     
     
         89 . The kit of  claim 82  further comprising a standard for the cardiac troponin. 
     
     
         90 . The method of  claim 1  wherein the method further comprises detecting a plurality of troponin molecules in the sample and determining a concentration of troponin in the sample. 
     
     
         91 . The method of  claim 90  further comprising detecting a plurality of troponin molecules in a second sample, determining the concentration of troponin in said second sample, and detecting a difference in concentration between said first and second samples, wherein said method is capable of a detecting a difference in concentration of less than about 40 pg/ml between said samples. 
     
     
         92 . The method of  claim 91  wherein said method is capable of detecting a change in the presence or absence of said label, wherein the difference is a difference of less than about 20 pg/ml. 
     
     
         93 . The method of  claim 91  wherein said method is capable of detecting a change in the presence or absence of said label, wherein the difference is a difference of less than about 5 pg/ml. 
     
     
         94 . The method of  claim 91  wherein said method is capable of detecting a change in the presence or absence of said label, wherein the difference is a difference of less than about 1 pg/ml. 
     
     
         95 . The method of  claim 91  wherein said method is capable of detecting a change in the presence or absence of said label, wherein the difference is a difference of less than about 0.1 pg/ml. 
     
     
         96 . The method of  claim 91  wherein detecting a difference in concentration of at least 1 pg/ml indicates a cardiac event. 
     
     
         97 . The method of  claim 33  wherein said diagnosis, prognosis or method of treatment is based on a comparison of concentrations of troponin in a series of samples. 
     
     
         98 . The method of  claim 97  wherein the individual samples of the series of samples are taken less than 4 hours apart. 
     
     
         99 . The method of  claim 97  further comprising detecting the concentration of troponin in a first sample, detecting the concentration of troponin in a second sample, determining whether a difference in the concentration of troponin between said first and second sample is less than about 40 pg/ml is present between samples, wherein the difference in concentration by less than about 40 pg/ml indicates a cardiac event. 
     
     
         100 . The method of  claim 97  further comprising detecting the concentration of troponin in a first sample, detecting the concentration of troponin in a second sample, determining whether a difference in the concentration of troponin between said first and second sample is less than about 20 pg/ml is present between samples, wherein the difference in concentration by less than about 20 pg/ml indicates a cardiac event. 
     
     
         101 . The method of  claim 97  further comprising detecting the concentration of troponin in a first sample, detecting the concentration of troponin in a second sample, determining whether a difference in the concentration of troponin between said first and second sample is less than about 5 pg/ml is present between samples, wherein the difference in concentration by less than about 5 pg/ml indicates a cardiac event. 
     
     
         102 . The method of  claim 97  further comprising detecting the concentration of cardiac troponin in a first sample, detecting the concentration of cardiac troponin in a second sample, and determining a difference in the concentration of cardiac troponin between said first and second samples. 
     
     
         103 . The method of  claim 102  wherein the difference in concentration of troponin between samples is an increase in concentration. 
     
     
         104 . The method of  claim 102  wherein the difference in concentration of troponin between samples is a decrease in concentration. 
     
     
         105 . The method of  claim 102  wherein the difference in concentration between said first and second samples is less than about 40 pg/ml between samples. 
     
     
         106 . The method of  claim 102  wherein the difference in concentration between said first and second sample is less than about 20 pg/ml between samples. 
     
     
         107 . The method of  claim 102  wherein the difference in concentration between said first and second sample is less than about 5 pg/ml between samples. 
     
     
         108 . The method of  claim 102  wherein the difference in concentration between said first and second sample is less than about 1 pg/ml between samples. 
     
     
         109 . The method of  claim 102  wherein the difference in concentration between said first and second sample is less than about 0.1 pg/ml between samples. 
     
     
         110 . The method of  claim 102  wherein said first and second samples are taken less than 4 hours apart. 
     
     
         111 . The method of  claim 102  further comprising detecting the concentration of troponin in a first sample, detecting the concentration of troponin in a second sample, detecting a difference in the concentration of troponin between said first and second samples of about 10% percent, wherein the difference in concentration between samples of about 10% percent indicates myocardial ischemia or infarct. 
     
     
         112 . The method of  claim 102  further comprising detecting the concentration of troponin in a first sample, detecting the concentration of troponin in a second sample, determining whether a difference in the concentration of troponin in said first and second samples by about 5% percent is present between samples, wherein the difference in concentration by about 5% percent indicates myocardial ischemia or infarct. 
     
     
         113 . The method of  claim 102  wherein a measured increase in concentration of more than about 1 pg/ml between samples indicates a cardiac event. 
     
     
         114 . The method of  claim 102  wherein a difference in concentration between samples of more than about 0.02 pg/ml between samples indicates myocardial ischemia or infarct. 
     
     
         115 . The method of  claim 102  wherein the samples are taken before, during, or after a cardiac stress test. 
     
     
         116 . The method of  claim 102  wherein the samples are taken from a patient diagnosed with a heart condition. 
     
     
         117 . The method of  claim 102  wherein the samples are taken from a patient who is a candidate for a heart transplant. 
     
     
         118 . A method for determining the effectiveness of a treatment for myocardial infarct comprising i) determining a concentration of cardiac troponin in a first sample, wherein said concentration is determined by a cardiac troponin assay with a limit of detection for said cardiac troponin in said sample of less than about 40 pg/ml; ii) determining a concentration of cardiac troponin in a second sample; and iii) detecting a decrease in the concentration of cardiac troponin between said first and second samples.