JP2020180974A - Highly sensitive system and method for analysis of troponin - Google Patents

Abstract

To provide a method of determining cardiovascular health of an individual.SOLUTION: A method disclosed herein comprises: a) measuring blood, serum, or plasma concentration of Cardiac Troponin I (cTnI), or Cardiac Troponin T (cTnT), from a blood sample collected from an individual; b) comparing the measured concentration to a predetermined threshold concentration representing the 99th percentile concentration of cTnI in a group of normal individuals with a corresponding coefficient variation (CV) of 10% or less; and c) determining cardiac damage in the individual when the concentration of cTnI is greater than the threshold concentration.SELECTED DRAWING: Figure 1A

Description

This application is a US patent provisional application 60/789304, filed April 19, 2006, filed April 4, 2006, which is incorporated herein by reference in its entirety under Article 119 of the US Patent Act. U.S. Patent Provisional Application 60/793664, U.S. Patent Provisional Application 60/808662 filed May 26, 2006, U.S. Patent Provisional Application 60/861498 filed November 28, 2006 and December 4, 2006 Claims the priority of US Patent Provisional Application 60/872986.

Background Technology In the United States, about 6,000,000 people are taken to the emergency department each year for chest pain. Only 15% to 20% of these patients are ultimately diagnosed with acute coronary syndrome (ACS), but about half are hospitalized for evaluation. Conversely, 2% of ACS patients are accidentally discharged. Patients with ACS have a relatively high risk of serious cardiovascular adverse events in the short term, so it is clear that there is a need for accurate objective means to identify them.

Currently used cardiac injury markers have the drawback of limiting their clinical usefulness. Cardiac enzyme assays form the basis for determining the presence or absence of damage to the myocardium. Unfortunately, the standard creatine kinase MB (CK-MB) assay is unreliable in removing infarcts by 10-12 hours after the onset of chest pain. Early diagnosis will have very special advantages with respect to fibrinolytic therapy and triage.

In one aspect, the invention provides a method.

In some embodiments, the present invention is a method for determining the presence or absence of a single molecule or fragment or complex of troponin in a sample, i) a molecule, fragment, or complex is present. In some cases, the step of labeling with a label and the step of ii) detecting the presence or absence of the label, and the detection of the presence of the label is a step of indicating the presence of a single molecule, fragment, or complex of troponin in the sample. Provide a method to include. In some embodiments of the method of the invention, troponin is a myocardial isoform of troponin. In some embodiments of the methods of the invention, the troponin can be myocardial troponin I (cTnI) or myocardial troponin C (cTnC). In some embodiments of the method of the invention, the troponin is cTnI. In some embodiments of the method of the invention, a single molecule of troponin can be detected with a detection limit of less than about 100 pg / ml. In some embodiments of the methods of the invention, single molecules or troponins can be detected at detection levels below about 20 pg / ml. In some embodiments of the method of the invention, the label comprises a fluorescent moiety. In some embodiments, the fluorescent moiety is capable of emitting at least about 200 photons when stimulated by a laser that emits at the excitation wavelength of that moiety, the laser having a diameter of about 5 microns including the fluorescent moiety. The total energy focused on the above spots and directed by the laser to the spots is about 3 microjoules or less. In some embodiments of the method of the invention, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, the substituent on the 3-carbon of the indolium ring being a chemically reactive or conjugated substance. Includes a conjugated substance group. In some embodiments of the method of the invention, the fluorescent moiety comprises a dye. Examples of dyes include, but are not limited to, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, and AlexaFluor 700. In some embodiments of the method of the invention, the fluorescent moiety comprises AlexaFluor647. In some embodiments, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, and the substituent on the 3-carbon of the indolium ring comprises a chemically reactive or conjugate group. In some embodiments of the methods of the invention, the label further comprises a binding partner for a molecule, fragment, or complex of troponin. In some embodiments of the methods of the invention, the binding partner comprises an antibody specific for a molecule, fragment, or complex of troponin. In some embodiments of the methods of the invention, the antibody is specific for a particular region of the troponin molecule. In some embodiments of the methods of the invention, the antibody is specific for the region of myocardial troponin I containing amino acids 27-41. In some embodiments of the methods of the invention, the antibody can be a polyclonal antibody. In some embodiments of the methods of the invention, the antibody is a monoclonal antibody. In some embodiments of the method of the invention, the method further comprises the step of capturing the troponin or troponin complex on a solid support. In some embodiments of the method of the invention, the solid support can be a microtiter plate or paramagnetic beads. In some embodiments of the methods of the invention, the solid support comprises a troponin or troponin complex-specific capture partner that binds to the solid support. In some embodiments of the methods of the invention, the binding of the capture partner to the solid support is non-covalent. In some embodiments of the methods of the invention, the binding of the capture partner to the solid support is covalent. In some embodiments of the method of the invention, the covalent bond of the capture partner is one in which the capture partner binds to the solid support in a particular direction. In some embodiments of the methods of the invention, the particular orientation helps maximize the specific binding of the troponin or troponin complex to the capture partner. In some embodiments of the methods of the invention, the capture partner comprises an antibody. In some embodiments of the methods of the invention, the antibody is a monoclonal antibody. In some embodiments of the methods of the invention, the antibody is specific for amino acids 87-91 of myocardial troponin I. In some embodiments of the methods of the invention, the antibody is specific for amino acids 41-49 of myocardial troponin I. In some embodiments of the method of the invention, the sample is a blood, serum, or plasma sample. In some embodiments of the method of the invention, the sample is a serum sample. In some embodiments of the method of the invention, the label comprises a fluorescent moiety and step ii) comprises passing the label through a single molecule detector. In some embodiments of the method of the invention, the single molecule detector is a) an electromagnetic radiation source for stimulating the fluorescent moiety, b) a capillary flow cell for passing the fluorescent moiety, and c) in a capillary flow cell. The driving source for moving the fluorescent part, d) the interaction space defined in the capillary flow cell for receiving the electromagnetic radiation emitted from the electromagnetic radiation source, e) the electromagnetic characteristics of the stimulated fluorescent part are measured. Includes an electromagnetic radiation detector operably connected to the interaction space, and a microscope objective lens located between the intervention space and the detector, which is a high numerical aperture lens.

In some embodiments, the invention is a method for determining a diagnosis, prognosis, or treatment method in an individual, i) determining the concentration of myocardial troponin in a sample obtained from the individual, or A step of determining the concentration of myocardial troponin in a series of samples, a step of determining the concentration by a myocardial troponin assay in which the detection limit of myocardial troponin in the sample is less than about 50 pg / ml, and ii) the concentration in the sample. Alternatively, a method comprising a step of determining a diagnosis, prognosis, or treatment method in an individual based on a concentration in a series of samples is provided. In some embodiments of the method of the invention, step ii) compares a concentration or series of concentrations to a normal value of that concentration, or compares a concentration or series of concentrations to a predetermined threshold level. Includes analysis such as comparing a concentration or set of concentrations to baseline values, and determining the rate of change in concentration for a set of concentrations. In some embodiments of the method of the invention, step ii) compares the troponin concentration in the sample to a predetermined threshold concentration, and if the sample concentration is higher than the threshold level, diagnosis, prognosis diagnosis, Or it involves deciding how to treat. In some embodiments of the method of the invention, the threshold concentration is determined by determining the 99th percentile concentration of troponin in a normal population and setting the threshold concentration at the 99th percentile concentration. In some embodiments of the methods of the invention, at least one sample is taken during or after a cardiac stress test. In some embodiments of the methods of the invention, myocardial troponin is selected from the group consisting of myocardial troponin I and myocardial troponin T. In some embodiments of the method of the invention, the myocardial troponin is myocardial troponin I. In some embodiments of the method of the invention, the concentration of myocardial troponin is the concentration of total myocardial troponin. In some embodiments of the methods of the invention, the concentration of myocardial troponin is the concentration of myocardial troponin complex, myocardial troponin fragment, phosphorylated myocardial troponin, oxidized myocardial troponin, or a combination thereof. In some embodiments of the method of the invention, the concentration of myocardial troponin is compared to total myocardial troponin. In some embodiments of the methods of the invention, the diagnostic, prognostic, or therapeutic method is a diagnostic, prognostic, or therapeutic method for myocardial infarction. In some embodiments of the methods of the invention, diagnostic, prognostic, or therapeutic methods include risk stratification of myocardial infarction risk levels. In some embodiments of the methods of the invention, the concentration or series of concentrations is when, or the like, an individual presents to a healthcare professional one or more symptoms that indicate myocardial ischemia or infarction or the possibility thereof. Will be decided nearby. In some embodiments, one or more symptoms can be chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels or shortness of breath. In some embodiments, the concentration is determined by a method comprising detecting a single molecule of troponin or a complex or fragment thereof. In some embodiments, the method of the invention comprises labeling the troponin or troponin complex with a label containing a fluorescent moiety. In some embodiments of the method of the invention, the fluorescent moiety is capable of emitting at least about 200 photons when stimulated by a laser that emits at the excitation wavelength of that moiety, the laser comprising the fluorescent moiety. Focused on a spot with a diameter of 5 microns, the total energy directed by the laser to the spot is less than about 3 microjoules. In some embodiments of the method of the invention, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, the substituent on the 3-carbon of the indolium ring being a chemically reactive or conjugate group. Including. In some embodiments of the methods of the invention, the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. In some embodiments of the method of the invention, the fluorescent moiety comprises AlexaFluor647. In some embodiments of the methods of the invention, the label further comprises a binding partner for troponin. In some embodiments, the binding partner comprises an antibody specific for troponin. In some embodiments, the antibody is a polyclonal antibody. In some embodiments of the method of the invention, the method further comprises the step of capturing the troponin or troponin complex on a solid support. In some embodiments of the method of the invention, the solid support can be a microtiter plate or paramagnetic beads. In some embodiments of the methods of the invention, the solid support comprises a troponin or troponin complex-specific capture partner that binds to the solid support. In some embodiments of the methods of the invention, the binding of the capture partner to the solid support is non-covalent. In some embodiments of the methods of the invention, the binding of the capture partner to the solid support is covalent. In some embodiments of the method of the invention, the covalent bond of the capture partner is one in which the capture partner binds to the solid support in a particular direction. In some embodiments of the methods of the invention, the particular orientation helps maximize the specific binding of the troponin or troponin complex to the capture partner. In some embodiments of the methods of the invention, step i) further comprises assessing another indicator of the individual, step ii) for assessing troponin concentration and another indicator of non-troponin markers in the sample. Includes determining a diagnosis, prognostic diagnosis, or treatment method in an individual based on or based on a concentration in a series of samples. In some embodiments, another indicator is a clinical indicator of myocardial ischemia or infarction. In some embodiments, another indicator is the concentration of one or more non-troponin markers in a sample or in a series of samples. In some embodiments of the methods of the invention, one or more markers are markers of cardiac ischemia or inflammation and plaque instability. In some embodiments, one or more cardiac ischemia markers are creatine kinase (CK) and its myocardial fraction CK myocardial band (MB), aspartate aminotransferase, lactate dehydrogenase (LDH), α-hydroxybutyric acid dehydrogenase, Must be myoglobin, oxaloacetate transaminase glutamate, glycogen phosphorylase BB, unbound free fatty acid, cardiac fatty acid binding protein (H-FABP), ischemia-modified albumin, myosin light chain 1 or myosin light chain 2. Can be done. In some embodiments of the methods of the invention, the one or more markers include one or more specific markers of myocardial injury. In some embodiments of the methods of the invention, the diagnostic, prognostic, or therapeutic method is a diagnostic, prognostic, or therapeutic method for a condition that is not a myocardial infarction. In some embodiments, the condition is cardiotoxic. In some embodiments, cardiotoxicity is associated with administration of the drug to the individual. In some embodiments of the methods of the invention, the conditions include acute rheumatic fever, amyloid deposits, cardiac trauma (contusion, ablation, pacing, firing, cardioversion, catheter insertion, and cardiac surgery. Includes), reperfusion injury, congestive heart failure, end-stage renal failure, type II glucose storage (Pompe's disease), heart transplantation, abnormal hemoglobinosis due to transfusion hemosiderosis, hypertension including gestational hypertension, often low with arrhythmia It is selected from the group consisting of hypertension, hypothyroidism, myocarditis, peritonitis, postoperative non-cardiac surgery, pulmonary arterial embolism and septicemia.

In another aspect, the invention comprises a composition.

In some embodiments, the invention comprises a composition for detecting troponin isoforms comprising a binding partner for the troponin isoform bound to the fluorescent moiety, the fluorescent moiety being a laser emitting at the excitation wavelength of the moiety. Can emit at least about 200 photons when stimulated by, the laser is focused on a spot with a diameter of about 5 microns or more, including a fluorescent portion, and the total energy directed by the laser to the spot is about 3 micron. It is less than Jules. In some embodiments of the compositions of the invention, the binding partner comprises an antibody against the troponin isoform. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the troponin isoform is a myocardial isoform. In some embodiments, the myocardial isoform is selected from the group consisting of cTnI and cTnT. In some embodiments, the myocardial isoform is cTnI. In some embodiments, the antibody is specific for a particular region of the troponin molecule. In some embodiments, the antibody is specific for a region of myocardial troponin I containing amino acids 27-41. In some embodiments of the compositions of the invention, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, the substituent on the 3-carbon of the indolium ring being a chemically reactive group or conjugated. Includes groups. In some embodiments, the fluorescent moiety comprises a dye that can be AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700. In some embodiments, the fluorescent moiety comprises AlexaFluor647.

In some embodiments, the present invention comprises a composition comprising a set of standards for determining the concentration of myocardial troponin, at least one standard having a myocardial troponin concentration of less than about 10 pg / ml.

In some embodiments, the invention comprises a kit comprising a composition comprising an antibody against myocardial troponin bound to a fluorochrome moiety, when the fluorochrome moiety is stimulated by a laser that emits light at the excitation wavelength of that moiety. Can emit at least about 200 photons, the laser is focused on a spot with a diameter of about 5 microns or more, including the fluorescent portion, and the total energy directed by the laser to the spot is less than about 3 microjoules. The composition is packaged in a suitable packaging material. In some embodiments of the kits of the invention, the myocardial troponin is myocardial troponin I or myocardial troponin T. In some embodiments, the myocardial troponin is myocardial troponin I. In some embodiments of the kit of the invention, the kit further comprises instructions. In some embodiments of the kit of the invention, the kit further comprises a composition comprising a capture antibody for myocardial troponin I bound to a solid support. In some embodiments, the solid support comprises a microtiter plate or paramagnetic microparticles. In some embodiments of the kits of the invention, the kit further comprises components selected from the group consisting of wash buffers, assay buffers, elution buffers and calibrator diluents. Some embodiments of the kits of the invention further include standards of myocardial troponin.

Incorporation by Reference All publications and patent applications pointed out herein are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated for inclusion by reference. Incorporated.

The novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the invention can be obtained by reference to the following detailed description, which describes exemplary embodiments in which the principles of the invention are utilized, as well as the accompanying drawings. There will be.

It is a figure which shows the schematic of the arrangement of the component of a single particle analyzer, and is the figure which shows the analyzer which contains one electromagnetic radiation source and one electromagnetic detector. It is a figure which shows the schematic of the arrangement of the component of a single particle analyzer, and is the figure which shows the analyzer which includes two electromagnetic radiation sources and one electromagnetic detector. It is a figure which shows the schematic of the capillary flow cell of a single particle analyzer, and is the figure which shows the flow cell of the analyzer which contains one electromagnetic radiation source. It is a figure which shows the schematic of the capillary flow cell of a single particle analyzer, and is the figure which shows the flow cell of the analyzer which contains two electromagnetic radiation sources. It is a figure which shows the schematic diagram which shows the conventional arrangement of the laser and the detector optics of a single particle analyzer, and is the figure which shows the arrangement of the analyzer which has one electromagnetic radiation source and one electromagnetic detector. It is a figure which shows the schematic diagram which shows the confocal arrangement of the laser and the detector optics of a single particle analyzer, and is the figure which shows the arrangement of the analyzer which has two electromagnetic radiation sources and two electromagnetic detectors. It is a figure which shows the linearized standard curve about the concentration range of cTnI. FIG. 5 shows that the biological threshold (cutoff concentration) of cTnI is at a cTnI concentration of 7 pg / ml when established at the 99th percentile with a corresponding CV of 10%. It is a figure which shows the correlation (R2 = 0.9999) of the assay result of cTnI determined using the analyzer system of this invention, and the standard product measurement value provided by the National Institute of Standards and Technology. It is a figure which shows the detection of cTnI in the continuous serum sample obtained from the patient who showed chest pain in the emergency room. It is a figure which shows the comparison between the measurement performed by the analyzer system of this invention, and the measurement performed by the assay which is commercially available. FIG. 5 shows the distribution of normal (non-ischemic) biological concentrations of cTnI and cTnI concentrations in serum samples obtained from patients exhibiting chest pain.

Overview I. Introduction II. Myocardial troponin III. Labeling of myocardial troponin A A. Troponin binding partner 1. Antibody 2. Cross-reaction antibody B. Fluorescent portion used with the binding partner 1. Dye 2. Quantum dot C.I. Binding partner Fluorescent partial composition IV. High-sensitivity analysis of myocardial troponin A. Sample B. Sample preparation C. Detection of troponin and determination of concentration V. Instruments and Systems Suitable for Sensitive Analysis of Troponin A. Equipment / System B. Single particle analyzer 1. Electromagnetic radiation source 2. Capillary flow cell 3. Driving force 4. Detector C.I. Sampling system D. Sample preparation system E. Sample recovery VI. Method using sensitive analysis of myocardial troponin A. Sample B. Diagnosis, prognosis, or determination of treatment method 1. Acute myocardial infarction 2. Pathophysiology other than AMI a. Cardiotoxicity C.I. Business method VII. Composition VIII. kit

I. Introduction

The present invention provides compositions and methods for sensitive detection of troponin, such as myocardial troponin. The release of myocardial isoforms of myocardial-specific troponins (myocardial troponin I and / or T) into the blood indicates damage to the myocardium and for their use as prognostic markers or to aid treatment decisions. Provides the basis for.

The intramuscular troponin complex consists of troponins I, C, and T. Troponin C has one from the myocardium and slow-twich muscle and one from the first-twich muscle.
Its use as a specific marker is limited because it exists as two isoforms from and is found in virtually all striated muscles. In contrast, troponins I and T are expressed as different isoforms in slow, fast, and myocardium. The unique myocardial isoforms of troponins I and T make it possible to immunologically distinguish them from other tropons in skeletal muscle. Therefore, the release of myocardial troponins I and T into the blood indicates damage to the myocardium and provides the basis for their use as markers of diagnosis and prognosis or to assist in treatment decisions.

The markers currently used for heart injury have the drawback of limited clinical utility. Cardiac enzyme assays form the basis for determining the presence or absence of myocardial damage. Unfortunately, the standard creatine kinase-MB (CK-MB) assay is unreliable in removing infarcts 10 to 12 hours after the onset of chest pain. Early diagnosis will have very special advantages with respect to fibrinolytic therapy and triage.

Troponin is a highly sensitive and specific marker of heart damage because the levels of troponin found in the blood circulation of healthy individuals are extremely low and heart-specific troponin does not originate from sources other than the heart. In addition to cardiac infarction, many other pathologies can cause damage to the myocardium, and early diagnosis of such damage will be shown to be useful to clinicians. However, current methods of detection and quantification have sufficient sensitivity to detect the release of myocardial troponin into the blood until an abnormally high concentration, eg, a level of 0.1 ng / ml or higher, is reached. Not.

Therefore, the methods and compositions of the present invention are methods and compositions for sensitive detection and quantification of myocardial troponin, as well as diagnostics, prognostic diagnosis, and / or based on such sensitive detection and quantification. Includes compositions and methods for therapeutic decisions.

II. Myocardial Troponin When two unique forms of myocardial troponin, myocardial troponin I (cTnl) and myocardial troponin (cTnT), are released from the myocardium into the blood, several species of each can be present in the blood. These include various complexes of two forms, with each other and / or with myocardial troponin C (cTnC). In addition, the two forms undergo substantially rapid proteolytic denaturation into various fragments. In addition, various phosphorylated and oxidized forms of troponin can be present in the blood. See, for example, U.S. Pat. No. 6,991,907, which is incorporated herein by reference in its entirety. Unless otherwise specified, "myocardial troponin" as described herein includes all forms of myocardial troponin, including.

In some embodiments, the present invention relates to the concentration of total myocardial troponin, i.e. a sample (eg, blood, serum), regardless of whether myocardial troponin is free, complex, proteolytic fragment, phosphorylated, oxidized, or modified. , Or plasma samples) provide methods and compositions for the detection and / or determination of the total of all or substantial portions of myocardial troponin. In some embodiments, the myocardial troponin is cTnl, in another it is cTnT, and in yet another embodiment the myocardial troponin is cTnl and cTnT. It is well understood that it is not necessary to make a complete full measurement as long as a consistent percentage of the total is determined and this can be compared to the standard value. It will also be well understood that if the form of troponin is a minority component of all, the lack or low detection level of that form does not significantly affect the measurement of total troponin. .. Thus, as used herein, "whole myocardial troponin" is a measurement intended to measure any or substantially all forms of a particular myocardial troponin, eg, all cTnl or all cTnT, in a sample. Pointing to a value, in this case, consistency between samples is such that clinically relevant conclusions can be drawn from a comparison between a sample and a standard or from a comparison between one sample and another.

In some embodiments, the present invention relates to one or more various forms of troponin as distinct entities in a sample, such as complexed cTnl, free cTnl, muddied c.
Provided methods and compositions for the detection and / or determination of the concentration of Tnl (eg, oxidation or phosphorylation), or complexed cTnT, free cTnT, muddy cTnT (eg, oxidation or phosphorylation), typically. Can provide the concentration of the form in the sample. In the latter embodiment, ratios or absolute values may be determined for different entities. Thus, in some embodiments, the invention is a method of detecting the concentration of one or more forms of complexed troponin, or one or more troponin fragments, or one or more oxidized or phosphorylated forms of troponin. And typically provides a method of providing a decision method. In some embodiments, two or more forms are detected, eg, by performing a multiplexed assay on different entities on a single sample, or separately on an aliquot obtained from the same or similar sample. By performing an assay, concentrations in various forms can be determined. Ratios of concentrations in various forms can be obtained. For example, the ratio of the concentration of myocardial troponin in a particular form, eg, fragment, complex, or modified form to the concentration of total myocardial troponin can be determined. These ratios and / or absolute values can provide meaningful clinical information. For example, the relative proportions of fragments of myocardial troponin can indicate the length of time from release into the blood and thus indirectly from the length of time (eg, from myocardial infarction). See, for example, U.S. Pat. No. 6,991,907, which is incorporated herein by reference in its entirety.

m. Labeling of Myocardial Troponin In some embodiments, the present invention provides methods and compositions comprising labeling for sensitive detection and quantification of myocardial troponin.

Those skilled in the art are well aware that many strategies can be used to label the target molecule and allow detection or identification in the mixture of particles. Labels can be combined by any known means, including methods that utilize unspecified or specific interactions between the label and the target. Labels can provide a detectable signal or affect the movement of particles in an electric field. In addition, labeling can be achieved either directly or through a binding partner.

In some embodiments, the label comprises a binding partner for troponin that is bound to the fluorescent moiety.

A. Troponin binding partner Any suitable binding partner having the required specificity for the morphology of myocardial troponin to be detected can be used. For example, binding partners specific for all or substantially all forms of cTnl can be used, or binding partners specific for all or substantially all forms of cTnT. Can be used. Typically, such binding partners bind to regions of myocardial troponin that are common to all or most of the different forms found in the sample. In some embodiments, one such as a complexed cTnl, free cTnl, muddy cTnl (eg, oxidation or phosphorylation), or a complexed cTnT, free cTnT, muddy cTnT (eg, oxidation or phosphorylation) binding partner. A binding partner specific for the above specific forms of myocardial troponin can be used. Binding partners are known in the art and include, for example, aptamers, lectins, and receptors. A useful and versatile binding partner is an antibody.

