JP2022172058A - Highly sensitive system and method for analysis of troponin - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

COPYRIGHT: (C)2023,JPO&INPIT

Description

No. 60/789,304, filed April 4, 2006, filed April 19, 2006, herein incorporated by reference in its entirety under 35 U.S.C. U.S. Provisional Application No. 60/793,664; U.S. Provisional Application No. 60/808,662 filed May 26, 2006; U.S. Provisional Application No. 60/861,498 filed November 28, 2006; claims priority from U.S. Provisional Patent Application No. 60/872,986.

BACKGROUND OF THE INVENTION Approximately 6,000,000 people in the United States are rushed to the emergency department each year for chest pain. Only 15% to 20% of these patients are eventually diagnosed with acute coronary syndrome (ACS), but about half are hospitalized for evaluation. Conversely, 2% of ACS patients are erroneously discharged. Since patients with ACS are at relatively high risk of serious adverse cardiovascular events in the short term, there is a clear need for an accurate and objective means of identifying them.

Cardiac damage markers currently in use suffer from drawbacks that limit their clinical utility. Cardiac enzyme assays form the basis for determining the presence or absence of damage to the myocardium. Unfortunately, standard creatine kinase MB (CK-MB) assays are unreliable in clearing infarcts by 10-12 hours after chest pain onset. Earlier diagnosis would have very particular advantages with respect to fibrinolytic therapy and triage.

In one aspect, the invention provides a method.

In some embodiments, the invention provides a method for determining the presence or absence of a single molecule of Troponin or a fragment or complex thereof in a sample, wherein i) the molecule, fragment, or complex is present optionally labeling with a label; and ii) detecting the presence or absence of the label, wherein detection of the presence of the label indicates the presence of a troponin single molecule, fragment, or complex in the sample. Provide a method that includes In some embodiments of the methods of the invention, the troponin is a cardiac muscle isoform of troponin. In some embodiments of the methods of the invention, the troponin can be cardiac troponin I (cTnI) or cardiac troponin C (cTnC). In some embodiments of the methods of the invention, the troponin is cTnI. In some embodiments of the methods of the invention, single molecules 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 of less than about 20 pg/ml. In some embodiments of the methods 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 emitting at the excitation wavelength of the moiety, the laser having a diameter of about 5 microns including the fluorescent moiety. The total energy focused to the spot and directed by the laser to the spot is less than about 3 microjoules. In some embodiments of the methods of the present invention, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring is a chemically reactive group or conjugated entity including conjugated substance groups. In some embodiments of the methods of the invention, the fluorescent moiety comprises a dye. Examples of dyes include, but are not limited to, AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, and AlexaFluor700. In some embodiments of the methods of the present invention, the fluorescent moiety comprises AlexaFluor647. In some embodiments, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring comprises a chemically reactive or conjugated group. In some embodiments of the methods of the invention, the label further comprises a binding partner of a troponin molecule, fragment, or complex. In some embodiments of the methods of the invention, the binding partner comprises an antibody specific for a troponin molecule, fragment, or complex. In some embodiments of the methods of the invention, the antibodies are specific for particular regions of the troponin molecule. In some embodiments of the methods of the invention, the antibody is specific for a region comprising amino acids 27-41 of cardiac troponin I. 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 methods of the invention, the method further comprises capturing the troponin or troponin complex on a solid support. In some embodiments of the methods 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 capture partner specific for troponin or troponin complex bound to the solid support. In some embodiments of the methods of the invention, 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 methods of the invention, the covalent attachment of the capture partner is such that the capture partner binds to the solid support in a specific orientation. In some embodiments of the methods of the invention, a particular orientation helps maximize 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 cardiac troponin I. In some embodiments of the methods of the invention, the antibody is specific for amino acids 41-49 of cardiac troponin I. In some embodiments of the methods of the invention, the sample is a blood, serum, or plasma sample. In some embodiments of the methods of the invention, the sample is a serum sample. In some embodiments of the methods 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 methods of the invention, the single molecule detector comprises a) a source of electromagnetic radiation for stimulating the fluorescent moiety, b) a capillary flow cell for passing the fluorescent moiety, c) within the capillary flow cell. d) an interrogation space defined within the capillary flow cell for receiving electromagnetic radiation emitted from an electromagnetic radiation source; e) measuring the electromagnetic properties of the stimulated fluorescent moiety. and f) a microscope objective lens positioned between the interrogation space and the detector and which is a high numerical aperture lens.