A. Antibodies In some embodiments, the binding partner is an antibody specific for myocardial troponin. As used herein, the term "antibody" is a broad term, but not limited to, natural antibodies, as well as unnatural antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, and humanized antibodies. , As well as their antigen-binding fragments, used in their usual sense. In some embodiments, the antibody is specific for cTnl. In some embodiments, the antibody is specific for cTnT. In some embodiments, the antibody is specific for cTnT. In some embodiments, the label comprises antibodies against both cTnl and cTnT. Antibodies are specific for all or substantially all forms of myocardial troponin, eg, all or substantially all forms of cTnl or all or substantially all forms of cTnT. In some embodiments, one or more specific forms of myocardial troponin, such as complexed cTnl, free cTnl, muddy cTnl (eg, oxidation or phosphorylation), or complexed cTnT, free cTnT, muddy cTnT (eg, oxidation or phosphorylation). For example, an antibody specific for a binding partner (oxidation or phosphorylation) can be used. Mixtures of antibodies, such as mixtures of antibodies against cTnl and cTnT, or mixtures of antibodies against various forms of troponin (free, complexed, etc.), or mixtures of mixtures are also included in the invention.

It is well understood that the selection of troponin epitopes or regions (which yield antibodies to them) determines, for example, their specificity for total troponins, certain fragments, complexed troponins, modified troponins. Has been done. In some embodiments, the antibody is specific for a particular amino acid region of myocardial troponin. In some embodiments, the antibody is specific for a particular amino acid 27-41 of human myocardial troponin I. Both monoclonal and polyclonal antibodies are useful as binding partners. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody specific for amino acids 27-41 of human myocardial troponin I. In some embodiments, the antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complex formation and does not cross-react with skeletal muscle troponin I.

The method for producing the antibody is well established. Specific sequences of the heart for troponin I and troponin T are described in FEBS Lett. 270, 57-61 (1990) and Genomics 21, 311-316 (1994). For example, Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, N. et al. Y. Those skilled in the art are well aware that many techniques are available for the production of antibodies, as described in. Those skilled in the art are also well aware that binding fragments or Fab fragments that mimic antibodies can be prepared from genetic information by a variety of techniques (Antibody Engineering: A Practical Application (Borrevac, C, ed.), 1995, Oxford University). Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Methods for producing antibodies to various complexed, fragmented, phosphorylated, and oxidized forms of troponin are described in US Pat. No. 5,579,687; 6,991,907; and US Pat. No. 2,050,164317 (US Pat. These are incorporated herein by reference in their entirety). A synthetic peptide consisting of 14 amino acids that mimics the specific sequence of the heart of troponin I and a method for preparing an antibody against the peptide are described in International Patent Application PCT / US94 / 05468. Monoclonal and polyclonal antibodies against free and complex myocardial troponins are also commercially available (HyTest, HyTest Ltd., Turku, Finland; Abeam Inc., Cambridge, Massachusetts, USA, Life Diagonosis, Inc., West Chester, Pennsylvania). , USA; Fitzgerald Industries International, Inc., Concord, Massachusetts 01742-3049 USA; BiosPacific, Emeryville, Canada).

In some embodiments, the antibody is a mammalian, eg, goat polyclonal anti-cTnl antibody. The antibody can be specific for a specific region of cTnl, eg, amino acids 27-41 of human myocardial troponin I. A pair of capture-binding partner and detection-binding partner, such as a pair of capture and detection antibody, can be used in embodiments of the present invention. Therefore, some embodiments typically use a heterogeneous assay protocol with two binding partners, eg, two antibodies. One binding partner is a capture partner that is normally immobilized on a solid support, and the other binding partner is typically a detection binding partner to which a detectable label is attached. In some embodiments, the paired capture-binding partner member is an antibody specific for all or substantially all forms of myocardial troponin. One example is an antibody specific for a cTnl-forming complex with free myocardial troponin I (cTnl) amino acids 41-49 and other troponin components, such as a monoclonal antibody. This antibody is preferably unaffected by heparin, phosphorylation, oxidation, and troponin complex formation and does not cross-react with skeletal muscle troponin I. Therefore, the antibody is believed to bind to total cTnl. Another example is a monoclonal antibody specific for myocardial troponin I (cTnl) amino acids 87-91, which does not cross-react with skeletal muscle troponin I. Such antibodies are commercially available from Bios Pacific, Emeryville, Canada. Pairs of other antibodies are known and can be designed.

In some embodiments, it is beneficial to use antibodies that cross-react with various species as capture antibodies, detection antibodies, or both. Such embodiments include measurement of drug toxicity, for example by determining the release of myocardial troponin into the blood as a marker of heart damage. Cross-reactive antibodies allow the study of toxicity in certain species, such as non-human species, to the study or clinical observation of other species, such as humans, using the same antibody or antibody pair in the assay reagents. And the results can be transferred directly, thus reducing inter-assay variability. Thus, in some embodiments, the one or more antibodies used as binding partners for markers such as myocardial troponin, such as myocardial troponin I, may be cross-reactive antibodies. In some embodiments, the antibody cross-reacts with a marker from at least two selected from the group consisting of humans, monkeys, dogs, and mice, such as myocardial troponin. In some embodiments, the antibody cross-reacts with a marker from all of the group consisting of humans, monkeys, dogs, and mice, such as myocardial troponin.

B. Fluorescent portion also used with a binding partner In some embodiments of the label used in the present invention, a binding partner, such as an antibody, is bound to the fluorescent moiety. The fluorescence of the fluorescent moiety is sufficient to allow detection with a single molecule detector such as the single molecule detector described herein. As used herein, the term "fluorescent moiety" is one or more fluorescent entities whose entire fluorescence is such that the moiety can be detected by the single molecule detector described herein. including. Thus, the fluorescent moiety can include a single entity (eg, quantum dots or fluorescent molecules) or multiple entities (eg, multiple fluorescent molecules). When the term "part" as used herein refers to a group of fluorescent entities, eg, multiple fluorochrome molecules, the individual entities are separately bound to a binding partner as long as the entity provides sufficient fluorescence to detect. It will be understood that they can be combined or entities can be combined together.

In general, the fluorescence of the fluorescent moiety contains a combination of sufficient quantum efficiency and lack of photobleaching for the fluorescent moiety to be detectable by a single molecule detector beyond the background level, the desired level of the assay. Has the required consistency for detection, accuracy and accuracy. For example, in some embodiments, the fluorescence of the fluorescent moiety is at a detection level of less than about 10, 5, 4, 3, 2, or 1 pg / ml and about 20, 15 in the instruments described herein. , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, for example, detection and / or quantification of troponin with a coefficient of variation of about 10% or less. It makes it possible. In some embodiments, the fluorescence of the fluorescent moiety provides detection and / or quantification of troponin with a detection limit of less than about 5 pg / ml and a coefficient of variation of less than about 10% in the instruments described herein. It makes it possible. As used herein, the term "detection limit" includes the lowest concentration at which a sample can be identified as containing molecules of the substance of interest, eg, the first non-zero value. This can be defined by the variability of zero and the slope of the standard curve. For example, the detection limit of an assay can be determined by drawing a standard curve, determining the zero value of the standard curve, and adding two standard deviations to that value. The concentration of the substance of interest that produces a signal equal to this value is the "lower detection limit" concentration.

In addition, the fluorescent moiety has properties consistent with its use in the assay of choice. In some embodiments, the assay is an immunoassay, where the fluorescent moiety is attached to the antibody. The fluorescent moiety must have properties such that it does not aggregate with other antibodies or proteins, or that it does not cause aggregation that matches the required accuracy and accuracy of the assay. In some embodiments, preferred fluorescent moieties are, for example, 1) high absorption coefficient, 2) high quantum yield, 3) high photostability (low photobleaching), and 4) the analyzer of the invention and Labeling the biomolecule of interest (eg, protein) so that it can be analyzed using the system (eg, without causing precipitation of the protein of interest and precipitation of proteins with fluorescent moieties attached). Fluorescent moieties such as dye molecules that have a combination of compatibility with.

Fluorescent moieties useful in some embodiments of the invention, such as a single fluorochrome molecule or multiple fluorochrome molecules, can be defined by their photon emission properties when stimulated by EM radiation. For example, in some embodiments, the present invention averages at least about 10, 20, 30, 40, 50, 75, 100, 125, 150, when stimulated by a laser that emits at the excitation wavelength of the fluorescent portion. 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800 900 or fluorescent dye moieties capable of emitting 900 or 1000 photons, such as a single fluorescent dye molecule or plural. Utilizing the fluorescent dye molecule of, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less. It will be appreciated that total energy can be achieved by many different combinations of laser power and dye portion exposure time. For example, a 1 mW output laser can be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms, and so on.

In some embodiments, the present invention is capable of emitting at least about 50 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety, eg, a single fluorochrome moiety. Utilizing a fluorescent dye molecule or a plurality of fluorescent dye molecules, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention is capable of emitting at least about 100 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety, eg, a single fluorochrome moiety. Utilizing a fluorescent dye molecule or a plurality of fluorescent dye molecules, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention is capable of emitting at least about 150 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety, eg, a single fluorochrome moiety. Utilizing a fluorescent dye molecule or a plurality of fluorescent dye molecules, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention is capable of emitting at least about 200 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety, eg, a single fluorochrome moiety. Utilizing a fluorescent dye molecule or a plurality of fluorescent dye molecules, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention is capable of emitting at least about 300 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety, eg, a single fluorochrome moiety. Utilizing a fluorescent dye molecule or a plurality of fluorescent dye molecules, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention is capable of emitting at least about 500 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety, eg, a single fluorochrome moiety. Utilizing a fluorescent dye molecule or a plurality of fluorescent dye molecules, the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed to the spot by the laser is about 3 microjoules or less.

In some embodiments, the fluorescent moiety comprises, on average, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fluorescent entities, such as fluorescent molecules. In some embodiments, the fluorescent moiety contains, on average, only about 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 fluorescent entities, such as fluorescent molecules. In some embodiments, the fluorescent moieties average about 1-11, or about 2-10, or about 2-8, or about 2-6, or about 2-5, or about 2-4, or about 3. -10, or about 3-8, or about 3-6, or about 3-5, or about 4-10, or about 4-8, or about 4-6, or about 2, 3, 4, 5, 6 It contains the above number of fluorescent entities. In some embodiments, the fluorescent moieties include an average of about 2-8 fluorescent moieties. In some embodiments, an average of about 2-6 fluorescent entities. In some embodiments, the fluorescent moiety contains an average of about 2-4 fluorescent entities. In some embodiments, the fluorescent moiety contains an average of about 3-10 fluorescent entities. In some embodiments, the fluorescent moiety contains an average of about 3-8 fluorescent entities. In some embodiments, the fluorescent moiety contains an average of about 3-6 fluorescent entities. "Average" constitutes a fluorescent moiety, as determined by standard analytical methods, in a given sample, which is a representative sample of the group of labels of the invention, wherein the sample contains multiple binding partner fluorescent moiety units. It means that the molar ratio of a particular fluorescent entity to a binding partner corresponds to a specified number or range of numbers. For example, in embodiments where the label comprises a binding partner that is an antibody and a fluorescent moiety that contains multiple fluorochrome molecules of a particular absorbance, the labeled solution is diluted to an appropriate level to determine the molar concentration of the protein (antibody). A spectrophotometric assay can be used to obtain the absorbance at 280 nm to obtain the absorbance at, for example, 650 nm (in the case of AlexaFluor647) to determine the molar concentration of the fluorescent dye molecule. The ratio of the latter molar concentration to the former represents the average number of fluorescent entities (dye molecules) in the fluorescent moiety that binds to each antibody.

1. 1. Dye In some embodiments, the present invention utilizes a fluorescent entity that comprises a fluorescent dye molecule. In some embodiments, the invention utilizes a fluorochrome molecule that can emit at least about 50 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorochrome molecule. It is focused on a spot containing the molecule and having a diameter of about 5 microns or more, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the invention utilizes a fluorochrome molecule that can emit at least about 75 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorochrome molecule. It is focused on a spot containing the molecule and having a diameter of about 5 microns or more, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the invention utilizes a fluorochrome molecule that can emit at least about 100 photons on average when stimulated by a laser that emits light at the excitation wavelength of the fluorochrome molecule. It is focused on a spot containing the molecule and having a diameter of about 5 microns or more, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the invention utilizes a fluorochrome molecule that can emit at least about 150 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorochrome molecule. It is focused on a spot containing the molecule and having a diameter of about 5 microns or more, and the total energy directed to the spot by the laser is about 3 microjoules or less. In some embodiments, the invention utilizes a fluorochrome molecule that can emit at least about 200 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorochrome molecule. It is focused on a spot containing the molecule and having a diameter of about 5 microns or more, and the total energy directed to the spot by the laser is about 3 microjoules or less.

A non-comprehensive list of useful fluorescent entities for use in the fluorescent moieties of the invention is shown in Table 2 below. In some embodiments, the fluorescent dye is selected from the group consisting of AlexaFlour 488, 532, 647, 700, 750, fluorescein, B-phycocyanin, allophycocyanin, PBXL-3 and Qdot 605. In some embodiments, the fluorescent dye is selected from the group consisting of AlexaFlour 488, 532, 700, 750, fluorescein, B-phycocyanin, allophycocyanin, PBXL-3, and Qdot 605.

Suitable dyes for use in the present invention include modified carbocyanine dyes. Modifications of the carbocyanine dye include modification of the indolium ring of the carbocyanine dye to accept a reactive group or conjugate substance at the 3-position. Modification of the indolium ring is more uniform and more uniform on proteins, nucleic acids, and other biopolymers than conjugates labeled with structurally similar carbocyanine dyes attached via the nitrogen atom at position 1. Provides a substantially stronger fluorescent dye conjugate. In addition to having stronger fluorescence emission than structurally similar dyes at substantially the same wavelength and reducing the effect of absorption spectrum when conjugated to biopolymers, modified carbocyanine dyes have peak absorbance. At wavelength, it has higher photostability and higher absorbance (absorbance coefficient) than structurally similar dyes. Therefore, modified carbocyanine dyes result in increased sensitivity of assays using modified dyes and their conjugates. Preferred modified dyes include compounds having at least one substituted indolium ring system, wherein the substituent on the 3-carbon of the indolium ring comprises a chemically reactive group or conjugate material. Other dye compounds include compounds into which an azabenzazolium ring moiety and at least one sulfonic acid moiety have been introduced. Modified carbocyanine dyes that can be used to detect individual particles in various embodiments of the invention are described in US Pat. No. 6,977,305, which is incorporated herein by reference in its entirety. Has been done. Thus, in some embodiments, the labeling of the invention comprises a fluorescent dye comprising a substituted indolium ring system, wherein the substituent on the 3-carbon of the indolium ring contains a chemically reactive or conjugate substance group. Use.

In some embodiments, the label comprises a fluorescent moiety containing one or more Alexa dyes (Molecular Probes, Eugene, OR). Alexa dyes are disclosed in US Pat. Nos. 6977305, 6974874, 6130101, and 6974305, which are incorporated herein by reference in their entirety. Some embodiments of the present invention utilize dyes selected from the group consisting of AlexaFluor 647, AlexaFluor 488, AlexaFluor 532, AlexaFluor 555, AlexaFluor 610, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750. Some embodiments of the present invention utilize dyes selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 700, and AlexaFluor 750. Some embodiments of the present invention utilize AlexaFluor647 molecules with an absorption maximum of about 650-660 nm and an emission maximum of about 660-670 nm. The AlexaFluor 647 dye is used alone or in combination with other AlexaFluor dyes.

Furthermore, currently available organic phosphors can be improved by weakening their hydrophobicity by adding hydrophilic groups such as polyethylene. Alternatively, at present, sulfonated organic phosphors such as AlexaFluor647 dye can weaken their acidity by making them zwitterionic. Particles, such as antibodies labeled with modified phosphors, are less likely to bind non-specifically to surfaces and proteins in immunoassays, thus allowing for more sensitive and lower background assays. .. Methods for modifying and improving the properties of fluorescent dyes for the purpose of increasing the sensitivity of systems that detect single particles are known in the art. Preferably, the modification improves the Stokes shift while maintaining a high quantum yield.

2. Quantum Dots In some embodiments, the fluorescently labeled moiety used to detect molecules in a sample using the analyzer system of the present invention is a quantum dot. Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals in the range of 2-10 nm containing 100 to 1,000 electrons. Some QDs can be 10 to 20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications. QDs are fluorescent groups that fluoresce by the formation of excitons, which are considered to be the excited states of traditional fluorescent groups but can have a much longer lifetime of up to 200 nanoseconds. This property provides QD with low photobleaching. The energy level of the QD can be controlled by varying the size and shape of the QD as well as the depth of the QD potential. One of the optical properties of the small excitatory QD is coloration, which is determined by the size of the dots. The larger the dots, the redder the fluorescence or the closer to the red end of the spectrum. The smaller the dot, the bluer or closer it is to the blue end. The bandgap energy that determines the energy and thus the color of the fluorescence emission is inversely proportional to the square of the magnitude of the QD. Larger QDs have more energy levels with closer spacing, thus allowing the QDs to absorb photons containing less energy, i.e. closer to the red edge of the spectrum. Since the emission frequency of dots is determined by the band gap, it is possible to control the output wavelength of dots with the highest accuracy. In some embodiments, the proteins detected by the single particle analyzer system are labeled with QD. In some embodiments, a single particle analyzer is used to detect a protein labeled with one QD with a filter that allows the detection of different proteins at different wavelengths. The QD has broad excitation and narrow emission properties, which, when used with color filtering, is a single electromagnetic radiation for multiple analysis of multiple targets in a single sample to decompose individual signals. Only the source is needed. Therefore, in some embodiments, the analyzer system comprises one continuous wave laser and particles each labeled with one QD. The colloidally prepared QD floats freely and can be attached to various molecules via a metal coordination bond functional group. These groups include, but are not limited to, thiols, amines, nitriles, phosphines, phosphine oxides, phosphonic acids, carboxylic acids, or other ligands. By attaching suitable molecules to the surface, the quantum dots can be dispersed or dissolved in almost any solvent, or introduced into a variety of inorganic and organic films. Quantum dots (QDs) can bind to streptavidin directly via a maleimide ester binding reaction or to an antibody via a maleimide-thiol binding reaction. As a result, a material having a biomolecule covalently bonded to the surface is produced, and a conjugate having high specific activity is produced. In some embodiments, the protein detected by the single particle analyzer is labeled with a single quantum dot. In some embodiments, the quantum dots are 10 to 20 nm in diameter. In other embodiments, the quantum dots are 2-10 nm in diameter. Useful quantum dots include QD 605, QD 610, QD 655 and QD 705. A particularly preferred quantum dot is QD 605.

C. Fluorescent Part Composition with Binding Partner The label of the invention is a binding partner (eg, an antibody) bound to the fluorescent moiety to provide the fluorescence required for detection and quantification with the instruments described herein. Generally includes. Any suitable combination of binding partner and fluorescent moiety for detection with the single molecule detector described herein can be used as a label in the present invention. In some embodiments, the invention provides a label for a molecule or fragment thereof, complex, phosphorylated, or form of oxidation of myocardial troponin, the label comprising an antibody against myocardial troponin and a fluorescent moiety. The antibody may be an antibody against any antibody as described herein, such as cTnT or cTnl. In some embodiments, the antibody is an antibody against cTnl. In some embodiments, the antibody is specific for a particular region of myocardial troponin, eg, amino acids 27-41 of human cTnl. In some embodiments, the present invention comprises a fluorescent moiety bound to an anti-cTnl antibody, eg, BiosPacific, a polyclonal antibody such as a goat polyclonal antibody obtained from one commercially available from Emeryville called G129C. I will provide a. Fluorescent moieties average at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, when the label is stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety. It can be coupled so that it can emit 400, 500, 600, 700, 800 900, or 1000 photons, and the laser is focused on a spot of about 5 microns or more in diameter containing its label, and the laser. The total energy directed by the spot is less than about 3 microjoules. In some embodiments, the fluorescent moiety is capable of emitting at least about 50, 100, 150, or 200 photons on average when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety. The laser is focused on a spot with a diameter of about 5 microns or more, including the fluorescent portion, and the total energy directed by the laser to the spot is about 3 microjoules or less. The fluorescent moiety may include a substituted indolium ring system, and the substituent on the 3-carbon of the indolium ring may be a fluorescent moiety containing one or more dye molecules having a structure containing a chemically reactive group or a conjugate substance group. The labeling composition can include a fluorescent moiety containing one or more dye molecules selected from the group consisting of AlexaFluor488, 532, 647, 700, or 750. The labeling composition can include a fluorescent moiety containing one or more dye molecules selected from the group consisting of AlexaFluor488, 532, 700, or 750. The labeling composition can include a fluorescent moiety containing one or more dye molecules that are AlexaFluor488. The labeling composition can include a fluorescent moiety containing one or more dye molecules which are AlexaFluor555. The labeling composition can include a fluorescent moiety containing one or more dye molecules that are AlexaFluor610. The labeling composition can include a fluorescent moiety containing one or more dye molecules that are AlexaFluor647. The labeling composition can include a fluorescent moiety containing one or more dye molecules that are AlexaFluor680. The labeling composition can include a fluorescent moiety containing one or more dye molecules that are AlexaFluor 700. The labeling composition can include a fluorescent moiety containing one or more dye molecules that are AlexaFluor750.

In some embodiments, the present invention relates to the AlexFluor molecule, amino acid 2 of human ctnI.
Provided are compositions for the detection of myocardial troponin I, comprising AlexaFluor molecules selected from the description group, such as AlexaFluor647 molecules bound to antibodies such as goat polyclonal anti-cTnl antibodies specific for 7-41. In some embodiments, the composition is bound to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI, averaging 1-11, or about 2-10, or about 2-8. , Or about 2-6, or about 2-5, or about 2-4, or about 3-10, or about 3-8, or about 3-6, or about 3-5, or about 4-10, or It contains about 4-8, or about 4-6, or about 2, 3, 4, 5, 6, or more than about 6 AlexaFluor 647 molecules. In some embodiments, the present invention binds to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI, averaging 1-11, or about 2-10, or about 2-8. , Or about 2-6, or about 2-5, or about 2-4, or about 3-10, or about 3-8, or about 3-6, or about 3-5, or about 4-10, or A composition for the detection of myocardial troponin I comprising from about 4-8, or about 4-6, or about 2, 3, 4, 5, 6, or more than about 6 AlexaFluor647 molecules is provided. In some embodiments, the present invention comprises the detection of myocardial troponin I, comprising an average of about 2-10 AlexaFluor647 molecules bound to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. The composition for is provided. In some embodiments, the present invention is a goat polyclonal anti-cTnl specific for amino acids 27-41 of human ctnI.
Provided is a composition for the detection of myocardial troponin I, which comprises an average of about 2-8 AlexaFluor647 molecules bound to an antibody, such as an antibody. In some embodiments, the present invention comprises the detection of myocardial troponin I comprising an average of about 2-6 AlexaFluor647 molecules bound to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. The composition for is provided. In some embodiments, the present invention comprises the detection of myocardial troponin I, comprising an average of about 2-4 AlexaFluor647 molecules bound to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. The composition for is provided. In some embodiments, the present invention relates to human ctnI amino acids 27-41.
Provided is a composition for the detection of myocardial troponin I, which comprises an average of about 3-8 AlexaFluor647 molecules bound to an antibody such as a goat polyclonal anti-cTnl antibody specific for. In some embodiments, the present invention comprises the detection of myocardial troponin I, comprising an average of about 3-6 AlexaFluor647 molecules bound to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. The composition for is provided. In some embodiments, the present invention comprises the detection of myocardial troponin I, comprising an average of about 4-8 AlexaFluor647 molecules bound to antibodies such as goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. The composition for is provided.

The binding of the binding partner, eg, the fluorescent moiety or the fluorescent entity that forms the fluorescent moiety to the antibody, can be by any suitable means. Such methods are known in the art and exemplary methods are shown in the examples. In some embodiments, filtration is performed after binding of the fluorescent moiety to the binding partner to form the label used in the method of the invention and before utilizing the label to label the protein of interest. It is useful to carry out the process. For example, antibody-dye labels can be filtered prior to use, for example through a 0.2 micron filter or any filter suitable for removing aggregates. Other reagents used in the assays of the invention can also be filtered through, for example, a 0.2 micron filter or any suitable filter. Without being bound by theory, it is believed that such filtration removes, for example, some of the antibody-dye-labeled aggregates. Such aggregates bind as units to the protein of interest, but are prone to degradation when released in elution buffers, which can lead to false positives. That is, some labels are detected in aggregates bound only to the single protein molecule of interest. Regardless of theory, filtration has been found to reduce false positives in subsequent assays and improve accuracy and accuracy.

IV. High-sensitivity analysis of myocardial troponin In one aspect, the present invention comprises the steps of i) labeling a molecule, fragment, or complex, if any, with a label, and ii) detecting the presence or absence of a label. Detection of the presence of the label is to determine the presence or absence of a single molecule, fragment or complex of myocardial troponin in the sample by a step indicating the presence of a single molecule, fragment, or complex of myocardial troponin in the sample. This is the method. As used herein, the term "molecule of myocardial troponin" refers to the entire natural amino acid sequence of a particular type of myocardial troponin, including post-translational modified forms (eg, phosphorylated forms) as well as oxidized or chemically altered forms. Includes molecules that are substantially contained. As used herein, a "fragment" of a molecule includes a molecule of myocardial troponin that is shorter than the entire natural amino acid sequence, including modifications for the entire molecule. A "complex" of myocardial troponin molecules as used herein includes a myocardial troponin molecule or fragment associated with one or more other molecules or substances (eg, associated with one or more other myocardial troponin molecules). included. In some embodiments, the method is about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, ,. It is possible to detect troponin with a detection limit of less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 pg / ml. is there. In some embodiments, the method is capable of detecting troponin with a detection limit of less than about 100 pg / ml. In some embodiments, the method is capable of detecting troponin with a detection limit of less than about 50 pg / ml. In some embodiments, the method is capable of detecting troponin with a detection limit of less than about 20 pg / ml. In some embodiments, the method is capable of detecting troponin with a detection limit below about 10 pg / ml. In some embodiments, the method is capable of detecting troponin with a detection limit below about 5 pg / ml. In some embodiments, the method is capable of detecting troponin with a detection limit below about 3 pg / ml. In some embodiments, the method is capable of detecting troponin with a detection limit below about 1 pg / ml. The detection limit can be determined by using a suitable standard, such as the National Institute of Standards and Technology standard, such as the cTnl standard.

The method also provides a method of determining the concentration of myocardial troponin in a sample by detecting a single molecule of troponin in the sample. "Detection" of a single molecule of troponin involves detecting the molecule directly or indirectly. In the case of indirect detection, a label corresponding to a single molecule of myocardial troponin, for example, a label bound to a single molecule of myocardial troponin can be detected.

The type of myocardial troponin for detection is as described herein, eg, cTnT, cTnl, total myocardial troponin (eg, total cTnl or total cTnT) or a free body, complex, or fragment of myocardial troponin. .. In some embodiments, total myocardial troponin is detected and / or quantified. In some embodiments, total cTnT is detected. In some embodiments, total cTnl is detected and / or quantified.

A. Sample The sample can be any suitable sample. Typically, the sample is a biological sample, such as a biological liquid. Such fluids include, but are not limited to, inhaled breath condensate (EBC), bronchoalveolar lavage fluid (BAL), blood, serum, plasma, etc.
Urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, sheep water, gastric fluid, lymph fluid, interstitial fluid, tissue homogenate, cell extract, saliva, sputum, stool, physiological secretions, tears, mucus, sweat, milk , Mucus, serum, vaginal secretions, fluids obtained from ulcers and other surface rashes, blisters, and abscesses, and tissue extracts containing biopsies of normal, malignant, and suspicious tissue, or target particles of interest. Includes all other components of the body that may contain. Cultures of cells or tissues, or other similar specimens such as culture medium are also included.

In some embodiments, the sample is a blood sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a serum sample.

B. Sample Preparation In general, any sample preparation method can be used that produces a label corresponding to the myocardial troponin for which measurement is desired, where the label is detectable in the instruments described herein. As is known in the art, the preparation of a sample that labels one or more particles can be done in a uniform or non-uniform form. In some embodiments, the sample preparation is formed in a uniform format. In an analyzer system that uses a uniform format, unbound labels are not removed from the sample. See, for example, U.S. Patent Application 11/048660, which is incorporated herein by reference in its entirety. In some embodiments, the particles or particles of interest are labeled by addition to the particles of interest or a labeled antibody or antibody that binds to the particles.

In some embodiments, a heterogeneous assay format is used, in which case a step is typically used to remove the unbound label. Such assay formats are well known in the art. One particularly useful assay format is a sandwich assay, such as a sandwich immunoassay. In this form, the molecule of interest, such as a marker of biological state, is captured on a solid support or the like using a capture binding partner. Unwanted molecules and other substances can then optionally be washed away, followed by binding of a detection binding partner and a label containing a detectable label (eg, fluorescent moiety). Further washing removes the unbound label and then releases, but not necessarily, the detectable label that is usually still bound to the detection binding partner. In an alternative embodiment, the sample and label are added in between (eg, simultaneously) to the capture binding partner without washing. Other variants will be apparent to those skilled in the art.

In some embodiments, the method for detecting troponin particles uses a sandwich assay with an antibody, such as a monoclonal antibody, as a capture binding partner. The method involves binding a troponin molecule in a sample to a capture antibody immobilized on the binding surface and binding a label containing the detection antibody to troponin to form a "sandwich" complex. Labels include detectable fluorescent labels as described herein, for example detected using a single molecule analyzer of the invention. Both the capture antibody and the detection antibody specifically bind to troponin. Many examples of sandwich immunoassays are known, some of which are described in US Pat. No. 4,168,146 of Grubb et al. And US Pat. No. 4,366,241 of Tom et al. (Both incorporated herein by reference). Further examples specific for myocardial troponin are described in the Examples.

The capture binding partner may be bound to a solid support such as a microtiter plate or paramagnetic beads. In some embodiments, the invention provides a binding partner for myocardial troponin, which is bound to paramagnetic beads. Any suitable binding partner specific for the type of myocardial troponin for which capture is desired can be used. The binding partner may be an antibody such as a monoclonal antibody. The antibody is specific for free myocardial troponin (cTnl or cTnT) or complex myocardial troponin, modified myocardial troponin, or fragments of myocardial troponin as described herein, or in a sample of interest. It can be specific for all or substantially all forms of myocardial troponin, such as cTnl or cTnT, that are believed to be found in. The production and sources of antibodies to myocardial troponin are described elsewhere herein. The preferred antibody form for measuring total troponin is that it is substantially unaffected by heparin, phosphorylation, oxidation, and troponin complex formation and does not cross-react with skeletal muscle troponin, such as troponin I. In some embodiments, the antibody is specific for a particular region of myocardial troponin. In some embodiments, the region comprises amino acids 41-49 of human myocardial troponin I. In some embodiments, the region comprises amino acids 87-91 of human myocardial troponin I. Such antibodies are well known in the art and are commercially available, for example, from BiosPacific, Emeryville, Canada. Examples of capture antibodies useful in embodiments of the invention are antibodies that react with cTnl, which forms a complex with free myocardial troponin I (cTnl) amino acids 41-49 and other troponin components, such as monoclonal antibodies. This antibody is preferably unaffected by heparin, phosphorylation, oxidation, and troponin complex formation and does not cross-react with skeletal muscle troponin I. An exemplary antibody of this type is monoclonal antibody clone number A34650228P commercially available from BiosPacific, Emeryville, Canada. Another example of a capture antibody useful in an embodiment of the invention is an antibody that reacts with free myocardial troponin I (cTnl) amino acids 87-91 and cTnl that forms a complex with other troponin components, such as a monoclonal antibody. .. This antibody is preferably unaffected by heparin, phosphorylation, oxidation, and troponin complex formation and does not cross-react with skeletal muscle troponin I. An exemplary antibody of this type is Monoclonal Antibody Clone No. A34440228P commercially available from BiosPacific, Emeryville, Canada. It will be appreciated that an antibody identified herein as useful as a capture antibody can also be useful as a detection antibody and vice versa.

The binding to the solid support of the binding partner such as an antibody may be a covalent bond or a non-covalent bond. In some embodiments, the bond is a non-covalent bond. An example of a non-covalent bond well known in the art is the biotin-avidin / streptavidin interaction. Thus, in some embodiments, solid supports such as microtiter plates or paramagnetic beads bind to capture binding partners such as antibodies by non-covalent binding such as biotin-avidin / streptavidin interactions. There is. In some embodiments, the bond is a covalent bond. Thus, in some embodiments, a solid support such as a microtiter plate or paramagnetic beads is covalently bound to a capture binding partner such as an antibody. Covalent bonds are particularly useful in which the orientation of the capture antibody optimizes the capture of the molecule of interest. For example, in some embodiments, a solid support such as a microtiter plate or paramagnetic microparticles can be used when the binding partner bond, such as an antibody, is a covalent bond or other alignment bond.

An exemplary protocol for orientation binding of the antibody to a solid support is as follows: IgG is dissolved in 0.1 M sodium acetate buffer, pH 5.5 to a final concentration of 1 mg / ml. Add ice-cooled 20 mM sodium periodate in an equal volume of 0.1 M sodium acetate (pH 5.5) solution. Oxidize IgG on ice for 1/2 hour. Excess periodate reagent is quenched by adding 0.15 volumes of 1M glycerol. Low molecular weight oxidation reaction by-products are removed by ultrafiltration. The oxidized IgG fraction is diluted to a suitable concentration (typically 0.5 micrograms per ml of IgG) and reacted with a hydrazide-activated multiwell plate at room temperature for at least 2 hours. Unbound IgG is removed by washing the multiwell plate with borate buffered saline or another suitable buffer. If desired, the plate may be dried for storage. If the material of the microbeads is suitable for such bonding, a similar protocol can be adopted for the microbeads.

In some embodiments, the solid support is a microtiter plate. In some embodiments, the solid support is paramagnetic beads. An exemplary paramagnetic bead is StreptavidinCl (Dynal, 650.01-03). Other suitable beads will be apparent to those skilled in the art. Methods for binding an antibody to paramagnetic beads are well known in the art. An example is shown in the examples.

The subject's myocardial troponin is brought into contact with a capture binding partner, such as a capture antibody immobilized on a solid support. Several sample preparations may be used, such as the preparation of serum from a blood sample or the concentration procedure prior to contacting the sample with a capture antibody. Protocols for binding proteins in immunoassays are well known in the art and are included in the Examples.

The expected time for binding varies depending on the conditions. It will be clear that shorter binding times are desirable in some environments, especially in clinical environments. The use of paramagnetic beads and the like can reduce the time required for bonding. In some embodiments, the expected time for binding of the protein of interest to a capture binding partner, such as an antibody, is less than or about 12, 10, 8, 6, 4, 3, 2, or 1 hour. Shorter than 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 60 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 40 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 30 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 20 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 15 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 10 minutes. In some embodiments, the time expected for binding of the protein of interest to a capture binding partner, such as an antibody, is less than about 5 minutes.

In some embodiments, the troponin particles bind to a capture binding partner, such as a capture antibody, after which the particles that can bind non-specifically and other unwanted substances in the sample are washed away and specifically bound. Virtually leaves only the troponin particles that are present. It will be appreciated that in other embodiments, no wash is used between the addition of the sample and the addition of the label, which further reduces sample preparation time. Thus, in some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is approximately 12, 10, 8, 6, Shorter than 4, 3, 2, or 1 hour, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is less than about 60 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is less than about 40 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is less than about 30 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is less than about 20 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and of the label to binding to the protein of interest is less than about 15 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is less than about 10 minutes. In some embodiments, the expected time for both binding of the protein of interest to a capture binding partner, such as an antibody, and binding of the label to the protein of interest is less than about 5 minutes.

Some immunoassay diagnostic reagents, including capture and signal antibodies used to measure the target analyte, can be derived from animal serum. Endogenous human heterophilic antibodies, or human anti-animal antibodies capable of binding to immunoglobulins of other species, are present in the serum or plasma of more than 10% of patients. These circulating heterophilic antibodies can interfere with immunoassay measurements. In a sandwich immunoassay, these heterophobic antibodies can crosslink capture and detection (diagnostic) antibodies, thereby producing false positive signals, or block the binding of diagnostic antibodies, thereby causing false negative signals. Can be generated. In a competitive immunoassay, the heterophobic antibody can bind to the analytical antibody and block its binding to troponin. They can also block or increase the separation of antibody-troponin complexes from free troponins, especially when using anti-species antibodies in the separation system. Therefore, it is difficult to predict the effects of interfering with these heterophobic antibodies. Therefore, it is advantageous to block the binding of any heterophobic antibody. In some embodiments of the invention, the immunoassay comprises the step of depleting the sample with an atypical antibody blocker using one or more atypical antibody blockers. Methods for removing heterologous antibodies from the samples tested in the immunoassay are known and include heating the sample in sodium acetate buffer (pH 5.0) for 15 minutes at 90 ° C. and centrifuging at 1200 g for 10 minutes. Alternatively, the heterophilic antibody can be precipitated with polyethylene glycol (PEG); including the step of immunoextracting interfering heterophilic immunoglobulin from the specimen using protein A or protein G. Alternatively, the step of adding non-immune mouse IgG is included. Embodiments of the methods of the invention contemplate preparing a sample prior to analysis with a single molecule detector. The appropriateness of the pretreatment method can be determined. Biochemicals for minimizing the interference of immunoassays caused by heterophobic antibodies are commercially available. For example, a product called MAK33, which is an IgG1 monoclonal antibody against h-CK-MM, is available from Boehringer Mannheim. MAK33 Plus products include a combination of IgG1 and IgG1-Fab. polyMAK33 contains IgG1-Fab polymerized with IgG1, and polyMAC2b / 2a contains polyMAC2b / 2a containing IgG2a-Fab polymerized with IgG2b. A second commercially available source of biochemicals for neutralizing heterophobic antibodies is Immunoglobulin Inhibiting Reagent sold by Bioreclamation Inc, East Meadow, NY. This product is a preparation of immunoglobulins (IgG and IgM) obtained from a large number of species, mainly mouse IgG2a, IgG2b, and IgG3 obtained from Balb / c mice (mice). In some embodiments, the heterologous antibody is sampled using methods known in the art, such as depleting the sample from the heterologous antibody by binding an antibody that interferes with protein A or G. Immune extraction can be performed from. In some embodiments, the heterophilic antibody is neutralized with one or more heterophilic antibody inhibitors. The heterophilic inhibitor can be selected from the group consisting of anti-isoform heterophilic antibody inhibitors, anti-idiotype heterophilic antibody inhibitors, and anti-anti-idiotype heterophilic antibody inhibitors. In some embodiments, a combination of heterophobic antibody inhibitors can be used.

Labels are added with or after sample addition and washing. Protocols for binding antibodies and other immune labels to proteins and other molecules are well known in the art. If the label binding step is separated from the capture binding, the time expected for labeling binding can be important, for example in a clinical environment. In some embodiments, the time expected to bind the protein of interest to a label (such as an antibody-dye) is less than or about 12, 10, 8, 6, 4, 3, 2, or 1 hour. Shorter than 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, the time expected to bind the protein of interest to a label (such as an antibody-dye) is less than about 60 minutes. In some embodiments, the time expected for binding of the protein of interest to a label, such as an antibody-dye, is less than about 40 minutes. In some embodiments, the time expected to bind the protein of interest to a label (such as an antibody-dye) is less than about 30 minutes. In some embodiments, the time expected to bind the protein of interest to a label (such as an antibody-dye) is less than about 20 minutes. In some embodiments, the time expected for binding of the protein of interest to a label, such as an antibody-dye, is less than about 15 minutes. In some embodiments, the time expected to bind the protein of interest to a label (such as an antibody-dye) is less than about 10 minutes. In some embodiments, the time expected to bind the protein of interest to a label (such as an antibody-dye) is less than about 5 minutes. Excess labeling is removed by washing.

The label is then eluted from the protein of interest. Preferred elution buffers are effective in releasing the label without creating a significant background. It is also useful if the elution buffer is bacteriostatic. The elution buffer used in the present invention includes chaotropes (eg, urea or guanidium compounds), buffers (eg, bovine buffered saline), protein carriers (eg, for coating the walls of capillary tubes of detection instruments). Albumin such as human albumin, bovine albumin, or fish albumin, or IgG), and surfactants (eg, ionic or nonionic surfactants selected to produce a relatively low background (eg,). , Tween20, TritonX-100, or SDS)).

The elution buffer / labeled aliquot collected in the single molecule detector is referred to as the "processing sample" to distinguish it from the original sample obtained from the individual.

In another embodiment, the solid phase binding assay can use a competitive binding assay format. One such method is: a) a capture antibody immobilized on the binding surface, i) troponin particles in a sample, and ii) a labeled analog of troponin particles containing a detectable label (detection reagent). Competitively combine, and b) measure the amount of label using a single particle analyzer. Another such method is: a) an antibody with a detectable label (detection reagent), i) troponin particles in a sample, and ii) an analog of troponin particles immobilized on a binding surface (capture reagent). ) Competitively, and b) measuring the amount of labeling using a single particle analyzer. As used herein, the term "troponin analog" means a species that competes with troponin for binding to a capture antibody. Examples of competitive immunoassays are disclosed in U.S. Pat. No. 4,235,601 by Deutsch et al., U.S. Pat. No. 4,442,204 by Liotta, and U.S. Pat. No. 5,208535 by Buechler et al., All of which are incorporated herein by reference. There is.

C. Detection of troponin and determination of concentration After elution, the label is passed through a single molecule detector, eg, in elution buffer. The processing sample may be unlabeled, may contain a single label, or may contain multiple labels. The number of labels corresponds to or is proportional to the number of molecules of myocardial troponin captured during the capture step (when using sample dilution or fractionation).

Any suitable single molecule detector capable of detecting the label used with the protein of interest can be used. Suitable single molecule detectors are described herein. Typically, the detector is part of a system that includes an automated sampling device for collecting the prepared sample and a recovery system for optionally collecting the sample.

In some embodiments, a molecular flow system is utilized and the capillary flow cell, a laser for illuminating the interrogation space in the interrogation space through which the processing sample passes, and the radiation emitted from the interrogation space are detected. The processing sample is analyzed with a single molecule analyzer, which includes a detector for the purpose and a driving source for moving the processing sample through the interrogation space. In some embodiments, the single molecule analyzer further comprises a microscope objective that collects the light emitted from the processing sample as it passes through the interrogation space, such as a high numerical aperture microscope objective. In some embodiments, the laser and detector are in a confocal arrangement. In some embodiments, the laser is a continuous wave laser. In some embodiments, the detector is an avalanche photodiode detector. In some embodiments, the driving force is a pump for supplying pressure. In some embodiments, the present invention includes an analyzer that includes a sampling system capable of automatically sampling multiple samples that provide flow communication between the sample container and the interrogation space. Provide a system. In some embodiments, the interrogation space is between about 0.001 and 500 pL, or between about 0.01 pL and 100 pL, or between about 0.01 pL and 10 pL, or about. Between 0.01pL and 5pL, or between about 0.01pL and 0.5pL, or between about 0.02pL and about 300pL, or between about 0.02pL and about 50pL, Or between about 0.02 pL and about 5 pL, or between about 0.02 pL and about 0.5 pL, or between about 0.02 pL and about 2 pL, or about 0.05 pL and about 50 pL. Between, or between about 0.05 pL and about 5 pL, or between about 0.05 pL and about 0.5 pL, or between about 0.05 pL and about 0.2 pL, or about 0 It has a volume between .1 pL and about 25 pL. In some embodiments, the interrogation space has a volume between about 0.004 pL and 100 pL. In some embodiments, the interrogation space has a volume between about 0.02 pL and 50 pL. In some embodiments, the interrogation space has a volume between about 0.001 pL and 10 pL. In some embodiments, the interrogation space has a volume between about 0.001 pL and 10 pL. In some embodiments, the interrogation space has a volume between about 0.01 pL and 5 pL. In some embodiments, the interrogation space has a volume between about 0.02 pL and about 5 pL. In some embodiments, the interrogation space has a volume between about 0.05 pL and 5 pL. In some embodiments, the interrogation space has a volume between about 0.05 pL and 10 pL. In some embodiments, the interrogation space has a volume between about 0.5 pL and about 5 pL. In some embodiments, the interrogation space has a volume between about 0.02 pL and about 0.5 pL.

In some embodiments, the single molecule detector used in the method of the invention utilizes a capillary flow system to illuminate the capillary flow cell, the interrogation space in the capillary through which the processing sample passes. Laser, high aperture microscope objective lens that collects the light emitted from the interrogation space as the processing sample passes through the interrogation space, avalanche photodiode for detecting the radiation emitted from the interrogation space It includes a detector and a pump to provide pressure to move the processing sample through the interrogation space, in which case the interrogation space is between about 0.02 pL and about 50 pL. In some embodiments, the single molecule detector used in the method of the invention utilizes a capillary flow system to irradiate the capillary flow cell, the antenna space in the capillary through which the processing sample passes, a continuous wave. Lasers, high numerical aperture microscope objectives that collect the light emitted from the interrogation space as the processing sample passes through the interrogation space (in this case, the lens has a numerical aperture of at least about 0.8), It includes an avalanche photodiode detector to detect radiation emitted from the interrogation space and a pump to provide pressure to move the processing sample through the interrogation space, in this case interlogate. The gatement space is between about 0.004 pL and about 100 pL. In some embodiments, the single molecule detector used in the method of the invention utilizes a capillary flow system to illuminate the capillary flow cell, the interrogation space in the capillary through which the processing sample passes. Lasers, high numerical aperture microscope objectives that collect the light emitted from the interrogation space as the processing sample passes through the interrogation space (in this case, the lens has a numerical aperture of at least about 0.8), It includes an avalanche photodiode detector to detect radiation emitted from the interrogation space and a pump to provide pressure to move the processing sample through the interrogation space, in this case interlogate. The gatement space is between about 0.05 pL and about 10 pL. In some embodiments, the single molecule detector used in the method of the invention utilizes a capillary flow system and is a continuous wave laser for illuminating the interrogation space in the capillary flow cell, the capillary through which the processing sample passes. A high numerical aperture microscope objective (in this case, the lens has a numerical aperture of at least about 0.8), which collects the light emitted from the interlogeat space as the processing sample passes through the interlogate space. It includes an avalanche photodiode detector for detecting radiation emitted from the logation space and a pump for providing pressure to move the processing sample through the interrogation space, in this case the interrogation space. Is between about 0.05 pL and about 5 pL. In some embodiments, the single molecule detector used in the method of the invention utilizes a capillary flow system and is a continuous wave laser for illuminating the interrogation space in the capillary flow cell, the capillary through which the processing sample passes. A high numerical aperture microscope objective (in this case, the lens has a numerical aperture of at least about 0.8), which collects the light emitted from the interlogeat space as the processing sample passes through the interlogate space. It includes an avalanche photodiode detector for detecting radiation emitted from the logation space and a pump for providing pressure to move the processing sample through the interrogation space, in this case the interrogation space. Is between about 0.5 pL and about 5 pL.

In some embodiments, the single molecule detector is capable of determining the concentration of the molecule of interest in the sample, in which case the range of concentration in the sample is at least about 100-fold, or 1000-fold, Alternatively, it may exceed the range of 10,000 times, 100,000 times, or 30,000 times, or 1,000,000 times, or 1,000,000 times, or 30,000,000 times.

In some embodiments, the methods of the invention are about 50%, 40%, 30%, in concentration of an analyzer between a first sample and a second sample introduced into a detector. Utilizing a single molecule detector capable of detecting a difference of less than 20%, 15%, or 10%, the volume of the first sample and the second sample introduced into the analyzer is about 100. , 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or less than 1 μl, and the analyte is about 100, 90, 80, 70, 60, 50. , 40, 30, 20, 15, 10, 5, 4, 3, 2, or in concentrations less than 1 femtomoll. In some embodiments, the methods of the invention are capable of detecting a difference of less than about 50% in the concentration of the analyte between the first and second samples introduced into the detector. Utilizing a single molecule detector, the volume of the first sample and the second sample introduced into the analyzer is less than about 100 μl and the analyte is present at a concentration of less than about 100 femtomols. In some embodiments, the methods of the invention are capable of detecting a difference of less than about 40% in the concentration of the analyte between the first and second samples introduced into the detector. Utilizing a single molecule detector, the volume of the first sample and the second sample introduced into the analyzer is less than about 50 μl and the analyte is present at a concentration of less than about 50 femtomols. In some embodiments, the method of the invention is capable of detecting a difference of less than about 20% in the concentration of the analyte between the first sample and the second sample introduced into the detector. Utilizing a single molecule detector, the volume of the first sample and the second sample introduced into the analyzer is less than about 20 μl and the analyte is present at a concentration of less than about 20 femtomols. In some embodiments, the method of the invention is capable of detecting a difference of less than about 20% in the concentration of the analyte between the first sample and the second sample introduced into the detector. Utilizing a single molecule detector, the volume of the first sample and the second sample introduced into the analyzer is less than about 10 μl and the analyte is present at a concentration of less than about 10 femtomols. In some embodiments, the method of the invention is capable of detecting a difference of less than about 20% in the concentration of the analyte between the first sample and the second sample introduced into the detector. Utilizing a single molecule detector, the volume of the first sample and the second sample introduced into the analyzer is less than about 5 μl and the analyte is present at a concentration of less than about 5 femtomols.