In some embodiments, the invention is a method for determining a diagnostic, prognostic, or therapeutic method in an individual by i) determining the concentration of cardiac troponin in a sample obtained from the individual, or ii) determining the concentration of cardiac troponin in a series of samples by a cardiac troponin assay having a detection limit of less than about 50 pg/ml for cardiac troponin in the sample; or determining a diagnostic, prognostic, or therapeutic method in an individual based on the concentrations in the series of samples. In some embodiments of the methods of the present invention, step ii) comprises comparing the concentration or series of concentrations to a normal value for that concentration, comparing the concentration or series of concentrations to a predetermined threshold level, Analyzes such as comparing a concentration or series of concentrations to a baseline value and determining the percent change in concentration for the series of concentrations. In some embodiments of the methods of the present invention, step ii) compares the troponin concentration in the sample to a predetermined threshold concentration and, if the sample concentration is above the threshold level, diagnostic, prognostic, or determining treatment methods. In some embodiments of the methods of the present 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 cardiac stress testing. In some embodiments of the methods of the present invention, the cardiac troponin is selected from the group consisting of cardiac troponin I and cardiac troponin T. In some embodiments of the methods of the present invention, the cardiac troponin is cardiac troponin-I. In some embodiments of the methods of the present invention, the concentration of cardiac troponin is the concentration of total cardiac troponin. In some embodiments of the methods of the present invention, the concentration of cardiac troponin is the concentration of cardiac troponin complex, cardiac troponin fragment, phosphorylated cardiac troponin, oxidized cardiac troponin, or a combination thereof. In some embodiments of the methods of the invention, the concentration of cardiac troponin is compared to total cardiac troponin. In some embodiments of the methods of the present invention, the diagnosis, prognosis or treatment is of myocardial infarction. In some embodiments of the methods of the invention, the diagnostic, prognostic, or therapeutic method comprises risk stratification of myocardial infarction risk levels. In some embodiments of the methods of the present invention, the concentration or series of concentrations is determined when, or when, the individual exhibits one or more symptoms indicative of myocardial ischemia or infarction or the possibility thereof. To be determined in the near future. In some embodiments, the 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 methods of the invention comprise labeling the troponin or troponin complex with a label comprising a fluorescent moiety. In some embodiments of the methods of the present invention, the fluorescent moiety is capable of emitting at least about 200 photons when stimulated by a laser emitting at the excitation wavelength of the moiety, the laser comprising the fluorescent moiety. Focused to a 5 micron diameter spot, the total energy directed at the spot by the laser is about 3 microjoules or less. In some embodiments of the methods of the present invention, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring comprises a chemically reactive or conjugated group. include. In some embodiments of the methods of the present invention, the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680 or AlexaFluor700. In some embodiments of the methods of the invention, the fluorescent moiety comprises AlexaFluor647. In some embodiments of the methods of the invention, the label further comprises a troponin binding partner. 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 methods of the invention, the method further comprises capturing the troponin or troponin complex on a solid support. In some embodiments of the methods 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 capture partner specific for troponin or troponin complex bound to the solid support. In some embodiments of the methods of the invention, binding of the capture partner to the solid support is non-covalent. In some embodiments of the methods of the invention, binding of the capture partner to the solid support is covalent. In some embodiments of the methods of the invention, the covalent attachment of the capture partner is such that the capture partner binds to the solid support in a specific orientation. In some embodiments of the methods of the invention, a particular orientation helps maximize 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, and step ii) comprises assessing another indicator of troponin concentration and non-troponin markers in the sample. determining a diagnostic, prognostic, or therapeutic method in an individual on the basis of, or on the basis of concentrations in a series of samples. In some embodiments, another indication is a clinical indication of myocardial ischemia or infarction. In some embodiments, another indicator is the concentration of one or more non-troponin markers in the sample or series of samples. In some embodiments of the methods of the invention, the one or more markers are a marker of cardiac ischemia or a marker of inflammation and a marker of plaque instability. In some embodiments, the one or more markers of cardiac ischemia are 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 acids, heart-type 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 comprises one or more specific markers of myocardial injury. In some embodiments of the methods of the present invention, the diagnosis, prognosis or treatment is for a condition other than myocardial infarction. In some embodiments, the condition is cardiotoxicity. In some embodiments, cardiotoxicity is associated with drug administration to an individual. In some embodiments of the methods of the invention, the condition is acute rheumatic fever, amyloidosis, cardiac trauma (contusion, ablation, pacing, firing, cardioversion, catheterization, and cardiac surgery). reperfusion injury, congestive heart failure, end-stage renal failure, type II glycogen storage disease (Pompe disease), heart transplantation, dyshemoglobinopathy due to transfusion hemosiderosis, hypertension including gestational hypertension, hypothermia, often with arrhythmia Selected from the group consisting of hypertension, hypothyroidism, myocarditis, pericarditis, postoperative non-cardiac surgery, pulmonary embolism and sepsis.

In another aspect, the invention includes compositions.