Single molecule detectors and systems are described in more detail below. Further embodiments of a single molecule analyzer useful in the methods of the invention, such as a detector with an interrogation window greater than one, a detector utilizing electrophoretic or electrophoretic flow, etc. It can be found in US Patent Application 11/048660, the full text of which is incorporated herein by reference.

Equipment may be cleaned during operation. In order to maintain the conditioning of the capillaries, i.e. to keep the surface of the capillaries relatively constant between the samples and reduce variability, some wash buffers that maintain the concentration of salt and surfactant in the samples It can be used in embodiments.

A feature that contributes to the very high sensitivity of the instruments and methods of the invention is a method of detecting and counting labels, which, in some embodiments, binds to or more than a single molecule to be detected. It typically corresponds to a single molecule to be detected. Briefly, a given interrogation space of a capillary is allowed to enjoy EM radiation from a laser that emits light of an excitation wavelength suitable for the fluorescent portion used for labeling for a predetermined period of time, during which time. By detecting the emitted photons, the processing sample flowing through the capillary is effectively separated into a series of detection events. Each predetermined time is a "bin". If the total number of photons detected in a given bin exceeds a predetermined threshold level, a detection event is registered for that bin; that is, a label has been detected. If the total number of photons is not at a predetermined threshold level, no detection event will be registered. In some embodiments, the concentration of the processing sample is sufficiently dilute that a detection event with a large percentage value indicates that the detection event has only one label that has passed through the window. This corresponds to a single target molecule in the original sample, that is, some number of detection events indicates that there are two or more labels in a single bin. In some embodiments, a higher concentration of label in the processing sample, i.e., a label with a concentration at which the probability that more than one label will be detected as a single detection event is no longer important. Further purification is applied to allow accurate detection.

Other bin times can be used without departing from the scope of the invention, but in some embodiments the bin time is selected in the range of about 1 microsecond to about 5 ms. In some embodiments, the bin times are approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, More than 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In some embodiments, the bin time is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200. , 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or less than 5000 microseconds. In some embodiments, the bin time is from about 1 to 1000 microseconds. In some embodiments, the bin time is from about 1 to 750 microseconds. In some embodiments, the bin time is from about 1 to 500 microseconds. In some embodiments, the bin time is from about 1 to 250 microseconds. In some embodiments, the bin time is from about 1 to 100 microseconds. In some embodiments, the bin time is about 1 to 50 microseconds. In some embodiments, the bin time is about 1-40 microseconds. In some embodiments, the bin time is about 1 to 30 microseconds. In some embodiments, the bin time is from about 1 to 500 microseconds. In some embodiments, the bin time is about 1 to 20 microseconds. In some embodiments, the bin time is about 1 to 10 microseconds. In some embodiments, the bin time is from about 1 to 500 microseconds. In some embodiments, the bin time is about 1 to 5 microseconds. In some embodiments, the bin time is about 5 to 500 microseconds. In some embodiments, the bin time is about 5 to 250 microseconds. In some embodiments, the bin time is about 5 to 100 microseconds. In some embodiments, the bin time is about 5 to 50 microseconds. In some embodiments, the bin time is about 5 to 20 microseconds. In some embodiments, the bin time is about 5 to 10 microseconds. In some embodiments, the bin time is about 10 to 500 microseconds. In some embodiments, the bin time is about 10 to 250 microseconds. In some embodiments, the bin time is about 10 to 100 microseconds. In some embodiments, the bin time is about 10 to 50 microseconds. In some embodiments, the bin time is about 10 to 30 microseconds. In some embodiments, the bin time is about 10 to 20 microseconds. In some embodiments, the bin time is about 5 microseconds. In some embodiments, the bin time is about 5 microseconds. In some embodiments, the bin time is about 6 microseconds. In some embodiments, the bin time is about 7 microseconds. In some embodiments, the bin time is about 8 microseconds. In some embodiments, the bin time is about 9 microseconds. In some embodiments, the bin time is about 10 microseconds. In some embodiments, the bin time is about 11 microseconds. In some embodiments, the bin time is about 12 microseconds. In some embodiments, the bin time is about 13 microseconds. In some embodiments, the bin time is about 14 microseconds. In some embodiments, the bin time is about 5 microseconds. In some embodiments, the bin time is about 15 microseconds. In some embodiments, the bin time is about 16 microseconds. In some embodiments, the bin time is about 17 microseconds. In some embodiments, the bin time is about 18 microseconds. In some embodiments, the bin time is about 19 microseconds. In some embodiments, the bin time is about 20 microseconds. In some embodiments, the bin time is about 25 microseconds. In some embodiments, the bin time is about 30 microseconds. In some embodiments, the bin time is about 40 microseconds. In some embodiments, the bin time is about 50 microseconds. In some embodiments, the bin time is about 100 microseconds. In some embodiments, the bin time is about 250 microseconds. In some embodiments, the bin time is about 500 microseconds. In some embodiments, the bin time is about 750 microseconds. In some embodiments, the bin time is about 1000 microseconds.

In some embodiments, the background noise level is determined from the average noise level or from the root mean square of the noise. In other cases, select typical noise or statistical values. In most cases, noise is expected to follow a Poisson distribution. Therefore, in some embodiments, determining the concentration of the particle-labeled complex in the sample comprises determining the background noise level.

Thus, as the label flows through the capillary flow cell, the label is irradiated by a laser beam to produce a burst of photons. The photons emitted by the label are background light or background by taking into account only bursts of photons with energies above a predetermined threshold energy level that cause the amount of background noise present in the sample. Identified from noise emission. Background noise is typically Raman, a low-frequency emission produced by fluorescence inherent in unlabeled particles present in buffers or diluents used to prepare samples, eg, samples for analysis. Includes scattering and electrical noise. In some embodiments, the value assigned to the background noise is calculated as the average of the background signal noise detected in multiple bins, which is an interrogation space over a given length of time. It is a measured value of the photon signal detected in. Therefore, in some embodiments, background noise is calculated for each sample as a sample-specific number.

Given a value for background noise, a threshold energy level can be assigned. As discussed above, a threshold is determined to identify the true signal (due to the fluorescence of the label) from the background noise. Care must be taken in choosing the threshold so that the number of false positive signals from random noise is minimized while the number of true signals rejected is also minimized. Methods for selecting a threshold include determining a fixed value above the noise level and calculating the threshold based on the distribution of the noise signal. In one embodiment, the threshold is set to a fixed number of standard deviations above the background level. Given the Poisson distribution of noise, this method can be used to assess the number of false positive signals over time in an experiment. In some embodiments, the threshold level is calculated as a value of 4 sigma above the background noise. For example, given an average background noise level of 200 photons, the analyzer system will have a threshold level of 4√200, which exceeds the average background / noise level of 200 photons, such as 256 photons. Establish. Therefore, in some embodiments, determining the concentration of the label in the sample comprises establishing a threshold level (above the threshold level, the photon signal indicates the presence of the label). Conversely, a photon signal with an energy level that does not exceed the threshold level energy level indicates unlabeled.

Many bottle measurements are taken to determine the concentration of the sample and each bottle measurement is checked for labeling. Typically, more than 60,000 measurements can be made per minute (eg, in embodiments where the bottle size is 1 ms, the smaller the bottle size, the larger the number of measurements correspondingly, eg, 10 microseconds. 6000000 measurements per minute for bottle size). Therefore, there is no definitive single measurement and the method introduces a high error limit. Bins determined to be unlabeled (“no” bins) are not taken into account, and only measurements made in bins determined to be labeled (“yes” bins) determine the concentration of the label in the processing sample. Take into account when doing so. By not taking into account measurements made in "no" bins or unmarked bins, the signal-to-noise ratio and measurement accuracy are increased. Therefore, in some embodiments, determining the concentration of the label in the sample comprises detecting a bin measurement that reflects the presence of the label.

The signal-to-noise ratio or the sensitivity of the analyzer system can be increased by minimizing the time to detect background noise during bin measurements where the particle-labeled complex is detected. For example, in a bin measurement lasting 1 millisecond in which a particle-labeled complex is detected as it passes through interrogation space within 250 microseconds, 750 microseconds of 1 millisecond emits background noise. Spent to detect. The signal-to-noise ratio can be improved by shortening the bin time. In some embodiments, the bin time is 1 millisecond. In other embodiments, the bin time is 750, 500, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds, or 10 microseconds. Other bin times are as described herein.

Other factors that affect the measurement are the brightness or dimness of the fluorescent area, the flow velocity, and the power of the laser. The various combinations of related factors that enable the detection of the label will be apparent to those skilled in the art. In some embodiments, the bin time is adjusted without changing the flow rate. Anyone skilled in the art will have to increase the laser output directed to the interrogation space to keep the total energy applied to the interrogation space constant during the bin time if the bin time is reduced. Will be understood. For example, if the bin time is reduced from 1000 microseconds to 250 microseconds, the laser output must be increased about four times as the first approximation. With these settings, the same number of photons as the number of photons counted during the 1000 μs given in the previous setting can be detected in 250 μs, allowing the sample to be detected faster with lower background and therefore greater sensitivity. You will be able to analyze. In addition, the flow rate may be adjusted to speed up the processing of the sample. These numbers are merely examples, and one of ordinary skill in the art can appropriately adjust the parameters to achieve the desired result.

In some embodiments, the interrogation space covers the entire cross-section of the stream of sample. If the interrogation space covers the entire cross section of the sample stream, then only the number of labels counted over a set length of time and the volume across the cross section of the sample stream are the labels in the processing sample. Required to calculate the concentration. In some embodiments, the interrogation space can be defined to be smaller than the cross-sectional area of the stream of sample, for example by defining the interrogation space by the size of the spot irradiated by the laser beam. In some embodiments, the interrogation space is adjusted by adjusting the analyzer openings 306 (FIG. 1A) or 358 and 359 (FIG. 1B) to reduce the irradiation volume imaged by the objective lens to the detector. Can be defined. In embodiments where the interrogation space is defined to be less than the cross-sectional area of the stream of samples, the concentration of the label is emitted by the complex from a standard curve generated using one or more samples with known standard concentrations. It can be determined by interrogating the signal. In yet another embodiment, the concentration of the label can be determined by comparing the measured particles to the internal standard of the label. In the embodiment of analyzing a diluted sample, the dilution factor is taken into account in calculating the concentration of the molecule of interest in the starting sample.

As discussed above, if the interrogation space covers the entire cross section of the sample stream, then the markers counted through the cross section of the sample stream for a set length of time (bin). Only the volume of interrogated sample in number and bin is needed to calculate the concentration of the sample. The total number of labels contained in the "yes" bin is determined and associated with the volume of the sample represented by the total number of bins used in the analysis to determine the concentration of the label in the processing sample. Thus, in one embodiment, determining the concentration of labels in a processing sample determines the total number of labels that have detected a "yes" bin, and the total number of labels detected is the total volume of the sample analyzed. Includes associating with. The total volume of the sample analyzed is the volume of the sample that passes through the capillary flow cell and across the interrogation space at specific time intervals. Alternatively, the signal emitted by the label in a large number of bins is interpolated from a standard curve created by determining the signal emitted by the label in the same number of bins with a standard sample containing a known concentration of label in the sample. Determine the concentration of the labeled complex of.

In some embodiments, the number of individual labels detected in the bottle is related to the relative concentration of particles in the processing sample. At relatively low concentrations, such as concentrations less than about ^10-16 M, the number of labels is proportional to the photon signal detected in the bin. Therefore, at low concentrations, the photon signal is given as a digital signal. At relatively high concentrations, such as concentrations above about ^10-16 M, as one becomes critical, two or more labels can be counted across the interrogation space at about the same time, with labels and photon signals. The proportional relationship of is lost. Therefore, in some embodiments, individual particles in a sample at a concentration greater than about ^10-16 M are degraded by reducing the length of the bin measurement time.

Alternatively, in another embodiment, the total photon signal emitted by multiple particles present in any one bin is detected. Depending on these embodiments, the dynamic range of the present invention exceeds at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or 8 logs. Single molecule detectors are acceptable.

As used herein, the term "dynamic range" refers to a range of sample concentrations that can be quantified instrumentally without the need for dilution or other treatment to change the concentration of continuous samples of different concentrations. , In this case, the concentration is determined with the accuracy appropriate for the intended use. For example, the microtiter plate contains a sample with a concentration of 1 femtomoll for the subject assay in the first well, a sample with a concentration of 10000 femtomoll for the analyte in another well, and a third. If the well contains a sample with a concentration of 100 femtomols relative to the analyte of interest, additional treatment to adjust the concentration, such as dilution, is required with a device that has a dynamic range of at least 4 logs and a lower limit of quantification of 1 femtomoll. It is possible to accurately quantify the concentration of all samples. Accuracy can be determined using a series of standard concentrations across the dynamic range and by standard methods such as constructing a standard curve. For example, r ² is about 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0 It can be used as a measure with an accuracy greater than .94, 0.95, 0.96, 0.97, 0.98, or 0.99.

Increased dynamic range is achieved by modifying the mode in which the data from the detector is analyzed and / or by using an attenuator between the detector and the interrogation space. The lower side, where each detection event, the photon of each burst above the threshold level in the bin (“event photons”), probably represents only one label because the processing sample is sufficiently diluted. In the low end range, the data is analyzed to count the detection events as a single molecule. That is, as described above, each bin is simply analyzed as "yes" or "no" for the presence of the label. In more concentrated processing samples, the possibility that two or more labels occupy a single bin becomes significant, and the number of event photons in a significant number of bins is expected for a single label. For example, the number of event photons in a significant number of bins is twice, three times, or more than three times the expected number of event photons for a single label. Equivalent to. For these samples, the instrument changes the method of data analysis to a method of integrating the total number of event photons per bottle of the processing sample. This total will be proportional to the total number of labels in all bins. In even more concentrated processing samples, where many labels are present in most bins, background noise becomes an insignificant portion of all signals obtained from each bin, and the instrument provides a data analysis method per bin. Change to a method (including background) that counts all photons. A further increase in dynamic range is when the intensity of the light reaching the detector exceeds the ability of the detector to count photons accurately, i.e. the concentration is such that the detector is saturated. This can be achieved by using an attenuator between the and the detector.

The instrument can include a data analysis system that receives input from the detector, determines the appropriate analytical method for the sample being performed, and outputs values based on such analysis. The data analysis system can further output instructions to use or not use the attenuator if it is included in the instrument.

By using such a method, the dynamic range of the device can be dramatically increased. Thus, in some embodiments, the device is approximately 1000 (3log), 10000 (4log), 100000 (5log), 350,000 (5.5log), 1000000 (6log), 3500000 (6.5log), 10000000 (7log). ), 35000000 (7.5 log), or 100000000 (8 log) or more, the concentration of the sample can be measured over a dynamic range. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range of over about 100,000 (5 log). In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range of more than about 1,000,000 (6log). In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range of greater than about 10,000,000 (7log). In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range from about 1-10 femtomols to at least about 1000, 10000, 100,000, 350,000, 1000000, 3500000, 10000000, or 35000000 femtomolls. is there. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range from about 1-10 femtomols to at least about 10000 femtomolls. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range from about 1-10 femtomoles to at least about 100,000 femtomols. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range from about 1-10 femtomols to at least about 1,000,000 femtomolls. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range from about 1-10 femtomols to at least about 10,000,000 femtomolls.

In some embodiments, the analyzer or analyzer system of the present invention may detect an analyte, such as a biomarker, at a detection limit of less than 1 nanomolar, or 1 picomole, or 1 femtomole, or 1 attomol, or 1 zeptmol. It is possible. In some embodiments, the analyzer or analyzer system is such that the biological marker is present in the sample at a concentration of less than 1 nanomolar, or 1 picomole, or 1 femtomol, or 1 atmol, or 1 zeptmol, and the size of each sample. Is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1, 0.01, 0.001, or 0.0001 μl, about 0.1, 1, 2 Changes from one sample to another at concentrations of 5, 10, 20, 30, 40, 50, 60, or less than 80% or multiple analyzers (eg, biological markers or multiple biological markers) It is possible to detect. In some embodiments, the analyzer or analyzer system is a first sample of less than about 20% when the analyte is present at a concentration of less than about 1 picomolar and when the size of each sample is less than about 50 μl. It is possible to detect a change in the concentration of the analyte from to the second sample. In some embodiments, the analyzer or analyzer system is a first sample of less than about 20% when the analyte is present at a concentration of less than about 100 femtomols and when the size of each sample is less than about 50 μl. It is possible to detect a change in the concentration of the analyte from to the second sample. In some embodiments, the analyzer or analyzer system is a first sample of less than about 20% when the analyte is present at a concentration of less than about 50 femtomols and when the size of each sample is less than about 50 μl. It is possible to detect a change in the concentration of the analyte from to the second sample. In some embodiments, the analyzer or analyzer system is a first sample of less than about 20% when the analyte is present at a concentration of less than about 5 femtomols and when the size of each sample is less than about 50 μl. It is possible to detect a change in the concentration of the analyte from to the second sample. In some embodiments, the analyzer or analyzer system is a first sample of less than about 20% when the analyte is present at a concentration of less than about 5 femtomols and when the size of each sample is less than about 5 μl. It is possible to detect a change in the concentration of the analyte from to the second sample. In some embodiments, the analyzer or analyzer system is a first sample of less than about 20% when the analyte is present at a concentration of less than about 1 femtomoll and when the size of each sample is less than about 5 μl. It is possible to detect a change in the concentration of the analyte from to the second sample.

V. Instruments and Systems Suitable for Sensitive Analysis of Troponin The method of the present invention utilizes sensitive analytical instruments such as single molecule detectors. Such single molecule detectors include embodiments described below.

Instrument / System In one aspect, the method described herein utilizes an analyzer system capable of detecting a single particle in a sample. In one embodiment, the analyzer system is capable of detecting a single particle of fluorescently labeled particles, and the analyzer system is defined in a capillary flow cell that communicates fluidly with the sampling system of the analyzer system. When a single particle is present in the gation space, it detects the energy emitted by the fluorescent label excited in response to exposure by an electromagnetic source. In a further embodiment of the analyzer system, a power source moves a single particle through the interrogation space of the capillary flow cell. In another embodiment of the analyzer system, an automated sampling system may be included in the analyzer system to introduce the sample into the analyzer system. In another embodiment of the analyzer system, a sample preparation system may be included in the analyzer system to prepare the sample. In a further embodiment, the analyzer system may include a recovery system to recover at least a portion of the sample after the analysis is complete.

In one aspect, the analyzer system consists of an electromagnetic irradiation source for exciting single particles labeled with a fluorescent label. In one embodiment, the electromagnetic irradiation source of the analyzer system is a laser. In a further embodiment, the electromagnetic irradiation source is a continuous wave laser.

In a typical embodiment, the electromagnetic radiation source excites a fluorescent moiety bound to the label as the label as it passes through the interrogation space of the capillary flow cell. In some embodiments, the fluorescently labeled moiety comprises one or more fluorescent dye molecules. In some embodiments, the fluorescently labeled portion is a quantum dot. Any fluorescent moiety described herein can be used in the label.

The label is exposed to electromagnetic radiation as it passes through the interrogation space localized within the capillary flow cell. The interrogation space typically has fluid communication with the sampling system. In some embodiments, the marker passes through the interrogation space of the capillary flow cell by a power source for advancing the marker through the analyzer system. The interrogation space is located to receive the electromagnetic irradiation emitted from the irradiation source. In some embodiments, the sampling system is an automated sampling system that allows sampling of multiple samples without the intervention of a human operator.

The label passes through the interrogation space and emits a detectable amount of energy when excited by an electromagnetic radiation source. In one embodiment, the electromagnetic irradiation detector is operably connected to the interrogation space. The electromagnetic irradiation detector is capable of detecting the energy emitted by a label, eg, the fluorescent portion of the label.

In a further embodiment of the analyzer system, the system further comprises a sample preparation mechanism in which the sample can be partially or completely prepared for analysis by the analyzer system. In some embodiments of the analyzer system, the sample is discarded after being analyzed by the system. In other embodiments, the analyzer system further comprises a sample recovery mechanism in which at least a portion, or substantially all, or substantially all of the sample can be recovered after analysis by a sample recovery mechanism. In one such embodiment, the sample can be returned to the original sample. In some embodiments, the sample can be returned to the microtiter well on the sample microtiter plate. The analyzer system typically further comprises a data acquisition system for collecting and reporting the detected signals.

B. Single Particle Analyzer As shown in FIG. 1A, an embodiment of the analyzer system 300 is described herein. The analyzer system 300 includes an electromagnetic radiation source 301, a mirror 302, a lens 303, a capillary flow cell 313, a microscope objective lens 305, an opening 306, a detector lens 307, a detector filter 308, a single photon detector 309, and a detector. Includes a processor 310 operably connected.

During operation, the electromagnetic radiation source 301 is aligned so that its output 311 reflects the front surface 312 of the mirror 302. The lens 303 focuses the light beam 311 on a single interrogation space in the capillary flow cell 313 (an exemplary example of the interrogation space 314 is shown in FIG. 2A). The microscope objective lens 305 collects light from the particles of the sample and forms an image of the light beam on the opening 306. The opening 306 affects the fractionation of the light emitted by the specimen in the interrogation space of the capillary flow cell 313 that can be collected. The detector lens 307 collects the light that passes through the opening 306 and focuses the light over the effective region of the detector 309 after the light has passed through the detector filter 308. The detector filter 308 minimizes anomalous noise signals due to light scattering or ambient light, while maximizing the signal emitted by the excited fluorescent moieties bound to the particles. Processor 310 processes and processes optical signals from particles according to the methods described herein.

In one embodiment, the microscope objective lens 305 is a high numerical aperture microscope objective lens. As used herein, a "high numerical aperture lens" includes a lens having a numerical aperture of 0.6 or greater. Numerical aperture is a measure of the number of highly diffracted image-forming rays captured by an objective lens. The larger numerical aperture allows more and more oblique light to enter the objective lens, thereby producing a more highly resolved image. In addition, larger numerical apertures increase the brightness of the image. High numerical aperture lenses are commercially available from various vendors, and any lens with a numerical aperture of about 0.6 or greater can be used in an analyzer system. In some embodiments, the lens has a numerical aperture of about 0.6 to about 1.3. In some embodiments, the lens has a numerical aperture of about 0.6 to about 1.0. In some embodiments, the lens has a numerical aperture of about 0.7 to about 1.2. In some embodiments, the lens has a numerical aperture of about 0.7 to about 1.0. In some embodiments, the lens has a numerical aperture of about 0.7 to about 0.9. In some embodiments, the lens has a numerical aperture of about 0.8 to about 1.3. In some embodiments, the lens has a numerical aperture of about 0.8 to about 1.2. In some embodiments, the lens has a numerical aperture of about 0.8 to about 1.0. In some embodiments, the lens has a numerical aperture of at least about 0.6. In some embodiments, the lens has a numerical aperture of at least about 0.7. In some embodiments, the lens has a numerical aperture of at least about 0.8. In some embodiments, the lens has a numerical aperture of at least about 0.9. In some embodiments, the lens has a numerical aperture of at least about 1.0. In some embodiments, the aperture of the microscope objective lens 305 is about 1.25. In one embodiment using a 0.8 microscope objective lens 305, a Nikon 60X / 0.8 NA Achromat lens (Nikon, Inc., USA) can be used.

In some embodiments, the electromagnetic radiation source 301 is a laser that emits light in the visible spectrum. In all embodiments, the electromagnetic radiation source is set so that the laser wavelength is set to a wavelength sufficient to excite the fluorescent label attached to the particles. In some embodiments, the laser is a continuous wave laser with a wavelength of 639 nm. In another embodiment, the laser is a continuous wave laser with a wavelength of 532 nm. In another embodiment, the laser is a continuous wave laser with a wavelength of 422 nm. In another embodiment, the laser is a continuous wave laser with a wavelength of 405 nm. Any continuous wave laser having a wavelength suitable for exciting the fluorescent moiety as used in the methods and compositions of the present invention can be used without departing from the scope of the present invention.

In the single particle analyzer system 300, the particles enter an excited state as each particle passes through the light beam 311 of the electromagnetic radiation source. A detectable burst of light is emitted as the particle relaxes from its excited state. The excitation-emission cycle is repeated many times by each particle for the length of time it takes for each particle to pass through the light beam, and the analyzer system 300 determines each particle as it passes through the interrogation space 314. It will be possible to detect tens of thousands of photons. The photons emitted by the fluorescent particles are recorded by the detector 309 (FIG. 1A) with a time delay indicating the time it takes for the particle labeled complex to traverse the interrogation space. Photon intensities are recorded by detector 309 and sampling times are divided into bins, which are uniform and arbitrary time segments with freely selectable time channel widths. Evaluate the number of signals contained in each bin. One or a combination of statistical analysis methods is used to determine when the particles are present. Such methods include determining the baseline noise of the analyzer system and setting the signal intensity of the fluorescent label to a statistical level above the baseline noise to remove false positive signals from the detector. ..

The electromagnetic radiation source 301 is focused on the capillary flow cell 313 of the analyzer system 300, and the capillary flow cell 313 is fluidly connected to the sample system. The interrogation space 314 is shown in FIG. 2A. The light beam 311 from the continuous wave electromagnetic radiation source 301 of FIG. 1A is optically focused at a specific depth in the capillary flow cell 313. The light beam 311 is directed at the capillary flow cell 313 filled with the sample at an angle perpendicular to the capillary flow cell 313. Ray 311 is manipulated at a predetermined wavelength selected to excite a particular fluorescent label used to label the particles of interest. The size or volume of the interrogation space 314 is determined by the diameter of the rays 311 and the depth at which the rays 311 are focused. Alternatively, the interrogation space can be determined by performing a calibration sample of known concentration through an analyzer system.

When detecting a single molecule at sample concentration, it sets the ray size and focal depth required for single molecule detection, thereby defining the size of the interrogation space 314. The interrogation space 314 is set at a suitable sample concentration so that only one particle is present in the interrogation space 314 during each time interval during which observations are made. It will be appreciated that the detection interrogation volume defined by the rays is not a perfect spherical shape, but is typically of the "bow-tie" type. However, for the purposes of definition, the "volume" of the interaction space is defined herein as the volume surrounded by a sphere with a diameter equal to the diameter of the focused spot of the ray. The focusing spots of the rays 311 can have various diameters without departing from the scope of the present invention. In some embodiments, the diameter of the focused spot of the ray is from about 1 to about 5, 10, 15, or 20 microns, or from about 5 to about 10, 15, or 20 microns, or about 10 to about 20 microns. Or about 10 to about 15 microns. In some embodiments, the diameters of the focused spots of the rays are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, It is 18, 19, or 20 microns. In some embodiments, the diameter of the ray focusing spot is about 5 microns. In some embodiments, the diameter of the ray focusing spot is about 10 microns. In some embodiments, the diameter of the ray focusing spot is about 12 microns. In some embodiments, the diameter of the ray focusing spot is about 13 microns. In some embodiments, the diameter of the ray focusing spot is about 14 microns. In some embodiments, the diameter of the ray focusing spot is about 15 microns. In some embodiments, the diameter of the ray focusing spot is about 16 microns. In some embodiments, the diameter of the ray focusing spot is about 17 microns. In some embodiments, the diameter of the ray focusing spot is about 18 microns. In some embodiments, the diameter of the ray focusing spot is about 19 microns. In some embodiments, the diameter of the ray focusing spot is about 20 microns.

In an alternative embodiment of the single particle analyzer system, two or more sources of electromagnetic radiation can be used to excite particles labeled with fluorescent labels of different wavelengths. In another alternative embodiment, only one interrogation space can be used within the capillary flow cell. In another alternative embodiment, multiple detectors can be used to detect different emission wavelengths from the fluorescent label. An explanatory diagram incorporated into each of these alternative embodiments of the analyzer system is shown in FIG. 1B. These embodiments are incorporated by reference from prior US patent application 11/0468660.

In some embodiments of the analyzer system 300, a driving force is required to move the particles through the capillary flow cell 313 of the analyzer system 300. In one embodiment, the driving force can be in the form of pressure. The pressure used to move the particles through the capillary flow cell can be created by the pump. In some embodiments, Scivex, Inc. An HPLC pump can be used. In some embodiments where a pump is used as the driving force, the sample can pass through the capillary flow cell at a rate of 1 μL / min to about 20 μL / min, or about 5 μL / min to about 20 μL / min. In some embodiments, the sample can pass through the capillary flow cell at a rate of about 5 μL / min. In some embodiments, the sample can pass through the capillary flow cell at a rate of about 10 μL / min. In some embodiments, the sample can pass through the capillary flow cell at a rate of about 15 μL / min. In some embodiments, the sample can pass through the capillary flow cell at a rate of about 20 μL / min. In some embodiments, electrokinetic forces can be used to move the particles through the analyzer system. Such methods have been previously disclosed and are incorporated by reference from prior US patent application 11/048660.

In one aspect of the analyzer system 300, the detector 309 of the analyzer system detects photons emitted by the fluorescent label. In one embodiment, the photon detector is a photodiode. In a further embodiment, the detector is an avalanche photodiode detector. In some embodiments, the photodiode can be a silicon photodiode with wavelength detection of 190 nm and 1100 nm. When a germanium photodiode is used, the wavelength of the detected light is between 400 nm and 1700 nm. In another embodiment, when an indium gallium arsenide photodiode is used, the wavelength of light detected by the photodiode is between 800 nm and 2600 nm. When a lead sulfide photodiode is used as a detector, the wavelength of the detected light is between 1000 nm and 3500 nm.

In some embodiments, the optics of the electromagnetic radiation source 301 and the optics of the detector 309 are arranged in a conventional optical arrangement. In such an arrangement, the electromagnetic radiation source and detector are aligned on different focal planes. The arrangement of laser and detector optics in the analyzer system shown in FIGS. 1A and 1B is a conventional optical arrangement.

In some embodiments, the optics of the electromagnetic radiation source and the optics of the detector are arranged in a confocal optical arrangement. In such an arrangement, the electromagnetic radiation source 301 and the detector 309 are aligned on the same focal plane. The confocal arrangement makes the analyzer more robust because the electromagnetic radiation source 301 and the detector optics 309 do not need to be rearranged when the analyzer system is moved. This arrangement also makes the analyzer easier to use because it eliminates the need to rearrange the components of the analyzer system. The confocal arrangements of the analyzer 300 (FIG. 1A) and the analyzer 355 (FIG. 1B) are shown in FIGS. 3A and 3B, respectively. FIG. 3A shows that the light rays 311 from the electromagnetic radiation source 301 are focused by the microscope objective lens 315 to form one interrogation space 314 (FIG. 2A) in the capillary flow cell 313. A dichroic mirror 316 that reflects the laser light but allows the fluorescent light to pass through is used to separate the fluorescent light from the laser light. A filter 317 located in front of the detector removes any non-fluorescent light in the detector. In some embodiments, the analyzer system configured in a confocal arrangement can include two or more interrogation spaces. Such methods have been previously disclosed and are incorporated by reference from prior US patent application 11/048660.

The laser can be a tunable dye laser such as a helium-neon laser. The laser can be set to emit a wavelength of 632.8 nm. Alternatively, the wavelength of the laser can be set to emit a wavelength of 543.5 nm or 1523 nm. Alternatively, the electromagnetic laser can be an argon ion laser. In such an embodiment, the argon ion laser can be operated as a continuous gas laser at about 25 different wavelengths in the visible spectrum, although the wavelength is set between 408.9 and 686.1 nm. Its optimum performance is set between 488 and 514.5 nm.

1. 1. In some embodiments of the electromagnetic radiation source analyzer system, chemiluminescent labels can be used. In such an embodiment, it is not always necessary to utilize an EM radiation source for detecting the particles. In another embodiment, the extrinsic or intrinsic property of the particle is a light interaction label or property, such as a fluorescent label or a light scattering label. In one such embodiment, an EM radiation source is used to irradiate the label and / or particles. An EM radiation source for exciting the fluorescent label is preferred.

In some embodiments, the analyzer system comprises an electromagnetic radiation source 301. Any number of radiation sources can be used in any one analyzer system 300 without departing from the scope of the present invention. Numerous electromagnetic radiation sources have been previously disclosed and are incorporated by reference from prior US patent application 11/048660. In some embodiments, all continuous wave electromagnetic (EM) radiation sources emit electromagnetic radiation of the same wavelength. In other embodiments, different sources emit EM radiation of different wavelengths.

In one embodiment, the EM radiation sources 301, 351, 352 are continuous wave lasers that generate wavelengths between 200 nm and 1000 nm. Such an EM radiation source has the advantages of being small, durable, and relatively inexpensive. Moreover, in general, they are capable of producing larger fluorescent signals than other light sources. Specific examples of suitable continuous wave EM radiation sources include, but are not limited to, argon, krypton, helium-neon, helium-cadmium type lasers, and tunable diode lasers (red to infrared region). Each has the potential to double the frequency. Lasers provide continuous illumination without the need for ancillary electronic or mechanical devices to block the irradiation, such as shutters. In one embodiment using a continuous wave laser, a 3 mW electromagnetic radiation source can be of sufficient energy to excite the fluorescent label. The diameter of the light beam from a continuous wave laser with such an energy output can be between 2 and 5 μm. The exposure time of the particles to the laser beam for exposure to 3 mW can be a time of about 1 msec. In an alternative embodiment, the exposure time to the laser beam can be about 500 μsec or less. In one alternative embodiment, the exposure time can be about 100 μsec or less. In one alternative embodiment, the exposure time can be about 50 μsec or less. In one alternative embodiment, the exposure time can be about 10 μsec or less.

LEDs are another low cost and reliable source of irradiation. Recent advances in bright LEDs and dyes with high absorbency cross sections and high quantum yields support the applicability of LEDs to single particle detection. Such lasers can be used alone or in combination with other light sources such as mercury lamps, basic arc lamps, halogen lamps, arc discharges, plasma discharges, light emitting diodes, or combinations thereof.

In other embodiments, the EM radiation source can be in the form of a pulse wave laser. In such embodiments, the pulse size of the laser is an important factor. In such embodiments, the size, focus spot, and total energy emitted by the laser are important and must be sufficient to excite the fluorescent label. When using a pulsed laser, longer duration pulses may be required. In some embodiments, a 2 nanosecond laser pulse can be used. In some embodiments, a laser pulse of 5 nanoseconds can be used. In some embodiments, pulses between 2 and 5 nanoseconds can be used.

Optimal laser intensity is the characteristic of photobleaching of a single dye and the length of time required to traverse the interrogation space (particle velocity, distance between interrogation spaces when used multiple times, and Depends on (including the size of the interrogation space). For maximum signal, it is desirable to irradiate the sample with the highest intensity that does not result in photobleaching of high percent dyes. The preferred intensity is such that the amount of dye that fades by the time the particles cross the interrogation space is 5% or less.

The power of the laser is set according to the type of dye molecule that needs to be stimulated, the length of time the dye molecule is stimulated, and / or the speed at which the dye molecule passes through the capillary flow cell. Laser power is defined as the rate at which energy is delivered by a ray and is measured in joule-seconds or watts. It will be appreciated that the higher the power of the laser, the shorter the time it takes for the laser to irradiate the particles, while providing a certain amount of energy to the interrogation space as the particles pass through the space. Therefore, in some embodiments, the combination of laser output and irradiation time receives approximately 0.1, 0.5, 1, 2, 3, 4, 5 total energy received by the interrogation space during the irradiation time. , 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 microjoules. In some embodiments, the combination of laser output and irradiation time receives approximately 0.5, 1, 2, 3, 4, 5, 6, 7, total energy received by the interrogation space during the irradiation time. It is less than 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 0.1 and 100 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 1 and 100 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 1 and 50 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 2 and 50 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 3 and 60 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 3 and 50 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 3 and 40 microjoules. In some embodiments, the combination of laser power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is between about 3 and 30 microjoules. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space of about 1 microjoule during the irradiation time. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space of about 3 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 5 microjoules during the irradiation time. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space of about 10 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 15 microjoules during the irradiation time. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space of about 20 microjoules during the irradiation time. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space of about 30 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 40 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 50 microjoules during the irradiation time. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space of about 60 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 70 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 80 microjoules during the irradiation time. In some embodiments, the combination of laser power and irradiation time results in a total energy received by the interrogation space of about 90 microjoules during the irradiation time. In some embodiments, the combination of laser output and irradiation time results in a total energy received by the interrogation space during the irradiation time of approximately 100 microjoules.

In some embodiments, the laser output is at least about 1mW, 2mW, 3mW, 4mW, 5mW, 6mW, 7mW, 8mW, 9mW, 10mW, 13mW, 15mW, 20mW, 25mW, 30mW, 40mW, 50mW, 60mW, 70mW. , 80mW, 90mW, 100mW, or more than 100mW. In some embodiments, the laser power is set to at least about 1 mW. In some embodiments, the laser power is set to at least about 3 mW. In some embodiments, the laser power is set to at least about 5 mW. In some embodiments, the laser power is set to at least about 10 mW. In some embodiments, the laser power is set to at least about 20 mW. In some embodiments, the laser power is set to at least about 30 mW. In some embodiments, the laser power is set to at least about 40 mW. In some embodiments, the laser power is set to at least about 50 mW. In some embodiments, the laser output is set to at least about 60 mW. In some embodiments, the laser power is set to at least about 90 mW.

The time that the laser irradiates the interrogation space is about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 150. , 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 microseconds or higher. The time that the laser irradiates the interrogation space is about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 150, 300. , 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 microseconds or less. The time that the laser irradiates the interrogation space can be set between about 1 and 1000 microseconds. The time that the laser irradiates the interrogation space can be set between about 5 and 500 microseconds. The time that the laser irradiates the interrogation space can be set between about 5 and 100 microseconds. The time that the laser irradiates the interrogation space can be set between about 10 and 100 microseconds. The time that the laser irradiates the interrogation space can be set between about 10 and 50 microseconds. The time that the laser irradiates the interrogation space can be set between about 10 and 20 microseconds. The time that the laser irradiates the interrogation space can be set between about 5 and 50 microseconds. The time that the laser irradiates the interrogation space can be set between about 1 microsecond and 100 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 1 microsecond. In some embodiments, the time the laser irradiates the interrogation space is about 5 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 10 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 25 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 50 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 100 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 250 microseconds. In some embodiments, the time it takes for the laser to irradiate the interrogation space is about 500 microseconds. In some embodiments, the time the laser irradiates the interrogation space is about 1000 microseconds.

For example, the time the laser irradiates the interrogation space is 1 millisecond, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds with a laser that provides an output of 3 mW, 4 mW, 5 mW, or more than 5 mW. , Or can be set to 10 microseconds. In some embodiments, the label is irradiated with a laser that provides an output of 3 mW and the label is irradiated for about 1000 microseconds. In another embodiment, a laser that provides an output of about 20 mW or less irradiates the label for less than 1000 milliseconds. In another embodiment, the label is irradiated with a laser output of 20 mW for about 250 microseconds or less. In some embodiments, a laser output of about 5 mW irradiates the label for about 1000 microseconds or less.

2. Capillary flow cell The capillary flow cell is fluidly connected to the sample system. In one embodiment, the interrogation space 314 of the analyzer system is determined by the cross-sectional area of the corresponding ray 311 and the segment of ray in the field of view of the detector 309. In one embodiment of the analyzer system, the interrogation space 314 is between about 0.01 and 500 pL, or between about 0.01 pL and 100 pL, or between about 0.01 pL and 10 pL, or about 0. Between 0.01pL and 1pL, or between about 0.01pL and 0.5pL, or between about 0.02pL and about 300pL, or between about 0.02pL and about 50pL, or about 0.02pL. Between about 5 pL, or between about 0.02 pL and about 0.5 pL, or between about 0.02 pL and about 2 pL, or between about 0.05 pL and about 50 pL, or about 0.05 pL. Between about 5 pL, or between about 0.05 pL and about 0.5 pL, or between about 0.05 pL and about 0.2 pL, or between about 0.1 pL and about 25 pL. Has the volume defined in. In some embodiments, the interrogation space has a volume between about 0.01 pL and 10 pL. In some embodiments, the interrogation space 314 has a volume between about 0.01 pL and 1 pL. In some embodiments, the interrogation space 314 has a volume between about 0.02 pL and about 5 pL. In some embodiments, the interrogation space 314 has a volume between about 0.02 pL and about 0.5 pL. In some embodiments, the interrogation space 314 has a volume between about 0.05 pL and about 0.2 pL. In some embodiments, the interrogation space 314 has a volume of about 0.1 pL. The volume of other useful interrogation space is as described herein. Those skilled in the art should understand that the interrogation space 314 can be selected for the maximum performance of the analyzer. While very small interrogation spaces have been shown to minimize background noise, large interrogation spaces have the advantage of being able to analyze low concentration samples in a reasonable amount of time. In embodiments that use the two interrogation spaces 370 and 371, volumes such as those described herein for a single interrogation space 314 can be used.

In one embodiment of the invention, the interrogation space is large enough to detect particles with concentrations ranging from about 1000 femtomol (fM) to about 1 zeptmol (zM). In one embodiment of the invention, the interrogation space is large enough to detect particles with concentrations ranging from about 1000 fM to about 1 atom (aM). In one embodiment of the invention, the interrogation space is large enough to detect particles with concentrations ranging from about 10 fM to about 1 atom (aM). In many cases, the large interrogation space allows the detection of particles with concentrations below about 1 fM without additional pre-concentrators or techniques. Those skilled in the art will appreciate that the size of the most suitable intervention space depends on the brightness of the particles to be detected, the level of background signal, and the concentration of the sample to be analyzed.

The size of the interrogation space 314 can be limited by adjusting the optics of the analyzer. In one embodiment, the diameter of the light beam 311 can be adjusted to change the volume of the interrogation space 314. In another embodiment, the field of view of the detector 309 can be changed. Therefore, the radiation source 301 and the detector 309 can be adjusted so that a single particle is irradiated and detected within the interrogation space 314. In another embodiment, the width of the opening 306 (FIG. 1A) that determines the field of view of the detector 309 can be varied. This configuration allows the interrogation space to be altered in near real time to fill more or less concentrated samples, ensuring the probability of two or more particles being present in the interrogation space at the same time. make low. Similar changes may be made to the two or more interrogation spaces 370 and 371.

In another embodiment, the interrogation space can be defined by using a calibration sample of known concentration that passes through the capillary flow cell before testing the actual sample. If only one single particle is detected in the calibration sample at one time as the sample passes through the capillary flow cell, then the depth of focus and the diameter of the rays of the electromagnetic radiation source will determine the interrogation space in the capillary flow cell. The size is determined.

Physical constraints on the interrogation space can also be provided by solid walls. In one embodiment, the wall is one or more walls of the flow cell 313 (FIG. 2A) if the sample liquid is contained within the capillary. In one embodiment, the cell is made of glass, but other substances that are transparent to light in the range of about 200 to about 1000 nm and above, such as quartz, fused polystyrene, and Teflon®. ), Nylon, plastics (eg, polyvinyl chloride, polystyrene, and polyethylene) or any combination thereof may be used without departing from the scope of the invention. Other cross-sectional shapes (eg, quadrilateral, columnar) can be used to deviate from the scope of the invention, but in one embodiment the capillary flow cell 313 has a square cross-section. In another embodiment, the interrogation space may be at least partially defined by channels (not shown) etched into the chip (not shown). Similar considerations apply to embodiments where two interrogation spaces are used (370 and 371 in FIG. 2B).

The interrogation space is submerged in liquid. In one embodiment, the liquid is aqueous. In other embodiments, the liquid is non-aqueous or a combination of aqueous and non-aqueous liquids. In addition, the liquid may contain a pH regulator, an ionic composition, or a sieving agent such as soluble macroparticles or polymers or gels. It is contemplated that valves or other devices may be present between the interrogation spaces to temporarily interrupt the liquid connection. Temporarily interrupted interrogation space is considered to be connected by a liquid.

In another embodiment of the invention, the interrogation space resides in a flow cell 313, also called a sheath flow, which is constrained by the size of the laminar flow of the sample material within the diluent volume. Is a single interrogation space. In these and other embodiments, the interrogation space can be defined by the sheath flow alone or in combination with the dimensions of the source or the field of view of the detector. Sheath flow can be shaped in many ways, including: the sample material is the inner material of the concentric laminar flow, the outer is the diluent volume; the diluent volume is one side of the sample volume. The diluent volume is on two sides of the sample material; the diluent volume is on multiple sides of the sample material but does not completely surround the sample material; the diluent volume completely surrounds the sample material; dilution The liquid volume completely surrounds the sample material concentrically; the sample material is the internal material of a series of discontinuous droplets, and the diluent volume completely surrounds each droplet of the sample material.

In some embodiments, the single molecule detector of the present invention comprises only one interrogation space. In some embodiments, a large number of interrogation spaces are used. Numerous interrogation spaces have been previously disclosed and are incorporated by reference from US Patent Application 11/048660. Those skilled in the art will appreciate that in some cases the analyzer will include two, three, four, five, six, or more than six distinct interrogation spaces.

3. 3. In one embodiment of the motive force analyzer system, the particles move through the interrogation space by motive force. In some embodiments, the driving force for moving the particles is pressure. In some embodiments, pressure is supplied by a pump, pneumatic source, vacuum source, centrifuge, or a combination thereof. In some embodiments, the driving force for moving the particles is an electrokinetic force. The use of electrokinetic forces as a driving force has been previously disclosed in prior applications and is incorporated by reference from US patent application 11/048660.

In one embodiment, pressure can be used as the driving force for moving particles through the interrogation space of the capillary flow cell. In a further embodiment, a pump supplies pressure to move the sample. Suitable pumps are known in the art. In one embodiment, SCIVAX, Inc. Pumps manufactured for HPLC applications, such as those manufactured by, can be used as the driving force. In other embodiments, when the pump is used on a smaller volume of sample, a pump manufactured for the application of microfluidics can be used. These pumps are described in US Pat. Nos. 5094594, 5730187, 6033628, and 65333553, which disclose a device capable of using the pumps for liquid volumes in the nanoliter or picolitre range. ing. It is preferred that all material in the pump that comes into contact with the sample is made of a highly inert material such as polyetheretherketone (PEEK), fused quartz, or sapphire.

A driving force is required to move the sample through the capillary flow cell and push the sample through the interrogation space for analysis. A driving force is still required to push the flushing sample through the capillary flow cell after the sample has passed. When collecting a sample, a driving force is still required to push the sample back into the sample collection container. Standard pumps come in a variety of sizes and the appropriate size can be selected to meet the expected sample size and flow requirements. In some embodiments, separate pumps are used for sample analysis and system flushing. The volume of the analytical pump is about 0.000001 mL to about 10 mL, or about 0.001 mL to about 1 mL, or about 0.01 mL to about 0.2 mL, or about 0.005, 0.01, 0.05, 0. .1 or 0.5 mL. The flush pump may have a larger capacity than the analytical pump. The volume of the flash pump is about 0.01 mL to about 20 mL, or about 0.1 mL to about 10 mL, or about 0.1 mL to about 2 mL, or about 0.05, 0.1, 0.5, 1, 5, Or it may be 10 mL. The size of these pumps is only an example, and for those skilled in the art, the size of the pump is the application, the size of the sample, the viscosity of the liquid to which the pump is used, the tubing dimensions, the flow rate, the temperature, and the art You will understand that the choice can be made according to other factors well known in the field. In some embodiments, the pump of the system is driven by a stepper motor, which is very easy to control with a microprocessor.

In a preferred embodiment, the flush pump and the analytical pump are continuously used while controlling the flow direction with a special check valve. The piping is designed so that the sample does not reach the pump itself when the analytical pump sucks up the maximum amount of sample. This is achieved by selecting the ID and length of the tube between the analytical pump and the analytical capillary so that the volume of the tube exceeds the stroke volume of the analytical pump.