In some embodiments, the invention comprises a composition for troponin isoform detection comprising a binding partner of that troponin isoform conjugated to a fluorescent moiety, wherein the fluorescent moiety is a laser that emits at the moiety's excitation wavelength. The laser can emit at least about 200 photons when stimulated by a laser, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent portion, and the total energy directed by the laser to the spot is about 3 microns. joules or less. In some embodiments of the compositions of the invention, the binding partner comprises an antibody against a 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 antibodies are specific to particular regions of the Troponin molecule. In some embodiments, the antibody is specific to a region comprising amino acids 27-41 of cardiac troponin I. In some embodiments of the compositions of the invention, the fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring is a chemically reactive group or a conjugated including groups. In some embodiments, the fluorescent moiety comprises a dye that can be AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, or AlexaFluor700. In some embodiments, the fluorescent moiety comprises AlexaFluor647.

In some embodiments, the invention includes a composition comprising a set of standards for determination of cardiac Troponin concentration, wherein at least one standard has a cardiac Troponin concentration of less than about 10 pg/ml.

In some embodiments, the invention includes a kit comprising a composition comprising an antibody to cardiac troponin conjugated to a fluorochrome moiety, the fluorochrome moiety when stimulated by a laser that emits at the excitation wavelength of the moiety can emit at least about 200 photons at a point, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, the total energy directed to the spot by the laser is no more than about 3 microjoules; The composition is packaged in suitable packaging. In some embodiments of the kits of the invention, the cardiac troponin is cardiac troponin I or cardiac troponin T. In some embodiments, the cardiac troponin is cardiac troponin-I. In some embodiments of the kit of the invention, the kit further comprises instructions. In some embodiments of the kit of the present invention, the kit further comprises a composition comprising a capture antibody for cardiac troponin I bound to a solid support. In some embodiments, the solid support comprises microtiter plates or paramagnetic microparticles. In some embodiments of the kit of the invention, the kit further comprises a component selected from the group consisting of wash buffers, assay buffers, elution buffers and calibrator diluents. Some embodiments of kits of the present invention further include standards of cardiac troponin.

INCORPORATION BY REFERENCE All publications and patent applications mentioned in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. be taken in.

The novel features of the invention are set forth with particularity in the appended claims. A more complete understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the invention are employed, and the accompanying drawings. be.

FIG. 2 shows a schematic representation of the layout of the components of a single particle analyzer, showing an analyzer comprising one electromagnetic radiation source and one electromagnetic detector; Fig. 2 shows a schematic representation of the arrangement of components of a single particle analyzer, showing an analyzer comprising two electromagnetic radiation sources and one electromagnetic detector; FIG. 2 shows a schematic representation of a capillary flow cell of a single particle analyzer, showing a flow cell of an analyzer containing one electromagnetic radiation source. FIG. 3 shows a schematic representation of a capillary flow cell of a single particle analyzer, showing a flow cell of an analyzer containing two sources of electromagnetic radiation. FIG. 2 shows a schematic diagram showing a conventional arrangement of laser and detector optics for a single particle analyzer, showing an analyzer arrangement with one electromagnetic radiation source and one electromagnetic detector; FIG. 3 shows a schematic diagram showing the confocal arrangement of the laser and detector optics of a single particle analyzer, showing the arrangement of an analyzer with two electromagnetic radiation sources and two electromagnetic detectors. FIG. 3 shows a linearized standard curve for a range of concentrations of cTnI. Figure 2 shows that the cTnI biological threshold (cut-off concentration) is at a cTnI concentration of 7 pg/ml when established at the 99th percentile with a corresponding CV of 10%. FIG. 2 shows the correlation between cTnI assay results determined using the analyzer system of the invention and standard measurements provided by the National Institute of Standards and Technology (R2=0.9999). FIG. 1 shows detection of cTnI in serial serum samples obtained from patients presenting with chest pain in the emergency room. FIG. 4 shows a comparison of measurements made with the analyzer system of the invention and measurements made by commercially available assays. FIG. 2 shows the distribution of cTnI concentrations in serum samples obtained from patients exhibiting normal (non-ischemic) biological concentrations of cTnI and chest pain.

Overview I. Introduction II. cardiac troponin III. Cardiac troponin A labeling A. Binding Partners of Troponin 1 . antibody2. Cross-reacting antibodies B. Fluorescent moieties used with binding partners1. dye2. quantum dots C.I. Binding Partner Fluorescent Moiety Composition IV. High Sensitivity Analysis of Cardiac TroponinsA. Sample B. Sample preparationC. Troponin Detection and Concentration DeterminationV. Instruments and Systems Suitable for Sensitive Analysis of TroponinA. Device/system B. Single Particle Analyzer1. source of electromagnetic radiation2. capillary flow cell3. Driving force 4. detector C. Sampling system D. Sample preparation systemE. Sample recovery VI. Method A. Using Sensitive Analysis of Cardiac Troponin. Sample B. Diagnosis, Prognosis, or Determination of Treatment 1. Acute myocardial infarction2. Conditions other than AMI a. cardiotoxicity C.I. Business Methods VII. Composition VIII. kit