4. Detector One embodiment detects light emitted by a fluorescent label after exposure to electromagnetic radiation (eg, light in the ultraviolet, visible, or infrared range). The detector 309 (FIG. 1A) or multiple detectors (364, 365, FIG. 1B) captures the amplitude and duration of the photon burst from the fluorescent portion, and further signals the amplitude and duration of the photon burst. It is possible to convert to. Images can be generated with continuous signals using detection devices such as CCD cameras, video input module cameras, and streak cameras. In another embodiment, devices such as bolometers, photodiodes, photodiode arrays, avalanche photodiodes, and photomultiplier tubes that generate continuous signals can be used. Any combination of the detectors described above can also be used. In one embodiment, an avalanche photodiode is used to detect photons.

Use specific optical optics between the interrogation space 314 (FIG. 2A) and its corresponding detector 309 (FIG. 1A) to determine the emission wavelength, emission intensity, burst size, burst duration, and fluorescence polarization. Several distinct features of the emitted electromagnetic radiation, including, can be detected. In some embodiments, the detector 309 is a photodiode used in reverse bias. Photodiodes set in reverse bias usually have very high resistance. This resistance is reduced when light of the appropriate frequency illuminates the P / N junction. Therefore, a reverse bias diode can be used as a detector by monitoring the current flowing through it. Circuits based on this effect are more sensitive to light than circuits based on zero bias.

In one embodiment of the analyzer system, the photodiode can be an avalanche photodiode, which can be operated with a much higher inverse bias than conventional photodiodes, thus each photogeneration carrier is avalanche. The effective responsiveness (sensitivity) of the device is increased by increasing it by breakdown to bring an internal gain in the photodiode. The choice of photodiode is determined by the energy or wavelength emitted by the fluorescently labeled particles. In some embodiments, the photodiode is a silicon photodiode that detects energy in the range 190 to 1100 nm, and in another embodiment the photodiode is a germanium photo that detects energy in the range 800 to 1700 nm. A diode, in another embodiment the photodiode is an indium gallium arsenic photodiode that detects energy in the range of 800-2600 nm, and in yet another embodiment the photodiode is between less than 1000 nm and 3500 nm. It is a lead sulfide photodiode that detects energy in the range of. In some embodiments, an avalanche photodiode is a single photon detector designed to detect energy in the wavelength range of 400 nm to 1100 nm. Single photon detectors are commercially available (eg, Perkin Elmer, Wellesley, MA).

In some embodiments, the detector is an avalanche photodiode detector that detects energies between 300 nm and 1700 nm. In one embodiment, a silicon avalanche photodiode can be used to detect wavelengths between 300 nm and 1100 nm. Wavelengths between 900 nm and 1700 nm can be detected using indium gallium arsenide photodiodes. In some embodiments, the analyzer system can include at least one detector; in other embodiments, the analyzer system can include at least two detectors, each detector having a particular wavelength. It can be selected and configured to detect a range of light energies. For example, two separate detectors can be used to detect particles labeled with different labels that emit photons with different spectral energies when excited by an EM radiation source. In one embodiment, the analyzer system is a first detector capable of detecting fluorescence energies in the 450-700 nm range, such as those emitted by a green dye (eg Alexa 546), and a near infrared dye (eg Alexa 647). ) Can include a second detector capable of detecting fluorescence energies in the range of 620-780 nm, such as those emitted by. Detectors for detecting fluorescence energies in the 400-600 nm range, such as those emitted by blue dyes (eg, Hoechst33342), and those in the range of 560-700 nm, such as those emitted by red dyes (Alexa546 and Cy3). A detector for detecting energy can also be used.

A system containing two or more detectors can be used to detect individual particles each labeled with two or more labels that emit light in different spectra. For example, two different detectors can detect antibodies labeled with two different dye labels. Alternatively, an analyzer system containing two detectors can be used to detect different types of particles, each of which is labeled with a different dye molecule or a mixture of two or more dye molecules. For example, two different detectors can be used to detect two different types of antibodies that recognize two different proteins, each type with a different dye label or a mixture of two or more dye labeled molecules. Label. By varying the ratio of two or more dye-labeled molecules, two or more different particle types can be individually detected using two detectors. It is understood that three or more detectors can be used without departing from the scope of the present invention.

One of ordinary skill in the art can arrange and configure one or more detectors in each interrogation space in which one or more interrogation spaces are defined in the flow cell, and each detector is described above. It should be understood that any of the listed electromagnetic radiation characteristics can be arranged and configured to detect. For example, the use of multiple detectors for multiple interrogation spaces has been previously disclosed in previous applications and is incorporated herein by reference from US Patent Application 11/048660. Once the particle is labeled to make it detectable (or if the particle has unique characteristics that make the particle detectable), any suitable detection mechanism known in the art (eg, for example). CCD cameras, video input module cameras, streak cameras, borometers, photodiodes, photodiode arrays, avalanche photodiodes, and photomultiplier tubes that generate continuous signals, and combinations thereof) deviate from the scope of the present invention. It can be used without any problems. Different characteristics of electromagnetic radiation can be detected, including radiation wavelength, radiation intensity, burst size, burst duration, fluorescent polarization, and any combination thereof.

C. Sampling System In a further embodiment, the analyzer system can include a sampling system for preparing a sample for introduction into the analyzer system. The included sampling system is capable of automatically sampling multiple samples and providing fluid communication between the sample container and the first interrogation space.

In some embodiments, the analyzer system of the present invention includes a sampling system for introducing an aliquot of a sample into a single particle analyzer for analysis. Any mechanism capable of introducing the sample can be used. The sample can be sucked up using a pump, or the pressure exerted on the sample that pushes the liquid into the tube, or any vacuum suction created by any other mechanism that acts to introduce the sample into the sampling tube. In general, but not necessarily, the sampling system introduces a sample of known sample volume into a single particle analyzer, and in some embodiments where the presence or absence of one or more particles is detected. It is not important to know the exact sample size. In a preferred embodiment, the sampling system provides automated sampling for a single sample or for multiple samples. In embodiments where a known volume of sample is introduced into the system, the sampling system is approximately 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 20, 30, 40, 50. , 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500, or more than 2000 μl. In some embodiments, the sampling system is about 2000, 1000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.1. , 0.01, or less than 0.001 μl of sample for analysis. In some embodiments, the sampling system is between about 0.01 and 1500 μl, or between about 0.1 and 1000 μl, or between about 1 and 500 μl, or between about 1 and 100 μl. , Or between about 1 and 50 μl, or between about 1 and 20 μl. In some embodiments, the sampling system provides an analytical sample between about 5 μl and 200 μl, or between about 5 μl and about 100 μl, or between about 5 μl and 50 μl. In some embodiments, the sampling system provides an analytical sample between about 10 μl and 200 μl, or between about 10 μl and 100 μl, or between about 10 μl and 50 μl. In some embodiments, the sampling system provides an analytical sample between about 0.5 μl and about 50 μl.

In some embodiments, the sampling system provides a sample size that may vary between samples. In these embodiments, the sample size may be any one of the sample sizes described herein and may vary from sample to sample or set of samples if desired.

The accuracy of the sample volume and the precision of the intersample volume of the sampling system are needed for analysis for the time being. In some embodiments, the precision of the sampling volume is determined by the pump used, typically about 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0. Represented by a CV of less than 5, 0.1, 0.05, or 0.01%. In some embodiments, the precision between the samples of the sampling system is about 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, Or represented by a CV of less than 0.01%. In some embodiments, the accuracy within the assay of the sampling system is represented by a CV of less than about 10, 5, 1, 0.5, or 0.1%. In some embodiments, the precision within the assay of the sampling system exhibits a CV of less than about 5%. In some embodiments, the accuracy between assays of the sampling system is represented by a CV of about 10, 5, or less than 1%. In some embodiments, the interassay precision of the sampling system exhibits a CV of less than about 5%.

In some embodiments, the sampling system is advantageous in that it results in low sample carryover and does not require additional cleaning steps between the samples. Therefore, in some embodiments, the carryover of the sample is about 1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or It is less than 0.001%. In some embodiments, the carryover of the sample is less than about 0.02%. In some embodiments, the carryover of the sample is less than about 0.01%.

In some embodiments, the sampling device provides a sample loop. In these embodiments, a large number of samples are continuously drawn into the tube, each separated from the others by a buffer "plug". Samples are typically read one after another without flushing in between. Flushing is done once at the end of the loop. In embodiments where a buffer "plug" is used, the plug can drain and retrieve the buffer plug into separate wells of the microtiter plate.

The sampling system may be adapted for use with standard assay equipment such as 96-well microtiter plates, preferably 384-well plates. In some embodiments, the system provides a 96-well plate positioner and a mechanism for immersing the sample tube in and out of the well, eg, a mechanism for providing movement along the X, Y, and Z axes. Including. In some embodiments, the sampling system provides a large number of sampling tubes from which the sample can be stored and removed when the test is initiated. In some embodiments, all samples from multiple tubes are analyzed with a single detector. In other embodiments, a large number of single molecule detectors may be connected to the sample tube. The sample can be prepared by a step involving the operation on the sample in the well of the plate prior to sampling by the sampling system, or the sample can be prepared in the analyzer system, or in some combination of both. Can be prepared.

D. Sample Preparation System Sample preparation involves steps that require the preparation of crude samples for analysis. These steps are, by way of example, separation steps such as centrifugation, filtration, distillation, chromatography; concentration, cell lysis, pH modification, addition of buffer, addition of diluent, addition of reagents, heating or cooling, labeling. Any other necessary to prepare the sample for addition of, labeling binding, cross-linking by irradiation, separation of unbound labeling, inactivation and / or removal of interfering compounds, and analysis by a single particle analyzer. It can involve one or more steps of steps. In some embodiments, blood is processed to separate plasma or serum. Further labeling, removal of unbound labels, and / or dilution steps can also be performed on serum or plasma samples.

In some embodiments, the analyzer system comprises a sample preparation system that performs some or all of the processes necessary to provide a sample ready for analysis by a single particle analyzer. The system can perform any or all of the steps listed above to prepare the sample. In some embodiments, the sample is partially processed by the sample preparation system of the analyzer system. Therefore, in some embodiments, the sample may first be partially processed outside the analyzer system. For example, the sample may be centrifuged first. The sample may then be partially processed in the analyzer by the sample preparation system. Processing within the analyzer includes labeling the sample, mixing the sample with the buffer, and other processing processes known to those of skill in the art. In some embodiments, the blood sample is processed outside the analyzer system to provide a serum or plasma sample, which is introduced into the analyzer system and further processed by the sample preparation system to produce the particles or plurality of objects of interest. Label the particles and optionally remove the unbound label. In other embodiments, sample preparation can include immunomodpletion of the sample to remove non-target particles or to remove particles that can interfere with the analysis of the sample. In yet another embodiment, the sample can be depleted of particles that can interfere with the analysis of the sample. For example, sample preparation can include depletion of heterologous antibodies known to interfere with immunoassays using non-human antibodies to detect particles of interest directly or indirectly. .. Similarly, other proteins that interfere with the measurement of the particles of interest can be removed from the sample using antibodies that recognize the interfering proteins.

In some embodiments, the sample can be subjected to solid phase extraction prior to the assay and analysis. For example, a serum sample assayed for cAMP can first be subjected to solid phase extraction using the c18 column to which it binds. Rinse other proteins such as proteases, lipases, and phosphatases from the column and elute cAMP, essentially eliminating proteins that can degrade cAMP or interfere with cAMP measurements. Solid-phase extraction can be used to remove the basic matrix of the sample, which can reduce the sensitivity of the assay. In yet another embodiment, the particles of interest present in the sample can be concentrated by drying or lyophilizing the sample and solubilizing the particles to a volume smaller than the volume of the original sample. For example, a sample of inspiratory concentrate (EBC) can be dried and resuspended in a small volume of a suitable buffer solution to enhance the detection of particles of interest.

In some embodiments, the analyzer system is a blood sample, saliva sample, urine sample, cerebrospinal fluid sample, lymph sample, BAL sample, inspiratory concentrate (EBC), biopsy sample, forensic sample, bioterrorism sample, etc. Provided is a sample preparation system that provides a complete preparation of a sample analyzed by the system, such as a complete preparation of. In some embodiments, the analyzer system provides a sample preparation system that provides some or all of the sample preparation. In some embodiments, the first sample is a blood sample that is further processed by an analyzer system. In some embodiments, the sample is a serum or plasma sample that is further processed by an analyzer system. Serum or plasma samples can be further processed, for example by contact with a particle of interest or a label that binds to multiple particles, and then the sample is used with or without the unbound label removed. Can be done.

In some embodiments, sample preparation is performed on one or more microtiter plates, such as 96-well plates, either outside the analysis system or in the sample preparation components of the analysis system. As is well known in the art, leather bars such as reagents and buffers can provide intermittent fluid communication with the wells of the plate by means of a tube or other suitable structure. Samples can be prepared separately in 96-well plates or tubes. The steps of sample isolation, labeling binding and, if necessary, labeling separation can be performed on a single plate. In some embodiments, the prepared particles are then released from the plate and transferred to a tube for sampling in a sample analysis system. In some embodiments, all steps of sample preparation are performed on one plate and the analytical system obtains the sample directly from the plate. Although this embodiment has been described for a 96-well plate, it will be appreciated that any vessel suitable for preparing the sample for containing one or more samples can be used. For example, a 384-well or 1536-well standard microtiter plate can be used. More generally, in some embodiments, the sample preparation system is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 1000, 5000. , Or more than 10,000 samples can be retained and prepared. In some embodiments, a large number of samples can be taken for analysis on a large number of analyzer systems. Therefore, in some embodiments, two samples, or more than about 2, 3, 4, 5, 7, 10, 15, 20, 50, or 100 or more samples are taken from the sample preparation system and a large number of sample analyzers. Perform in parallel on the system.

Microfluidic systems are used as sample preparation systems that are part of an analyzer system for sample preparation, especially for samples that are suspected of containing particles that are suspected of containing sufficiently high concentrations that detection requires a small amount of sample. You may use it. The principles and techniques of microfluidic manipulation are known in the art. U.S. Pat. Nos. 4979824, 57770029, 5755942, 5746901, 5681751, 5658413, 5565939, 55653859, 5645702, 5605662, 5571410, 5543838, No. 5480614, No. 5716825, No. 5603351, No. 5858195, No. 5863801, No. 5955028, No. 5989402, No. 6041515, No. 6071478, No. 6355420, No. 6495104, No. 6386219, No. 6606609 No. 6802342, No. 6479734, No. 6623613, No. 6554744, No. 6361671, No. 6143152, No. 6132580, No. 5274240, No. 6689323, No. 6783992, No. 65337437, No. 6599436, See No. 68116668 and published PCT patent application WO9955461 (A1). Samples can be prepared continuously or in parallel for use in a single or multiple analyzer system.

The sample preferably contains a buffer solution. The buffer may be mixed with the sample outside the analyzer system or provided by a sample preparation mechanism. Any suitable buffer can be used, but the preferred buffer has a low background of fluorescence, is inert to detectable labeled particles, can maintain the pH of use, and is the driving force. In the electrodynamic embodiment, the ionic strength is suitable for electrophoresis. The buffer concentration can be any suitable concentration, such as in the range of about 1 to about 200 mM. Any buffer system can be used as long as it imparts solubility, function, and detectability of the molecule of interest. For applications using pumping, phosphate, glycine, acetate, citrate, acidulate, carbonate / hydrogen carbonate, imidazole, triethanolamine, glycine, amide, borate, MES , Bis-Tris, ADA, axes, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trisma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, It is preferred to select the buffer from the group consisting of HEPBS, TAPS, APPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS. The buffer solution is Gly-Gly, bicine, tricine, 2-morpholine ethanesulfonic acid (
You can also choose from the group consisting of MES), 4-morpholine propanesulfonic acid (MOPS), and 2-amino-2-methyl-1-propanol hydrochloride (AMP). A useful buffer is 2 mM Tris / boric acid with pH 8.1, but Tris / glycine and Tris / HCl are also acceptable. Other buffers are as described herein.

Buffers useful for electrophoresis have been disclosed in previous applications and are incorporated herein by reference from US patent application 11/048660.

E. Sample Recovery One highly useful feature of the analyzer and analytical system embodiments of the present invention is that the sample can be analyzed without consumption. This can be especially important when the sample material is limited. By collecting the sample, it is also possible to perform another analysis or reanalyze it. The advantage of this feature for applications where sample size is limited and / or where it is desirable to be able to reanalyze the sample, such as for court use, drug screening, and clinical diagnostic applications, is to those skilled in the art it is obvious.

Therefore, in some embodiments, the analyzer system of the present invention further provides a sample recovery system for recovering a sample after analysis. In these embodiments, the system includes a mechanism and method of drawing a sample into an analyzer, analyzing it, and then returning it to a sample holder, such as a sample tube, by, for example, the same route. The sample remains uncontaminated because it is not destroyed and does not enter either the valve or any other tube. Moreover, all materials in the sample pathway are highly inert, such as PEEK, fused quartz, or sapphire, so there is little contamination from the sample pathway. By using a stepper motor control pump (particularly an analytical pump), the volume drawn up and the volume drawn back can be precisely controlled. This allows the sample to be completely or almost completely recovered with a small amount of dilution, even if diluted with flash buffer. Thus, in some embodiments, about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99. More than 0.9% of samples are collected after analysis. In some embodiments, the recovered sample is undiluted. In some embodiments, the recovered sample is about 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, 1.05 times, 1.01 times, 1 Diluted less than .005 times or 1.001 times.

Any mechanism for transporting the liquid sample from the sample vessel to the analyzer can be used to collect and / or collect the sample. In some embodiments, the inlet end of the analytical capillary can be immersed in a sample vessel (eg, a test tube or sample well) or held over a waste vessel (eg, a short length tube). It is connected to PEEK tube). When flushing to cleanly remove the previous sample from the device, place this tube over a waste container to receive the flushed waste. If the sample is drawn in, place the tube in the sample well or test tube. Typically, the sample is pulled in quickly and then slowly extruded while observing the particles in the sample. Alternatively, in some embodiments, the sample may be slowly withdrawn during at least part of the withdrawal cycle and the sample may be analyzed during the slow withdrawal. After this, the sample can be quickly returned and flushed quickly. In some embodiments, the sample can be analyzed in both inward (pull-in) and outward (pull-out) cycles, which improves counting of statistics, such as small diluted samples, and confirmation of results. To. If it is desirable to store the sample, the sample can be pushed back into the same original sample well or another. If you do not want to store the sample, place the tube on the waste container.

VI. Method Using High Sensitivity Analysis of Myocardial Troponin The method of the present invention allows measurement of myocardial troponin levels at concentrations much lower than previously measured concentrations. Myocardial troponin is an accepted marker for myocardial injury, but its usefulness is due to the fact that current analytical methods can only detect after significant damage to the myocardium due to the insensitivity of current methods. Gender is limited. The European Association of Cardiology / American Cardiology Commission for Redefining Myocardial Infarction has described increased levels of myocardial troponin as a measure above the extremely low threshold of 99th percentile distribution of myocardial troponin levels in the reference group. Recommended to define. The total imprecision (CV) at this <10% determination limit is recommended. However,
The inaccuracy of the analysis obtained by the currently available immunoassays for myocardial troponin is not uniform, mainly in the low concentration range. In addition, currently available assays lack sufficient sensitivity to detect troponin levels in nonclinical (normal) subjects, and troponin levels defined in the true baseline or normal population are not defined. The analyzer system of the present invention has been shown to be able to consistently detect cTnI levels at concentrations below 10 pg / ml with a total accuracy of less than 10% (see Examples). Therefore, the present invention provides a diagnostic, prognostic, or therapeutic method based on sensitive detection of myocardial troponin in an individual.

In some embodiments, the invention is i) a step of determining the myocardial troponin concentration in a sample obtained from an individual or in a series of samples from an individual, wherein the sample contains. With the step of determining the concentration in a myocardial troponin assay where the detection limit of myocardial troponin is less than about 50, 40, 30, 10, 5, 4, 3, 2, or 1 pg / ml, for example less than about 20 pg / ml; ii ) A method for determining a diagnosis, prognosis, or treatment method in an individual by the step of determining a diagnosis, prognosis, or treatment method in the individual based on a concentration in a sample or a concentration in a series of samples. I will provide a. Methods of determining the concentration of myocardial troponin include any suitable method having the required sensitivity, such as the methods described herein. In some embodiments, the method utilizes a method of determining the concentration of myocardial troponin in a sample, including the detection of a single molecule or complex or fragment of troponin.

In some embodiments, a sample (eg, blood sample, serum sample, or plasma sample) obtained from an apparently healthy population is analyzed for myocardial troponin (eg, myocardial troponin I), and 80%, 90 of the population. The threshold concentration of troponin is determined by determining the level at which%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% is below that level (concentration). .. This value is a threshold. In some embodiments, the threshold is set at the 99th percentile. In some embodiments, the analysis is performed using a method having a detection level of myocardial troponin of less than about 50, 20, 10, 5, or 1 pg / ml, such as less than about 5 pg / ml.

In some embodiments, the present invention is a step of comparing the concentration value of myocardial troponin in a sample obtained from an individual with a normal value of myocardial troponin or a range of normal values, and detection of myocardial troponin in the sample. The step of determining normal or range of normal values in a myocardial troponin assay where the limits are less than about 50, 40, 30, 10, 5, 4, 3, 2 or 1 pg / ml, eg less than about 20 pg / ml, and ii. ) Provided is a method of determining a diagnosis, prognosis, or treatment method in an individual by a step of determining a diagnosis, prognosis, or treatment method in the individual based on comparison.

In some embodiments, the myocardial troponin is myocardial troponin I or myocardial troponin T. In some embodiments, the myocardial troponin is myocardial troponin T. In some embodiments, the myocardial troponin is myocardial troponin I. In this method, total troponins, such as total cTnI, or cTnT, or total cTnI + cTnT described herein can be used in diagnosis, prognosis, or determination of treatment method. In some embodiments, the method can use concentrations of frees, complexes, or fragments of myocardial troponin or comparisons (eg, ratios) thereof to determine diagnostic, prognostic, or therapeutic methods. ..

A. Sample The sample or series of samples may be any suitable sample; in some embodiments, the sample is blood, serum, or plasma. In some embodiments, the sample or series of samples is a serum sample. The individual may be an animal, such as a mammal, such as a human.

A single sample may be taken, or a series of samples may be taken. When a series of samples are taken, they can be taken at any suitable interval, eg, at intervals of minutes, hours, days, weeks, months, or years. In an acute clinical environment, a series of samples are typically taken over multiple hours and multiple days, at most a few hours apart. For longer follow-up of individuals, the sample interval can be multiple months or multiple years. Diagnosis, prognosis, or treatment method can be determined from a single sample or from one or more series of samples, or from changes in a series of samples, eg, a constant rate of concentration increase is a serious condition. On the other hand, a slower rate of increase or non-increase can indicate a relatively benign or less severe condition. The rate of change can be measured over multiple hours, multiple days, multiple weeks, multiple months, or multiple years. The rate of change of a given individual can in some cases be relative rather than absolute. In an acute environment, a very rapid rate of change, such as a "spike," can indicate an imminent, ongoing, or recent cardiac event. In other environments, multi-day, multi-week, multi-month, or multi-year elevations in individuals may cause ongoing and exacerbating cardiac damage, such as cardiac conditions (eg, cardiac hypertrophy or congestive heart failure). ) May indicate cardiac injury or non-cardiac pathology (eg, toxicity from drug exposure).

In some embodiments, at least one sample is taken during or after the cardiac stress test. For example, one sample is taken before the stress test and one or more samples are taken during the test. Deviations in myocardial troponin levels between samples taken before the test and those taken during the test can provide diagnostic or prognostic information, such as coronary artery disease or other diseases associated with the myocardium. The possibility can be shown. Other comparisons can also be made, such as comparing any sample to normal or threshold levels or determining the rate of change in myocardial troponin concentration in the sample, all of which are described herein as well as cardiac and cardiovascular health. It can provide useful information about the other pathologies described.