I. Introduction

The present invention provides compositions and methods for sensitive detection of troponins, such as cardiac troponins. The release of myocardial isoforms of troponin (cardiac troponin I and/or T) into the blood that are unique to the myocardium is indicative of damage to the myocardium, for their use as prognostic markers or to aid in treatment decisions. provide the basis for

The troponin complex in muscle consists of troponins I, C, and T. Troponin C, one from cardiac and slow-twich muscle, one from first-twich muscle
It exists as two isoforms from , and is found in virtually all striated muscles, thus limiting its use as a specific marker. In contrast, troponin I and T are expressed as different isoforms in slow, fast, and cardiac muscle. The unique cardiac isoforms of troponin I and T allow them to be immunologically distinguished from other troponins in skeletal muscle. Thus, the release of cardiac troponins I and T into the blood is indicative of damage to the myocardium and provides the basis for their use as diagnostic and prognostic markers or to aid in therapeutic decisions.

Currently used markers of cardiac injury suffer from the drawback that their clinical usefulness is limited. 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 clearing infarcts by 10 to 12 hours after chest pain onset. Earlier diagnosis would have very particular advantages with respect to fibrinolytic therapy and triage.

Troponin is an extremely sensitive and specific marker of cardiac injury 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 source. In addition to cardiac infarction, many other conditions can cause damage to the myocardium, and early diagnosis of such damage could prove useful to clinicians. However, current methods of detection and quantification have sufficient sensitivity to detect the release of cardiac troponin into the blood until levels of abnormally high concentrations, e.g., 0.1 ng/ml or higher, are reached. not

Accordingly, the methods and compositions of the present invention provide methods and compositions for sensitive detection and quantification of cardiac troponin, as well as diagnostic, prognostic, and/or therapeutic methods based on such sensitive detection and quantification. It includes compositions and methods for therapeutic determination.

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

In some embodiments, the present invention provides a concentration of total cardiac Troponin, i.e., a sample (e.g., blood, serum Methods and compositions are provided for the detection and/or determination of the sum of all or a substantial portion of cardiac troponin in a plasma sample). In some embodiments, the cardiac troponin is cTnl, in others cTnT, and in yet other embodiments the cardiac troponin is cTnl and cTnT. It is well understood that a complete total measurement need not be performed as long as a consistent percentage of total can be determined and compared to a standard value. It will also be appreciated that when that form of troponin is a minority component of the total, no or low levels of detection of that form will not significantly affect the measurement of total troponin. . Thus, as used herein, "total cardiac troponin" is a measurement intended to measure all or substantially all forms of a particular cardiac troponin in a sample, such as all cTnl or all cTnT. Refers to a value, where sample-to-sample consistency is one in which clinically relevant conclusions can be drawn from a comparison of a sample to a standard or from a comparison of one sample to another.

In some embodiments, the present invention provides one or more different forms of troponin, such as complexed cTnl, free cTnl, muddied c, as separate entities in a sample.
Methods and compositions are provided for detecting and/or determining the concentration of Tnl (e.g., oxidized or phosphorylated), or complexed cTnT, free cTnT, Madid cTnT (e.g., oxidized or phosphorylated), 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 provides methods for 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 provide a method of providing a determination method. In some embodiments, two or more forms are detected, e.g., by performing multiplexed assays for different entities on a single sample, or separate on aliquots from the same or similar samples. Assays can be performed to determine the concentration of the various forms. Various forms of concentration ratios can be obtained. For example, the ratio of the concentration of a particular form, eg, a fragment, complex, or modified form of cardiac troponin, to the concentration of total cardiac troponin can be determined. These ratios and/or absolute values can provide meaningful clinical information. For example, the relative proportions of cardiac troponin fragments 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, US Pat. No. 6,991,907, which is incorporated herein by reference in its entirety.

m. Labeling of Cardiac Troponins In some embodiments, the present invention provides methods and compositions comprising labels for sensitive detection and quantification of cardiac Troponins.

Those skilled in the art are well aware that many strategies can be used to label target molecules to enable detection or discrimination in a mixture of particles. Labels can be attached by any known means, including methods that utilize non-specific or specific interactions of label and target. A label can provide a detectable signal or affect the movement of particles in an electric field. Additionally, labeling can be accomplished directly or through a binding partner.

In some embodiments, the label comprises a troponin binding partner attached to a fluorescent moiety.

A. Binding Partners of Troponin Any suitable binding partner having the required specificity for the form of cardiac 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. can be used. Typically, such binding partners bind to a region of cardiac troponin common to all or most of the different forms likely to be found in the sample. In some embodiments, one, such as a complexed cTnl, free cTnl, maded cTnl (e.g., oxidized or phosphorylated), or a binding partner of complexed cTnT, free cTnT, maded cTnT (e.g., oxidized or phosphorylated) Binding partners specific for the above specific forms of cardiac 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 cardiac troponin. As used herein, the term "antibody" is a broad term and is not limited to naturally occurring antibodies, as well as non-naturally occurring antibodies, including, for example, single-chain antibodies, chimeric antibodies, bifunctional antibodies, and humanized antibodies. , as well as antigen-binding fragments thereof, in their ordinary 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 includes antibodies to both cTnl and cTnT. The antibodies are specific for all or substantially all forms of cardiac 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 cardiac troponin, e.g., complexed cTnl, free cTnl, maded cTnl (e.g., oxidized or phosphorylated), or Antibodies specific for binding partners (eg, oxidized or phosphorylated) can be used. Mixtures of antibodies, for example, against cTnl and cTnT, or against various forms of troponin (free, conjugated, etc.), or mixtures of mixtures, are also encompassed by the present invention.

It is well understood that the choice of the troponin epitopes or regions against which antibodies are raised determines its specificity for e.g. whole troponin, certain fragments, conjugated troponins, modified troponins. It is In some embodiments, the antibody is specific to a particular amino acid region of cardiac troponin. In some embodiments, the antibody is specific to specific amino acids 27-41 of human cardiac 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 cardiac troponin I. In some embodiments, the antibody is not affected by heparin, phosphorylation, oxidation, and troponin complex formation and does not cross-react with skeletal muscle troponin-I.

Methods for making antibodies are well established. Cardiac specific sequences for Troponin I and Troponin T are described in FEBS Lett. 270, 57-61 (1990) and Genomics 21, 311-316 (1994). See, for example, Antibody, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, N.G. 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 antibody-mimicking binding or Fab fragments can be prepared from genetic information by a variety of techniques (Antibody Engineering: A Practical Approach (Borrebaeck, C, ed.), 1995, Oxford University). Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Methods for generating antibodies to various conjugated, fragmented, phosphorylated, and oxidized forms of Troponin are described in U.S. Pat. Nos. 5,579,687; 6,991,907; , which are incorporated herein by reference in their entirety. A synthetic peptide of 14 amino acids that mimics the heart specific sequence of Troponin I and methods for preparing antibodies to the peptide are described in International Patent Application PCT/US94/05468. Monoclonal and polyclonal antibodies against free and complexed cardiac troponin are also commercially available (HyTest, HyTest Ltd., Turku, Finland; Abeam Inc., Cambridge, MA, USA; Life Diagonosis, Inc., West Chester, PA). BiosPacific, Emeryville, Canada).

In some embodiments, the antibody is a mammalian, eg, goat polyclonal anti-cTnl antibody. The antibody can be specific for a particular region of cTnl, eg, amino acids 27-41 of human cardiac troponin I. Capture binding partner and detection binding partner pairs, eg, capture and detection antibody pairs, can be used in embodiments of the invention. Thus, in some embodiments, a heterogeneous assay protocol is used, typically using two binding partners, eg, two antibodies. One binding partner is a capture partner, usually immobilized on a solid support, and the other binding partner is a detection binding partner, typically to which a detectable label is attached. In some embodiments, the capture binding partner member of the pair is an antibody specific for all or substantially all forms of cardiac troponin. One example is an antibody, eg, a monoclonal antibody, specific for free cardiac troponin I (cTnl) amino acids 41-49 and cTnl forming complexes with other troponin components. The antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complex formation, and preferably does not cross-react with skeletal muscle troponin-I. Therefore, the antibody is believed to bind all cTnl. Another example is a monoclonal antibody specific for cardiac 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. Other antibody pairs are known and can be designed.

In some embodiments, it is beneficial to use antibodies that cross-react with different species as capture antibodies, detection antibodies, or both. Such embodiments include measurement of drug toxicity, eg, by determining the release of cardiac troponin into the blood as a marker of heart damage. Cross-reacting antibodies allow toxicity studies to be performed in species other than humans, e.g., to studies or clinical observations in other species, e.g. humans, using the same antibody or pair of antibodies in the reagents of the assay. and its results can be directly transferred, thus reducing assay-to-assay variability. Thus, in some embodiments, one or more antibodies used as binding partners to a marker, eg, cardiac troponin, eg, cardiac troponin I, may be cross-reactive antibodies. In some embodiments, the antibody cross-reacts with a marker, eg, cardiac troponin, from at least two species selected from the group consisting of human, monkey, dog, and mouse. In some embodiments, the antibody cross-reacts with markers, eg, cardiac troponin, from all of the group consisting of human, monkey, dog, and mouse.