In some embodiments, at least one sample is taken when the individual presents or is close to one or more symptoms indicating a condition that may be associated with heart damage to the healthcare professional. The environment in which an individual can come to a healthcare professional is not limited, but in an emergency such as emergency, emergency management, clinical management, centralized management, monitoring unit, hospitalization, outpatient department, doctor's office, clinic, ambulance, etc. Corresponding environment and health screening environment are included. In some embodiments, it is taken from one or more sample w individuals and assayed for myocardial troponin locally, i.e. in or near the sampled environment. For example, an individual who comes to the hospital can have one or more samples assayed for myocardial troponin in the hospital. For example, an individual who comes to the hospital can have one or more samples to be assayed for myocardial troponin in the hospital. In some embodiments, one or more samples are taken from an individual and assayed for myocardial troponin in the CLIA laboratory. In some embodiments, the individual exhibits one or more symptoms consistent with acute coronary syndrome. In some embodiments, the individual exhibits one or more symptoms that match the AMI. Such symptoms include, but are not limited to, chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath.

B. Diagnosis, Prognosis Diagnosis, or Determination of Treatment Method In some embodiments, step ii) compares the concentration or series of concentrations with a normal value of the concentration, and predetermines the concentration or series of concentrations. It includes comparing with a threshold level, comparing the concentration or series of concentrations with baseline values, or determining the rate of change of concentration for the series of concentrations.

In some embodiments, step ii) compares the troponin concentration in the sample to a predetermined threshold concentration, and if the sample concentration is above a threshold level, diagnosis, prognostic diagnosis, or treatment. Including determining the method. The threshold concentration can be determined, for example, by determining the 99th percentile concentration of troponin in the population and setting the threshold concentration at the 99th percentile concentration. An example of this is provided in the Examples.

Normal values, thresholds, rates of change, ratios of values, and other useful diagnostic and prognostic indicators can be established by methods well known in the art. For example, these values compare samples obtained from the case population and the control population, where the case population indicates the biological condition for which a diagnosis, prognosis, or treatment method is desired, and the control population does not exhibit the biological condition. Can be determined by doing. In some embodiments, long-term testing can be performed. For example, the case population may be a subset of the control population that exhibits biological status over time. It will be appreciated that data from multiple studies can be used to determine the normal level or prognosis or the agreed value or range of values for the diagnostic level.

In developing a diagnostic or prognostic test, data on one or more possible markers can be obtained from the study group. The target group is divided into at least two sets, and it is preferable that the first set and the second set each have approximately the same number of subjects. The first set includes subjects who have a disease or, more generally, have been identified as having a first condition. For example, this first patient set may be a patient who has recently developed a disease or a patient who has a particular type of disease, such as AMI. Confirmation of pathology can be performed by more rigorous and / or costly tests such as MRI or CT. Hereinafter, the subject of this first set will be referred to as a "sick person". The objects in the second set are simply objects that do not fall into the first set. The subject of this second set may be a "non-diseased person" who is a normal subject. Alternatively, the subject of this second set can be selected to exhibit the symptoms exhibited by the "disease" subject or one symptom or group of symptoms similar to those symptoms. As yet another alternative, this second set may present them at a time different from the onset of the disease. It is preferred that the same marker set data be available to each patient. This marker set may include all candidate markers that may be suspected to be associated with the detection of a particular disease or condition. No actual known relevance is required. The compositions, methods, and embodiments of the system described herein can be used to determine which of the candidate markers is most relevant to the diagnosis of a disease or condition. The levels of each marker in the two sets of subjects can be widely distributed, for example as a Gaussian distribution. However, no distribution matching is required.

1. 1. Acute Myocardial Infarction The methods of the invention are particularly useful for diagnosis, prognosis, and / or treatment choice in patients with suspected acute myocardial infarction (AMI). Single or continuous myocardial troponin measurements in patients with suspected AMI provide increased prognostic information indicating appropriate and early interventions to improve prognosis and minimize the risk of adverse consequences.

Accordingly, the present invention assay samples obtained from individuals, such as blood samples, plasma samples, and / or serum samples, for myocardial troponin, such as cTnI, at about 50, 40, 30, 20, 15, 10, 9, ,. Myocardial troponin concentration in the sample at a detection limit of less than 8, 7, 6, 5, 4, 3, 2, or 1 pg / ml, for example less than about 20 pg / ml (in this case, the myocardial troponin concentration in the sample indicates AMI). Predicting) provides a method of diagnosing, predicting, and / or preventing or treating AMI in an individual. Myocardial troponin may be cTnI or cTnT, and may be total troponin or an indicator of a particular form (eg, free form, complex, or fragment); some embodiments. In, ratios of one or more forms of troponin are used, as described herein. In some embodiments, total cTnI in the sample or in a series of samples is measured. In some embodiments, the total cTnT in the sample or in a series of samples is measured. In some embodiments, total cTnI + cTnT in the sample or in a series of samples is measured. In some embodiments, myocardial troponin levels are determined at or near when the individual presents a AMI-indicating symptom to a healthcare professional. Such symptoms include, but are not limited to, chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath.

In some embodiments, a series of measurements are made and spikes in myocardial troponin concentration in the sample provide, predict, or provide a basis for the prognosis of AMI. In some embodiments, spikes greater than 50%, greater than 100%, greater than 150%, greater than 200%, greater than 250%, greater than 300%, greater than 400%, or greater than 500% of the baseline relate to the prognosis of AMI. The basis is shown, predicted, or provided. In some embodiments, when obtained, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 in a single sample. , Or myocardial troponin levels above 50 pg / ml, provide, predict, or provide a basis for the prognosis of AMI. In some embodiments, about 1-10, or about 5-15, or about 10-50, about 10-200, about 10-100, or about 10-40, or about 15-50, or about 15-. Myocardial troponin levels of 40, or about 20-200, about 20-150, about 20-100, about 20-50, or about 20-40, or about 20-30 pg / ml provide the basis for the prognosis of AMI. , Predicted, or provided.

In some embodiments, diagnosis or prognostic diagnosis involves stratification of individuals based on myocardial troponin concentration in a sample or series of samples. Such stratification includes the concentration of myocardial troponin in a single sample, the presence and / or magnitude of spikes from baseline in a series of samples, the proportion of different forms of myocardial troponin, and the presence of different forms of myocardial troponin. Absolute value, rate of change in concentration of myocardial troponin in a series of samples, or rate of change in concentration of one or more forms of myocardial troponin, change in proportion of different forms of myocardial troponin over time in a series of samples, sample or It can be based on any other information that is at least partially based on myocardial troponin concentration in a series of samples. Stratification can be based on values obtained from the normal and diseased populations described herein. Appropriate treatment can also be determined based on individual stratification.

In some embodiments, the concentration of myocardial troponin is determined in combination with one or more other markers, such as myocardial ischemia, myocardial infarction, or stroke, and the concentration of each marker is diagnostic. It is considered in prognostic diagnosis or in determining treatment methods. Other clinical indicators, such as EKG, symptoms, history, etc., will usually be taken into account, as will be apparent to those of skill in the art. Appropriate algorithms for diagnosis, prognosis, or treatment can be constructed based on the combination of such markers and clinical indicators combined with troponin levels.

Markers useful for combination with myocardial troponin in the methods of the invention are, but are not limited to, creatine kinase (CK) and its myocardial fraction CK myocardial band (MB), aspartate aminotransferase, lactate dehydrogenase (LDH). ), Α-Hydroxybutyrate dehydrogenase, myoglobin, glutamate oxaloacetate transaminase, glycogen phosphorylase BB, unbound free fatty acid, cardiac fatty acid binding protein (H-FABP), ischemic modified albumin, myosin light chain 1, myosin light chain 2. included. Markers of inflammation and plaque instability useful in combination with myocardial troponin in the methods of the invention are, but are not limited to, C-reactive proteins, white blood cell counts, soluble CD40 ligands, myeloperoxidase, monocyte chemotactic proteins. 1. Whole blood cholinergic and pregnancy-related blood protein A are included. Other markers of inflammation can also be detected and include IL-8, IL-1β, IL6, IL10, TNF, and IL-12p70, as well as combinations of other cytokines or markers apparent to those of skill in the art.

In some embodiments, myosin troponin, such as cTnI, is creatine kinase (CK) and its myocardial fraction CK myocardium, eg, in the same sample, or in a sample from the same individual taken at or near the same time point. Band (MB), aspartate aminotransferase, lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase, myoglobin, glutamate oxaloacetate transaminase, glycogen phosphorylase BB, unbound free fatty acid, cardiac fatty acid binding protein (H-FABP), imaginary It is measured with a marker selected from the group consisting of blood-modified albumin, myosin light chain 1, and myosin light chain 2. In some embodiments, myocardial troponin, such as cTnI, is measured with CK-MB, for example, in the same sample, or in a sample from the same individual taken at or near the same time point.

In some embodiments, myocardial troponin, measured as described herein, alone or in combination with other markers or clinical signs, is used to determine reinfarction. In some embodiments, myocardial troponin, measured as described herein, alone or in combination with other markers or clinical manifestations, is characterized by infarct characteristics, such as size, or duration from infarction. Is used to determine. In the latter case, fragments of troponin produced by proteolysis in the blood can be compared to total troponin; the higher the proportion of fragments, the longer the infarct.

2. Pathological conditions other than AMI The methods of the present invention also include diagnostic, prognostic, and therapeutic methods based on myocardial troponin concentration in samples useful for pathological conditions other than AMI.

Many conditions can occur or include actual heart damage, and measuring myocardial troponin at the levels described herein allows early detection and intervention of such damage. The myocardial troponin concentration measured by this method and the knowledge of the composition of the present invention are useful for diagnosing such pathological conditions, prognostic diagnosis, and therapeutic decision. Pathologies include percutaneous coronary intervention, cardiac surgery, heart failure, acute rheumatic fever, amyloid deposits, cardiac trauma (contusion, resection, pacing, firing, electrodefibrillation, catheter insertion and cardiac surgery, etc.), recanalization. Flow injury, cardiotoxicity due to cancer therapy, congestive heart failure, end-stage renal failure, type II glucose storage (Pompe's disease), cardiac transplantation, abnormal hemoglobinosis due to transfusion hemosiderosis, hypertension including pregnancy hypertension, often low with arrhythmia Includes blood pressure, hypothyroidism, myocarditis, peritonitis, postoperative non-cardiac surgery, pulmonary arterial embolism, and septicemia.

In these embodiments, troponin levels can be determined simultaneously with levels of markers specific for non-cardiac disease or other symptoms or clinical signs of the disease; determining diagnosis, prognostic diagnosis, and / or treatment method. To be done, the concentration and / or information of markers of other symptoms or clinical signs is combined with the information of myocardial troponin concentration determined as described herein. For example, in embodiments of the invention, in addition to determining myocardial troponin concentration, the concentration of one or more of the polypeptides described above, or other protein markers useful for diagnosing, prognosing, or identifying a disease. Can be used. In some embodiments, a group of markers for the disease is provided, which includes the myocardial troponin concentration described herein, and at least one other marker for the disease. The group can include one or more individual markers of myocardial troponin, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more, including total cTnI. Analysis of a single marker or a subset of markers can be performed by one of ordinary skill in the art to optimize clinical sensitivity or specificity in a variety of clinical environments. These include, but are not limited to, emergency, emergency management, crisis management, centralized management, monitoring units, inpatients, outpatients, doctor's offices, clinics, and health screening environments. In addition, one of ordinary skill in the art can use a single marker or a subset of markers in combination with the adjustment of diagnostic thresholds in each of the environments described above to optimize clinical sensitivity or specificity.

a. Cardiac Toxicity The compositions and methods of the present invention are particularly useful in determining and monitoring the cardiotoxicity resulting from treatment, eg, cardiotoxicity of drug treatment. Thus, for example, the invention is i) a step of determining the myocardial troponin concentration in a sample from an individual or determining the myocardial troponin concentration in a series of samples, during or after the individual being treated. At least one sample is taken from an individual and the detection limit of myocardial troponin in the sample is less than about 50, 40, 30, 10, 5, 4, 3, 2 or 1 pg / ml, for example, less than about 20 pg / ml. By measuring myocardial troponin in an individual by a step of determining one or more concentrations in a myocardial troponin assay; ii) a step of assessing the degree of cardiotoxicity of treatment based on the one or more concentrations. Provided is a method for assessing the cardiotoxicity of treatment. In some embodiments, the treatment is drug treatment. In some embodiments, the treatment is non-drug treatment. Methods for determining myocardial troponin concentration include any suitable method having the required sensitivity, eg, the method described herein. In some embodiments, the method utilizes a method of determining myocardial troponin concentration in a sample, including detection of a single molecule of troponin, or a complex or fragment thereof.

A method for measuring cardiotoxicity using a cross-reactive antibody described herein, i.e., an antibody that reacts with troponin from at least two species, such as human and rat, dog, mouse, or another species such as monkey. Is especially useful. Such antibodies can be used in animal studies of drug toxicity, and individuals evaluated for toxicity include mammals such as rats, mice, dogs, monkeys, or other animals used in such studies. It is an animal. Toxicity in various species can be compared directly if the antibodies used in the assay are the same antibody and thus variability can be reduced.

To monitor cardiotoxicity, it will be recognized that the compositions and methods of the invention can be used in the context of certain drugs whose side effects include cardiotoxicity. Therefore, the present invention is a step of determining the concentration of myocardial troponin in one or more samples obtained from an individual, and the detection limit of myocardial troponin in the one or more samples is about 50, 40, 30. 10, 5, 4, 3, 2 or less than 1 pg / ml, eg, less than about 20 pg / ml, with the step of determining one or more concentrations by myocardial troponin assay; ii) based on the one or more concentrations. Provided is a method for monitoring the cardiotoxicity of an individual receiving a drug known to cause cardiotoxicity by a step of assessing the degree of cardiotoxicity of drug treatment. In some embodiments, the method further comprises the step of determining whether to continue drug treatment based on the evaluation of iii) step ii). Drugs whose side effects include cardiotoxicity are well known in the art.

C. Business Methods The present invention establishes markers of myocardial troponin that can be used to diagnose, prognose, or determine therapeutic methods of biological conditions or conditions in an organism, and provides diagnostics based on such markers. Regarding diagnostics and systems and methods (including business methods) for commercializing / marketing services that utilize such diagnostics. The biological condition can be the acute myocardial infarction described herein, or cardiac injury due to drug toxicity, or a non-AMI condition.

In one embodiment, the business method herein is a biological sample by detecting a single molecule of one or more markers at a detection level of about 50, 20, 10, 5, or less than 1 pg / ml. One or more using a method comprising establishing a concentration range of the one or more markers in a biological sample obtained from a first population by measuring the concentration of one or more markers in. Includes a step of establishing a myocardial troponin marker; for example, a step of commercializing one or more markers established at the above steps in a diagnostic product. The diagnostic products herein emit at least about 200 photons when stimulated by one or more antibodies that specifically bind to myocardial troponin markers and a laser that emits at the excitation wavelength of the fluorescent moiety. It can include a fluorescent moiety that can be made, the laser is focused on a spot containing the fluorescent moiety and having a diameter of about 5 microns or more, and the total energy directed by the laser to the spot is about 3 microjoules or less.

In one embodiment, the business method herein measures the marker concentration of a biological sample by detecting a single molecule of the marker at a detection level of less than about 50, 20, 10, 5 or 1 pg / ml. By establishing a concentration range of said myocardial troponin marker in a biological sample obtained from a first population; the organism is in a condition or condition of interest, such as AMI, cardiotoxicity by drug treatment, or non- Includes the step of establishing a range of normal values for myocardial troponin markers using a system that includes providing diagnostic services to determine if a person has an AMI condition. The diagnostic services herein can be provided by CLIA-approved laboratories licensed under the business, or the business itself. The diagnostic services herein can be provided directly to a healthcare provider, health insurance company, or patient. Therefore, the business methods herein can generate revenue, for example, from diagnostic services or diagnostic products.

The business method herein also considers providing diagnostic services to, for example, a healthcare provider, health insurance company, or patient. The business herein may provide diagnostic services by outsourcing to a service laboratory or by establishing a service laboratory (under Clinical Laboratory Improvement Amendment (CLIA) or other regulatory approval). Such service laboratories can then carry out the methods disclosed herein to determine if myocardial troponin markers are present in the sample.

VII. Composition The present invention provides a composition useful for the detection and quantification of myocardial troponin. The composition is labeled with a label suitable for detection by the method of the invention. A binding partner, one or both of the binding partners of myocardial troponin is labeled with a label suitable for detection by the method of the invention. The binding partner being coupled, in some embodiments, includes a solid support to which the capture binding partner is attached, having a detection binding partner.

A typical embodiment comprises a composition for detecting myocardial troponin comprising a binding partner of myocardial troponin bound to a fluorescent moiety, the fluorescent moiety being at least when stimulated by a laser emitting at the excitation wavelength of that moiety. It can emit about 200 photons, the laser is focused on a spot with a diameter of about 5 microns or more, including a fluorescent portion, and the total energy directed by the laser to the spot is less than about 3 microjoules. In some embodiments, the binding partner comprises an antibody against myocardial troponin. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody cross-reacts with a cross-reactive antibody, eg, myocardial troponin from at least two species selected from the group consisting of at least two species, eg, human, monkey, dog and mouse. It is an antibody. In some embodiments, the antibody cross-reacts with myocardial troponin from all human, monkey, dog and mouse sources. In some embodiments, myocardial troponin is selected from the group consisting of cTnI and cTnT. In some embodiments, the myocardial troponin is cTnI. In some embodiments, the myocardial troponin is cTnT. The antibody is specific for a specific region of the troponin molecule, eg, a region of myocardial troponin I containing amino acids 27-41. The fluorescent moiety can contain one or more molecules containing at least one substituted indolium ring system, the substituent on the 3-carbon of the indolium ring being a chemically reactive or conjugated substance group. Contains. Labeling compositions may include fluorescent moieties containing one or more dye molecules selected from the group consisting of AlexaFluor488, 532, 647, 700, or 750. Labeling compositions can include fluorescent moieties containing one or more dye molecules selected from the group consisting of AlexaFluor 488, 532, 700, or 750. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, AlexaFluor488. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, AlexaFluor555. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, AlexaFluor610. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, AlexaFluor647. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, AlexaFluor680. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, which are AlexaFluor 700. Examples of the labeling composition include fluorescent moieties containing one or more dye molecules, AlexaFluor750e.

In some embodiments, the present invention provides a composition comprising a set of standards for determining myocardial troponin concentration, at least one of which is about 20, 15, 10, 5, 4 Myocardial troponin concentration is less than 3, 2, or 1 pg / ml. In some embodiments, the present invention provides a composition comprising a set of standards for determining myocardial troponin concentration, at least one of which is at a myocardial troponin concentration of less than about 20 pg / ml. is there. In some embodiments, the present invention provides a composition comprising a set of standards for determining myocardial troponin concentration, at least one of which is a myocardial troponin concentration of less than about 10 pg / ml. .. In some embodiments, the invention provides a composition comprising a set of standards for determining myocardial troponin concentration, at least one of which is at a myocardial troponin concentration of less than about 5 pg / ml. is there. In some embodiments, the invention provides a composition comprising a set of standards for determining myocardial troponin concentration, at least one of which is at a myocardial troponin concentration of less than about 1 pg / ml. is there.

Other compositions of the present invention are as described herein.

VIII. Kits The present invention further provides kits. The kit of the present invention contains one or more compositions useful for sensitive detection of myocardial troponin described herein in suitable packaging. In some embodiments, the kits of the invention provide a binding partner, such as a label, eg, an antibody specific for myocardial troponin, which is bound to a fluorescent moiety. In some embodiments, the kits of the invention provide a binding partner, eg, an antibody pair that is specific for myocardial troponin, with at least one of the binding partners for the myocardial troponin described herein. It is a sign. In some embodiments, the binding partner, eg, antibody, is provided in separate containers. In some embodiments, the binding partner, eg, antibody, is provided in the same container. In some embodiments, one of the binding partners, eg, an antibody, is immobilized on a solid support, eg, a microtiter plate or paramagnetic beads. In some of these embodiments, other binding partners, such as antibodies, are labeled with the fluorescent moieties described herein.

Fluorescent labels for binding partners, such as antibodies, solid supports, and components of the kit, can be any suitable component, such as the components described herein.

In addition, the kit contains reagents useful for the methods of the invention, such as buffers and other reagents used in binding reactions, cleaning agents, buffers or other reagents for preconditioning equipment to perform the assay, equipment. It can contain an elution buffer or other reagent for moving the sample through.

The kit is used in the assay of the present invention, such as one or more standards, such as highly purified standards, such as recombinant human cTnI or human cTnT, or various fragments, complexes thereof. Can include standard products. The kit can further include instructions for use.

Examples The following examples are provided for illustrative purposes without any intent to limit disclosure of the rest.

Unless otherwise specified, sample processing in the examples was analyzed in a single molecule detector (SMD) described herein using the following parameters: laser: focused to a spot size of approximately 2 microns. (0.004 pL interrogation space described herein) continuous wave gallium arsenite diode laser with a wavelength of 639 nm (Blue Sky Research, Milpitas, CA). Flow velocity = 5 microliters / minute through a fused quartz capillary of 100 micron square ID and 300 micron square OD; non-confocal arrangement of lenses (see eg FIG. 1A); 0.8 caliber focal lens (Olympus) Silicon avalanche photodiode detector (Perkin Elmer, Waltham, Massachusetts).

Biomarker sandwich assay: myocardial troponin I (cTnI)
Assay: The purpose of this assay was to detect the presence of myocardial troponin I (cTNI) in human serum. The assay mode was a two-step sandwich immunoassay based on a mouse monoclonal capture antibody and a goat polyclonal detection antibody. A 10 microliter sample was required. The range of action of the assay is 0-900 pg / ml, and detection of 1-3 pg / ml is a typical analytical limit. It took about 4 hours bench time to complete the assay.

Materials: In the procedure below, the following materials were used: Assay plate: Nunc Maxisorp, product 464718, 384 wells, clear, monoclonal antibody BiosPacific A34440228P, passive coating with lot number A0316 (5 μg / ml in 0.05 M sodium carbonate) , PH 9.6, overnight at room temperature); blocked by 5% sucrose in PBS, 1% BSA and stored at 4 ° C. Human myocardial troponin (BiosPacific Catalog No. J3403522) was used as the standard curve. Diluted solution for standard concentration was human serum immunodepleted with endogenous cTNI, aliquoted and stored at −20 ° C. Dilution of the standard was made in 96 wells, conical, polypropylene (Nunc product number 249944). The following buffers and solutions were used: (a) Buffer for assay: BBS with 1% BSA and 0.1% Triton X-100; (b) 2 mg / ml mouse IgG (Equitech Bio); Passive blocking solution in assay buffer containing 2 mg / ml goat IgG (Equitech Bio); and 2 mg / ml MAK33poly, Roche # 11939661; (c) detection antibody (Ab); goat The polyclonal antibody is affinity purified against peptide 3 (BiosPacific G129C), labeled with the fluorescent dye AlexaFluor647 and stored at 4 ° C .; detection antibody dilution: 50% assay buffer, 50% passive blocking solution. Wash buffer: borate buffer physiological saline Triton buffer (BBST) (1.0 M borate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); elution buffer Solution: BBS containing 4M urea, 0.02% Triton X-100, and 0.001% BSA.

Preparation of AlexaFluor647-labeled antibody: First, by dissolving 100 μg of the detection antibody G-129-C in 400 μL of coupling buffer (0.1 M NaHCO ₃ ), the detection antibody G-129- C was conjugated to AlexaFluor647. The antibody solution was then transferred into a YM-30 filter and the antibody solution was concentrated to 50 μl by subjecting the solution and filter to centrifugation. The YM-30 filter and antibody were then washed 3 times by adding 400 μl of coupling buffer. 50
The antibody was recovered by adding l to the filter, inverting the filter, and centrifuging at 5,000 xg for 1 minute. The antibody solution obtained was 1-2 μg / μl. The AlexaFluor647NHS ester was reconstituted by adding 20 μl DMSO to one vial of AlexaFluor647, and the solution was stored at −20 ° C. for up to 1 month. 3 μl of AlexaFluor647 stock solution was added to the antibody solution, which was then mixed and incubated in the dark for 1 hour. After 1 hour, 7.5 μl of 1M Tris was added to the antibody AlexaFluor 647 solution and mixed. The solution was ultrafiltered through YM-30 to remove low molecular weight components. The volume of concentrated water containing the antibody conjugated to AlexaFluor647 was adjusted to 200-400 liters by adding PBS. 3 μl of 10% NaN3 was added to the solution and the resulting solution was transferred to the Ultrafree 0.22 centrifuge unit and spun at 12,000 xg for 2 minutes. The filtrate containing the conjugated antibody was collected and used in the assay.