B. Fluorescent Moieties for Use with Binding Partners In some embodiments of the labels used in the invention, the binding partner, eg, an antibody, is attached to a fluorescent moiety. The fluorescence of the fluorescent moiety is sufficient to allow detection with a single molecule detector, such as the single molecule detectors described herein. As used herein, the term "fluorescent moiety" refers to one or more fluorescent entities whose total fluorescence is such that the moiety can be detected with the single molecule detectors described herein. including. Thus, a fluorescent moiety can include a single entity (eg, quantum dots or fluorescent molecules) or multiple entities (eg, multiple fluorescent molecules). When the term "moiety" as used herein refers to a group of fluorescent entities, e.g. a plurality of fluorophores, each individual entity may be separately bound to a binding partner so long as the entities as a group provide sufficient fluorescence to be detected. It will be appreciated that the entities can be joined together or joined together.

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

Additionally, the fluorescent moiety has properties consistent with its use in the assay of choice. In some embodiments the assay is an immunoassay and 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 undergo aggregation consistent with the required accuracy and precision 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 analyzers and Labeling a biomolecule of interest (e.g., protein) so that it can be analyzed using the system (e.g., without causing precipitation of the protein of interest and precipitation of proteins conjugated with fluorescent moieties) Fluorescent moieties such as dye molecules that have a combination of compatibility with

Fluorescent moieties, eg, a single fluorophore or multiple fluorophores, useful in some embodiments of the present invention can be defined by their photon emission properties when stimulated by EM radiation. For example, in some embodiments, the present invention provides an average of at least about 10, 20, 30, 40, 50, 75, 100, 125, 150, when stimulated by a laser emitting at the excitation wavelength of the fluorescent moiety. fluorophore moieties, such as a single fluorophore or a plurality of fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less. It will be appreciated that the total energy can be achieved by many different combinations of laser power and dye moiety exposure time. For example, a laser with an output of 1 mW can be used for 3 milliseconds, 3 mW for 1 millisecond, 6 mW for 0.5 milliseconds, 12 mW for 0.25 milliseconds, and so on.

In some embodiments, the present invention provides a fluorochrome moiety, such as a single Utilizing a fluorophore or fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less. In some embodiments, the present invention provides a fluorochrome moiety, such as a single Utilizing a fluorophore or fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less. In some embodiments, the present invention provides a fluorochrome moiety, such as a single Utilizing a fluorophore or fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less. In some embodiments, the present invention provides a fluorochrome moiety, such as a single Utilizing a fluorophore or fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less. In some embodiments, the present invention provides a fluorochrome moiety, such as a single Utilizing a fluorophore or fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less. In some embodiments, the present invention provides a fluorochrome moiety, such as a single Utilizing a fluorophore or fluorophores, the laser is focused to a spot of about 5 microns or more in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less.

In some embodiments, the fluorescent moiety comprises an average of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, eg, fluorescent molecules. In some embodiments, the fluorescent moiety includes an average of only about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 fluorescent entities, eg, 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 comprise 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 comprises 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 the fluorescent moiety, determined by standard analytical methods, in a given sample, where the sample is representative of the group of labels of the invention, wherein the sample contains a plurality of binding partner fluorescent moiety units. It is meant that the molar ratio of the particular fluorescent entity to the binding partner corresponds to the specified number or range of numbers. For example, in embodiments where the label includes a binding partner that is an antibody and a fluorescent moiety that includes multiple fluorophores of specific absorbance, the solution of label is diluted to an appropriate level and the molarity of the protein (antibody) is determined. A spectrophotometric assay can be used to obtain the absorbance at 280 nm to determine the molar concentration of the fluorophore, for example at 650 nm (for AlexaFluor 647). The molar concentration ratio of the latter to the former represents the average number of fluorescent entities (dye molecules) in the fluorescent moiety bound to each antibody.

1. Dyes In some embodiments, the present invention utilizes fluorescent entities that include fluorophores. In some embodiments, the present invention utilizes fluorophores capable of emitting an average of at least about 50 photons when stimulated by a laser emitting at the excitation wavelength of the fluorophore, wherein the laser is Focused to a spot of about 5 microns or greater in diameter containing the molecule, the total energy directed at the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention utilizes fluorophores capable of emitting an average of at least about 75 photons when stimulated by a laser emitting at the excitation wavelength of the fluorophore, wherein the laser is Focused to a spot of about 5 microns or greater in diameter containing the molecule, the total energy directed at the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention utilizes fluorophores capable of emitting an average of at least about 100 photons when stimulated by a laser emitting at the excitation wavelength of the fluorophore, wherein the laser is Focused to a spot of about 5 microns or greater in diameter containing the molecule, the total energy directed at the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention utilizes fluorophores capable of emitting an average of at least about 150 photons when stimulated by a laser emitting at the excitation wavelength of the fluorophore, wherein the laser is Focused to a spot of about 5 microns or greater in diameter containing the molecule, the total energy directed at the spot by the laser is about 3 microjoules or less. In some embodiments, the present invention utilizes fluorophores capable of emitting an average of at least about 200 photons when stimulated by a laser emitting at the excitation wavelength of the fluorophore, wherein the laser is Focused to a spot of about 5 microns or greater in diameter containing the molecule, the total energy directed at the spot by the laser is about 3 microjoules or less.