Procedure: Preparation and analysis of cTnI standards and samples:
The standard curve was prepared as follows: Working standard by serial dilution of the cTnI stock into standard diluents or to reach the cTnI concentration range of 1.2 pg / ml to 4.3 μg / ml. Was prepared (0 to 900 pg / ml).

10 μl of passive blocking solution and 10 μl of standard or sample were added to each well. The standard products were implemented four by four (in quadruplicate). The plates were sealed with Axyseal sealing film, centrifuged at 3000 RPM for 1 minute and incubated at 25 ° C. for 2 hours with shaking. The plate was washed 5 times and centrifuged on a paper towel in the inverted position until the rotor reached 3000 RPM. A 1 nM working dilution of the detection antibody was prepared and 20 μl of the detection antibody was added to each well. The assay was incubated at 25 ° C. for 1 hour with the plates sealed, centrifuged and shaken. 30 μl of elution buffer was added per well, the plates were sealed and the assay was incubated at 25 ° C. for 1/2 hour. Plates were stored at 4 ° C. for up to 48 hours prior to analysis or samples were analyzed immediately.

In the analysis, 20 μl per well was captured at 40 μl / min and 5 μl was analyzed at 5 μl / min. Data were analyzed based on a 4-sigma threshold. The unmodified signal and the standard concentration were plotted. Linear adaptation was performed for the low concentration range and non-linear adaptation was performed for the perfect standard curve. The detection limit (LoD) was calculated as LOD = (3 x zero standard deviation) / linear conformance gradient. The concentration of the sample was determined from an equation (linear or non-linear) suitable for the sample signal.

The aliquot was pumped into the analyzer. Antibodies labeled individually during the capillary flow were measured by setting the intervention volume so that the emission of only one fluorescent label was detected in the defined space after laser excitation. Each signal represents a digital event, and this configuration allows for extremely high analytical sensitivity. The sum of the fluorescent signals is determined as the sum of the individual digital events. Each molecule counted is a positive data point with hundreds to thousands of DMC events / samples. The detection limit of the cTnI assay of the present invention was determined by the mean + 3SD method.

Results: Table 3 shows data for a typical cTnI standard curve measured four by four using the assay protocol.

The sensitivity of the analyzer system was tested in 15 runs and it was determined to detect sub-femtomol / l (fM) levels of calibrators, as shown in Table 4. The accuracy was 10% at 4 pg / ml and 12 pg / ml cTnI.

A linearized standard curve for the concentration range of cTnI is shown in FIG.

The analytical limit (LoD) of detection was determined by 15 consecutive assays. LoD is the average of 0 standard + 3SD (n = 4) in-assay measurements. The average LoD was 1.7 pg / ml (range 0.4-2.8 pg / ml).

Sample recovery was determined by immunoremoving cTnI and analyzing a sample of serum spiked with a known amount of cTnI. Table 5 shows data on sample recovery by the system analyzed over 3 days.

Assay linearity was determined in human pool serum spiked with cTnI and diluted with standard dilutions. The results in Table 6 show% of the expected signal for dilutions and corresponding diluents.

These data indicate that the analyzer system of the present invention allows the performance of sensitive laser-guided immunoassays for cTnI at sub-femtomole concentrations.

TnI Sandwich Bead-Based Assay The same microtiter plate format is used in the above assay, using a plastic surface to anchor the target molecule. The single particle analyzer system is also compatible with assays performed in solution with microparticles or beads to achieve separation of bound material from unbound material.

Material: MyOne Streptavidin C1 microparticles (MPs) are obtained from Dynamic (650.01-03, 10 mg / ml stock). The buffers used in the assay were 10 x bovine buffered physiological saline Triton Buffer (BBST) (1.0 M bovine, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3). ); 2 mg / ml normal goat IgG in 0.1 M Tris (pH 8.1), 2 mg / ml normal mouse IgG, and 0.2 mg / ml MAB-33-IgG-polymer, assay buffer. 0.025M EDTA, 0.15M NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.1% NaN3, stored at 4 ° C.); and elution buffer (4M). Urea, BBS containing 0.02% Triton X-100, and 0.001% BSA, stored at 2-8 ° C.). Antibodies used in sandwich bead-based assays include Bio-Ab (A34650228P (BiosPacific) containing 1-2 biotins per IgG) and Det-Ab (G-129-C (BiosPacific) bound to A647). , 2-4 phosphors per IgG). The standard product is recombinant human myocardial troponin I (BiosPacific, catalog number J34120352). The calibrator diluent is BSA in 30 mg / ml TBS wEDTA.

Microparticle coating: Place 100 μl of MPs stock in an Eppendorf tube. By applying a magnet, MPs are washed 3 times with 100 μl of BBST wash buffer, the supernatant is removed, the magnet is removed and resuspended in wash buffer. After washing, MPs are resuspended in 100 μl assay buffer and 15 μg of Bio-Ab is added. The mixture is then incubated for 1 hour at room temperature with continuous mixing. As described above, MPs are washed 5 times with 1 ml of washing buffer. After washing, MPs are resuspended in 15 ml assay buffer (or 100 μl for storage at 4 ° C).

Standard and sample preparation: Dilute the standard with calibrator diluent to create a suitable standard curve (typically 200 pg / ml to 0.1 pg / ml). Frozen serum and plasma samples need to be centrifuged at 13 Krpm for 10 minutes at room temperature. Carefully remove the clear serum / plasma so that it does not become a potential pellet or suspension and place it in a fresh tube. Pipette 50 μl of each standard or sample into a suitable well.

Capture target: Add 150 μl of MPs (after resuspension in 15 ml of assay buffer + 400 mM NaCl) to each well. The mixture is incubated on JitterBug, 5 at room temperature for 1 hour.

Cleaning and detection: The plate is placed on a magnet, and after confirming that all MPs are captured by the magnet, the supernatant is removed. After removing the plate from the magnet, 250 μl of cleaning buffer is added. The plate is then placed on the magnet, and after confirming that all MPs are captured by the magnet, the supernatant is removed. Add 20 μl of Det-Ab per well (dilute Det-Ab to 500 ng / ml in assay buffer + 400 mM NaCl). The mixture is incubated on JitterBug, 5 at room temperature for 30 minutes.

Washing and Elution: Place the plate on a magnet and wash 3 times with wash buffer. After confirming that all MPs are captured by the magnet, remove the supernatant and add 250 μl of wash buffer. After washing, transfer the sample into a new 96-well plate. Then, a new plate is placed on the magnet, and after confirming that all MPs are captured by the magnet, the supernatant liquid is removed. After removing the plate from the magnet, 250 μl of cleaning buffer is added. The plate is then placed on the magnet, and after confirming that all MPs are captured by the magnet, the supernatant is removed. Then 20 μl of elution buffer is added and the mixture is incubated on JitterBug, 5 at room temperature for 30 minutes.

Filter out MPs and transfer to 384-well plate: Transfer standards and samples into a 384-well filter plate placed on top of the 384-well assay plate. The plate is then centrifuged using a plate rotor at 3000 rpm at room temperature. Remove the filter plate and add a suitable calibrator. The plate is coated and prepared for implementation in SMD.

SMD: Aliquot is pumped into the analyzer. After laser excitation, the individually labeled antibodies during the capillary flow are measured by setting the intervention volume so that the emission of only one fluorescent molecule is detected in the defined space. Each signal represents a digital event, and this configuration allows for extremely high analytical sensitivity. The sum of the fluorescent signals is determined as the sum of the individual digital events. Each molecule counted is a positive data point with hundreds to thousands of DMC events / samples. The detection limit of the cTnI assay of the present invention was determined by the mean + 3SD method.

Concentration Range of cTnI in Normal Non-Disease Target Population A reference range or normal range of cTnI concentration in human serum was established using serum samples obtained from 88 clearly healthy subjects (non-disease). The sandwich assay described in Example 1 was performed and the number of signals or events described above was counted using the single particle analyzer system of the present invention. The concentration of serum troponin I was determined by correlating the signal detected by the analyzer with the standard curve described above. All assays were performed four by four.

Following current recommendations from the European Society of Cardiology (ESC / ACC), the troponin assay is less than 10% assay to reliably distinguish between ACS patients and patients without ischemic heart disease. The 99th percentile in the normal range must be accurately quantified by assay imprecision (CV) and by stratifying the risk of adverse cardiac events. The assay shows that the TnI biological threshold (cutoff concentration) is a TnI concentration of 7 pg / ml, which is established at the 99th percentile with a corresponding 10% CV (FIG. 5). At 10% CV levels, the accuracy profile refers to TnI concentrations of 4 pg / ml and 12 pg / ml.

In addition, the assay correlates well with troponin I standards measurements provided by the National Institute of Standards and Technology (Figure 6).

The assays of the invention are sensitive and accurate enough to meet the requirements of ESC / ACC when compared to the assays described by Koerbin et al. (Ann Clin Biochem, 42: 19-23 (2005)). It is the most sensitive assay for myocardial troponin I. The assays of the invention are 10 to 20 times more sensitive than currently available assays for which the biological threshold range has been determined to be cTnI of 111 pg / ml to 333 pg / ml.

Detection test of early release of TnI into the blood circulation of patients suffering from acute myocardial infarction (AMI) Test 1: 47 samples from 18 patients who showed chest pain in the emergency department (ED) in succession Obtained. All of these patients had non-ST elevation ECG and were diagnosed with AMI. Concentrations of cTnI in initial samples from all 18 patients were determined by a commercial assay to be <350 pg / ml (10% cut point) upon entry into the emergency room, and 12 were <100 pg / ml (10% cut point). 99%) It was the percentile. These samples were later tested using the same commercial assay and tested positive for cTnI. The same serum samples were assayed for TnI according to the assays of the invention described in Examples 1 and 3 and the results were compared to the results obtained using a commercial assay.

Blood was first collected when the patient showed chest pain (Sample 1), followed by 4-8 hour intervals (Sample 2 at 12 hours; Sample 3 at 16 hours; Sample 4; 30 at 24 hours). Sample 5 at the hour; sample 6 at the 36th hour; sample 7 at the 42nd hour; and sample 8) at the 48th hour. Serum was analyzed by the method of the present invention and the current commercially available method, and the results obtained are shown in FIG. The analyzer of the present invention detected TnI at the time when the patient showed chest pain (Sample 1), whereas the commercially available assay detected cTnI for the first time at a much later time (Sample 6 at 36 hours). The concentration of TnI in Sample 3 exceeds the biological threshold level (7 pg / ml, see FIG. 5) established using the analyzer of the present invention, Sample 3 is positive for TnI, and the occurrence of cardiac events. It was shown to suggest. The biological threshold for commercial assays is between 111 pg / ml and 333 pg / ml. Therefore, Sample 3 would not be considered to show a positive cardiac event.

The methods and compositions of the present invention allow much earlier diagnosis and possible intervention based on myocardial troponin levels, as evidenced by the results for the first sample taken from the patient. In the three cases where the cTnI value of the first commercial assay was between 100 ng / ml and 350 ng / ml, all were positive for the cTnI value depending on the analytical method of the present invention (ie, cTnI of 7 pg / ml or more). ). Of the 12 cases where the cTnI value of the first commercial assay was less than 100 pg / ml, 5 cases were tested positive for cardiovascular events by the assay of the invention (ie, cTnI greater than or equal to 7 pg / ml). Predicting the use of the assay of the present invention would have detected 53% more AMI cases than current commercial assays when hospitalized samples were tested.

Test 2: 50 additional serum samples that were negative in the commercial assay test were tested using the analyzers and assays of the invention. The result is shown in FIG. Of the 50 samples, 36 were within 99% and were determined to be within the normal range established by the assay of the invention. However, testing of the remaining 14 samples determined to be within the commercially available "normal" or non-disease range was above the biological threshold established by the present invention.

Therefore, the sensitive cTnI assay of the present invention allows the detection of myocardial damage in patients when serum levels of cTnI are below the threshold according to commercially available techniques. The use of the sensitive and accurate cTnI assay of the present invention allows the detection of AMI to occur faster than with existing cTnI assays, thereby providing appropriate diagnosis and early medical intervention to improve results. Opportunity is provided.

Claims (89)

A method for determining the presence or absence of a single molecule or fragment or complex of troponin in a sample.
i) A step of labeling the molecule, fragment, or complex, if any, with a label.
ii) A method for detecting the presence or absence of the label, wherein the detection of the presence of the label includes a step indicating the presence of the single molecule, fragment, or complex of troponin in the sample. The method of claim 1, wherein the troponin is a myocardial isoform of troponin. The method of claim 2, wherein the troponin is selected from the group consisting of myocardial troponin I (cTnI) and myocardial troponin C (cTnC). The method of claim 3, wherein the troponin is cTnI. The method of claim 1, wherein the single molecule of troponin can be detected in the detection step with a detection limit of less than about 50 pg / ml. The method of claim 1, wherein the troponin can be detected at a detection level of less than about 20 pg / ml. The method of claim 1, wherein the label comprises a fluorescent moiety. When the fluorescent portion is stimulated by a laser that emits light at the excitation wavelength of the portion, it can emit at least about 200 photons, and the laser is focused on a spot having a diameter of about 5 microns or more including the fluorescent portion. The method of claim 1, wherein the total energy directed by the laser to the spot is about 3 microjoules or less. The method of claim 1, wherein the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, and the substituent on the 3-carbon of the indolium ring comprises a chemically reactive or conjugate group. The method of claim 1, wherein the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, or AlexaFluor700. The method of claim 1, wherein the fluorescent moiety comprises AlexaFluor647. The method of claim 1, wherein the label further comprises a binding partner for the troponin molecule, fragment, or complex. 12. The method of claim 12, wherein the binding partner comprises an antibody specific for the troponin molecule, fragment, or complex. 13. The method of claim 13, wherein the antibody is specific for a particular region of the troponin molecule. 14. The method of claim 14, wherein the antibody is specific for a region of myocardial troponin I containing amino acids 27-41. 13. The method of claim 13, wherein the antibody comprises a polyclonal antibody. 13. The method of claim 13, wherein the antibody is a monoclonal antibody. The method of claim 1, further comprising capturing the troponin or troponin complex on a solid support. 18. The method of claim 18, wherein the solid support is selected from the group consisting of microtiter plates and paramagnetic beads. 18. The method of claim 18, wherein the solid support comprises a capture partner specific for the troponin or troponin complex that binds to the solid support. 20. The method of claim 20, wherein the bond of the capture partner to the solid support is a non-covalent bond. The method of claim 20, wherein the bond of the capture partner to the solid support is a covalent bond. 22. The method of claim 22, wherein the covalent bond of the capture partner is one in which the capture partner binds to the solid support in a particular direction. 23. The method of claim 23, wherein the particular orientation helps maximize the specific binding of the troponin or troponin complex to the capture partner. The method of claim 20, wherein the capture partner comprises an antibody. 25. The method of claim 25, wherein the antibody is a monoclonal antibody. 25. The method of claim 25, wherein the antibody is specific for amino acids 87-91 of myocardial troponin I. 25. The method of claim 25, wherein the antibody is specific for amino acids 41-49 of myocardial troponin I. The method of claim 1, wherein the sample is a blood, serum, or plasma sample. 29. The method of claim 29, wherein the sample is a serum sample. The method of claim 1, wherein the label comprises a fluorescent moiety and step ii) passes the label through a single molecule detector. The single molecule detector
a) An electromagnetic radiation source for stimulating the fluorescent portion,
b) Capillary flow cell for passing the fluorescent portion,
c) A driving source for moving the fluorescent portion within the capillary flow cell,
d) An interrogation space defined within the capillary flow cell for receiving electromagnetic radiation emitted from the electromagnetic radiation source.
e) An electromagnetic radiation detector operably connected to the interrogation space for measuring the electromagnetic properties of the stimulated fluorescent portion, and f) a location between the interrogation space and the detector. 31. The method of claim 31, comprising a microscope objective lens having a numerical aperture of 0.6 or greater. A method for determining a diagnosis, prognosis, or treatment method in an individual.
i) A step of determining the concentration of myocardial troponin in a sample obtained from the individual or determining the concentration of myocardial troponin in a series of samples, wherein the detection limit of the myocardial troponin in the sample is about 50 pg /. The step of determining the concentration in a myocardial troponin assay of less than ml and
ii) A method comprising the step of determining a diagnosis, prognosis, or treatment method in the individual based on the concentration in the sample or the concentration in the series of samples. Step ii) compares the concentration or series of concentrations with a normal value of the concentration, compares the concentration or series of concentrations with a predetermined threshold level, and compares the concentration or series of concentrations with a baseline value. 33. The method of claim 33, comprising an analysis selected from the group consisting of comparing with and determining the rate of change of concentration for the series of concentrations. A claim comprising step ii) comparing the troponin concentration in the sample to a predetermined threshold concentration and determining a diagnosis, prognosis, or treatment method if the sample concentration is above a threshold level. Item 34. 35. The method of claim 35, wherein the threshold concentration is determined by determining the 99th percentile concentration of troponin in a normal population and setting the threshold concentration at the 99th percentile concentration. 33. The method of claim 33, wherein at least one sample is taken during or after the cardiac stress test. 33. The method of claim 33, wherein the myocardial troponin is selected from the group consisting of myocardial troponin I and myocardial troponin T. 33. The method of claim 33, wherein the myocardial troponin is myocardial troponin I. 38. The method of claim 38, wherein the concentration of myocardial troponin is the concentration of total myocardial troponin. 40. The method of claim 40, wherein the concentration of myocardial troponin is the concentration of myocardial troponin complex, myocardial troponin fragment, phosphorylated myocardial troponin, oxidized myocardial troponin, or a combination thereof. 41. The method of claim 41, wherein the concentration of myocardial troponin is compared to total myocardial troponin. 33. The method of claim 33, wherein the diagnosis, prognosis, or treatment method is a diagnosis, prognosis, or treatment method for myocardial infarction. 43. The method of claim 43, wherein the diagnosis, prognosis, or treatment method comprises risk stratification for myocardial infarction risk levels. 43. The concentration or series of concentrations is determined at or near the time when the individual presents or near the health care worker with one or more symptoms indicating myocardial ischemia or infarction or the possibility thereof. The method described. 45. The method of claim 45, wherein the one or more symptoms are selected from the group consisting of chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath. 33. The method of claim 33, wherein the concentration is determined by a method comprising detecting a single molecule of troponin or a complex or fragment thereof. 47. The method of claim 47, comprising labeling the troponin or troponin complex with a label comprising a fluorescent moiety. When the fluorescent portion is stimulated by a laser that emits light at the excitation wavelength of the portion, it can emit at least about 200 photons, and the laser is focused on a 5 micron diameter spot containing the fluorescent portion. 33. The method of claim 33, wherein the total energy directed by the laser to the spot is about 3 microjoules or less. 33. The method of claim 33, wherein the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, and the substituent on the 3-carbon of the indolium ring comprises a chemically reactive or conjugate group. 33. The method of claim 33, wherein the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, or AlexaFluor700. 51. The method of claim 51, wherein the fluorescent moiety comprises AlexaFluor647. 33. The method of claim 33, wherein the label further comprises a binding partner for the troponin. 53. The method of claim 53, wherein the binding partner comprises an antibody specific for the troponin. 54. The method of claim 54, wherein the antibody comprises a polyclonal antibody. 33. The method of claim 33, further comprising capturing the troponin or troponin complex on a solid support. 56. The method of claim 56, wherein the solid support is selected from the group consisting of microtiter plates and paramagnetic beads. 56. The method of claim 56, wherein the solid support comprises a capture partner specific for the troponin or troponin complex that binds to the solid support. Step i) further comprises evaluating another index of the individual, step ii) based on or based on the evaluation of the troponin concentration and the other index of the non-troponin marker in the sample, or the series. 33. The method of claim 33, comprising determining a diagnostic, prognostic, or therapeutic method in the individual based on the concentration in the sample. The method of claim 59, wherein the other indicator is a clinical indicator of myocardial ischemia or infarction. The method of claim 60, wherein the other indicator is the concentration of one or more non-troponin markers in the sample or in the series of samples. 59. The method of claim 59, wherein the one or more markers are markers of cardiac ischemia or inflammation and plaque instability. One or more of the above cardiac ischemic markers are creatine kinase (CK) and its myocardial fraction CK myocardial band (MB), aspartate aminotransferase, lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase, myoglobin, glutamate oxaloacetate transaminase. 62, which is selected from the group consisting of glycogen phosphorylase BB, unbound free fatty acid, heart-type fatty acid binding protein (H-FABP), ischemic modified albumin, myosin light chain 1, and myosin light chain 2. Method. 63. The method of claim 63, wherein the one or more markers comprises one or more specific markers of myocardial injury. 33. The method of claim 33, wherein the diagnosis, prognosis, or treatment is a diagnosis, prognosis, or treatment of a condition other than myocardial infarction. 65. The method of claim 65, wherein the condition is cardiotoxic. The method of claim 66, wherein the cardiotoxicity is associated with drug administration to the individual. These conditions include acute rheumatic fever, amyloid deposits, cardiac trauma (including contusion, resection, pacing, firing, electrodefibrillation, catheter insertion and cardiac surgery), reperfusion injury, congestive heart failure, end-stage renal failure. , Type II glucose storage (Pompe's disease), heart transplantation, abnormal hemoglobinosis due to transfusion hemosiderosis, hypertension including gestational hypertension, hypotension often accompanied by arrhythmia, hypothyroidism, myocarditis, peritonitis, 65. The method of claim 65, selected from the group consisting of post-operative non-cardiac surgery, pulmonary arterial embolism, and hypertension. A composition for the detection of a troponin isoform comprising a binding partner of a troponin isoform bound to a fluorescent moiety, wherein the fluorescent moiety is at least about 200 when stimulated by a laser emitting at the excitation wavelength of the moiety. A composition capable of emitting a number of photons, wherein the laser is focused on a spot having a diameter of about 5 microns or more including a fluorescent portion, and the total energy directed by the laser to the spot is about 3 microjoules or less. The composition according to claim 69, wherein the binding partner comprises an antibody against the troponin isoform. The composition according to claim 70, wherein the antibody is a polyclonal antibody. The composition according to claim 70, wherein the antibody is a monoclonal antibody. The composition according to claim 69, wherein the troponin isoform is a myocardial isoform. The composition according to claim 73, wherein the myocardial isoform is selected from the group consisting of cTnI and cTnT. The composition according to claim 74, wherein the myocardial isoform is cTnI. The composition of claim 75, wherein the antibody is specific for a particular region of the troponin molecule. The composition of claim 76, wherein the antibody is specific for a region of myocardial troponin I containing amino acids 27-41. The composition of claim 69, wherein the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, and the substituent on the 3-carbon of the indolium ring comprises a chemically reactive or conjugate group. The composition according to claim 69, wherein the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700. The composition of claim 79, wherein the fluorescent moiety comprises AlexaFluor647. A composition comprising a set of standards for determining the concentration of myocardial troponin, at least one standard having a myocardial troponin concentration of less than about 10 pg / ml. A kit containing a composition comprising an antibody against myocardial troponin bound to a fluorescent dye moiety, the fluorescent dye moiety emitting at least about 200 photons when stimulated by a laser emitting at the excitation wavelength of that moiety. The laser can be focused on a spot with a diameter of about 5 microns or more, including a fluorescent portion, the total energy directed by the laser to the spot is about 3 microjoules or less, and the composition is a suitable packaging material. Wrapped in a kit. The kit according to claim 82, wherein the myocardial troponin is myocardial troponin I or myocardial troponin T. The kit according to claim 82, wherein the myocardial troponin is myocardial troponin I. 28. The kit of claim 82, further comprising instructions. 28. The kit of claim 82, further comprising a composition comprising a capture antibody for said myocardial troponin I bound to a solid support. The kit of claim 86, wherein the solid support comprises a microtiter plate or paramagnetic microparticles. 28. The kit of claim 82, further comprising a component selected from the group consisting of a wash buffer, an assay buffer, an elution buffer, and a calibrator diluent. The kit of claim 82, further comprising a standard of myocardial troponin.