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

Dyes suitable for use in the present invention include modified carbocyanine dyes. Modification of carbocyanine dyes involves modification of the indolium ring of carbocyanine dyes to accept a reactive group or conjugating agent at the 3-position. Modification of the indolium ring is more uniform and , providing dye conjugates with substantially more fluorescence. In addition to having stronger fluorescence emission than structurally similar dyes at substantially the same wavelengths and having reduced absorption spectral effects upon conjugation to biopolymers, the modified carbocyanine dyes exhibit peak absorbance peak absorbance. It has higher photostability and higher absorbance (extinction coefficient) than structurally similar dyes at wavelengths. Thus, modified carbocyanine dyes provide increased sensitivity in 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-position carbon of the indolium ring comprises a chemically reactive group or conjugating agent. Other dye compounds include compounds incorporating an azabenzazolium ring moiety and at least one sulfonic acid moiety. Modified carbocyanine dyes that can be used to detect individual particles in various embodiments of the present invention are described in US Pat. No. 6,977,305, which is incorporated herein by reference in its entirety. It is Thus, in some embodiments, the labels of the invention are fluorescent dyes comprising a substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring comprises a chemically reactive group or a conjugated substance group. use.

In some embodiments, the label comprises a fluorescent moiety comprising one or more Alexa dyes (Molecular Probes, Eugene, Oreg.). Alexa dyes are disclosed in US Pat. Nos. 6,977,305, 6,974,874, 6,130,101, and 6,974,305, which are incorporated herein by reference in their entirety. Some embodiments of the present invention utilize dyes selected from the group consisting of AlexaFluor647, AlexaFluor488, AlexaFluor532, AlexaFluor555, AlexaFluor610, AlexaFluor680, AlexaFluor700, and AlexaFluor750. Some embodiments of the present invention utilize dyes selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor700, and AlexaFluor750. Some embodiments of the present invention utilize the AlexaFluor647 molecule, which has an absorption maximum of about 650-660 nm and an emission maximum of about 660-670 nm. AlexaFluor647 dye is used alone or together with other AlexaFluor dyes.

Additionally, currently available organic phosphors can be improved by weakening their hydrophobicity by adding hydrophilic groups such as polyethylene. Alternatively, sulfonated organic fluorophores, such as the AlexaFluor 647 dye, can now be made less acidic by making them zwitterionic. Particles such as antibodies labeled with modified fluorophores are less likely to bind non-specifically to surfaces and proteins in immunoassays, thus allowing higher sensitivity and lower background assays. . Methods are known in the art for modifying and improving the properties of fluorescent dyes for the purpose of increasing the sensitivity of single particle detection systems. Preferably, the modification improves the Stokes shift while maintaining a high quantum yield.

2. Quantum Dots In some embodiments, the fluorescent labeling moieties used to detect molecules in a sample using the analyzer systems of the invention are quantum dots. Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals ranging from 2-10 nm containing 100-1,000 electrons. Some QDs can be 10-20 nm in diameter. QDs have a high quantum yield, which makes them particularly useful for optical applications. QDs are fluorophores that fluoresce through the formation of excitons that can be thought of as excited states of traditional fluorophores but have much longer lifetimes of up to 200 ns. This property provides QDs with low photobleaching. The energy level of the QDs can be controlled by varying the size and shape of the QDs and the depth of the QD potential. One of the optical properties of small excitable QDs is coloration, which is determined by the dot size. The larger the dot, the redder the fluorescence or closer to the red end of the spectrum. The smaller the dot, the bluer it is or closer to the blue end. The bandgap energy, which determines the energy and thus the color of the fluorescence emission, is inversely proportional to the square of the QD size. Larger QDs have more energy levels that are more closely spaced, thus allowing the QDs to absorb photons that contain less energy, ie, those closer to the red end of the spectrum. Since the emission frequency of the dots is determined by the bandgap, it is possible to control the output wavelength of the dots with the highest precision. In some embodiments, proteins detected with a single particle analyzer system are labeled with QDs. In some embodiments, to detect one QD-labeled protein, a single particle analyzer is used with filters that allow detection of different proteins at different wavelengths. QDs have broad excitation and narrow emission characteristics, which, when used with color filtering, require a single electromagnetic radiation for multiplexed analysis of multiple targets in a single sample to resolve individual signals. Requires only a source. Thus, in some embodiments, the analyzer system includes one continuous wave laser and particles labeled with one QD each. Colloidally prepared QDs are free-floating and can bind to a variety of molecules through metal coordinating functional groups. 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 their surfaces, quantum dots can be dispersed or dissolved in almost any solvent or incorporated into a variety of inorganic and organic films. Quantum dots (QDs) can be conjugated directly to streptavidin via a maleimide ester conjugation reaction or to antibodies via a maleimide-thiol conjugation reaction. This results in a material with biomolecules covalently attached to the surface, producing conjugates with high specific activity. In some embodiments, one quantum dot labels the protein that is detected by the single particle analyzer. In some embodiments, the quantum dots are 10-20 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 QD605.

C. Binding Partner Fluorescent Moiety Compositions The labels of the present invention are composed of a binding partner (e.g., an antibody) attached to a fluorescent moiety to provide the required fluorescence for detection and quantification on the instruments described herein. generally includes Any suitable combination of binding partners and fluorescent moieties for detection in the single molecule detectors described herein can be used as labels in the present invention. In some embodiments, the invention provides a label for a molecule or fragment, complex, phosphorylated, or oxidized form of cardiac troponin, the label comprising an antibody to cardiac troponin and a fluorescent moiety. The antibody can be any antibody as described herein, eg, an antibody against cTnT or cTnl. In some embodiments, the antibody is an antibody to cTnl. In some embodiments, the antibody is specific to a particular region of cardiac troponin, eg, to amino acids 27-41 of human cTnl. In some embodiments, the invention provides a composition comprising a fluorescent moiety attached to an anti-cTnl antibody, e.g. I will provide a. Fluorescent moieties have an average of 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, the laser is focused to a spot about 5 microns or more in diameter containing the label, and the laser The total energy directed to the spot by is less than about 3 microjoules. In some embodiments, a fluorescent moiety is capable of emitting an average of at least about 50, 100, 150, or 200 photons when stimulated by a laser emitting at the excitation wavelength of the fluorescent moiety. , the laser is focused to a spot about 5 microns or greater in diameter containing the fluorescent portion, and the total energy directed at the spot by the laser is about 3 microjoules or less. The fluorescent moiety comprises a substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring may be a fluorescent moiety comprising one or more dye molecules having a structure comprising a chemically reactive group or a conjugated substance group. The labeling composition can comprise a fluorescent moiety comprising one or more dye molecules selected from the group consisting of AlexaFluor 488, 532, 647, 700, or 750. The labeling composition can comprise a fluorescent moiety comprising one or more dye molecules selected from the group consisting of AlexaFluor 488, 532, 700, or 750. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor488. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor555. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor610. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor647. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor680. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor700. The labeling composition can include a fluorescent moiety that includes one or more dye molecules that are AlexaFluor750.

In some embodiments, the invention provides an AlexFluor molecule, amino acid 2 of human ctnI.
Compositions are provided for the detection of cardiac Troponin I comprising an AlexaFluor molecule selected from the described group such as the AlexaFluor647 molecule conjugated to an antibody such as the goat polyclonal anti-cTnl antibody specific for 7-41. In some embodiments, the composition has an average of 1-11, or about 2-10, or about 2-8, bound antibodies, such as goat polyclonal anti-cTnl antibodies specific for amino acids 27-41 of human ctnI. or about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4-8, or about 4-6, or about 2, 3, 4, 5, 6, or greater than about 6 AlexaFluor 647 molecules. In some embodiments, the invention provides an average of 1-11, or about 2-10, or about 2-8, bound antibodies such as goat polyclonal anti-cTnl antibodies specific for amino acids 27-41 of human ctnI. or about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or Compositions are provided for the detection of cardiac Troponin I comprising about 4-8, or about 4-6, or about 2, 3, 4, 5, 6, or greater than about 6 AlexaFluor647 molecules. In some embodiments, the invention provides for the detection of cardiac 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. provides a composition for In some embodiments, the invention provides goat polyclonal anti-cTnl specific for amino acids 27-41 of human ctnI.
A composition for the detection of cardiac Troponin I is provided comprising an average of about 2-8 AlexaFluor647 molecules conjugated to an antibody, such as an antibody. In some embodiments, the invention provides for the detection of cardiac troponin I comprising an average of about 2-6 AlexaFluor647 molecules conjugated to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. provides a composition for In some embodiments, the invention provides for the detection of cardiac troponin I comprising an average of about 2-4 AlexaFluor647 molecules conjugated to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. provides a composition for In some embodiments, the invention provides amino acids 27-41 of human ctnI.
compositions for the detection of cardiac troponin I comprising an average of about 3-8 AlexaFluor 647 molecules conjugated to an antibody such as a goat polyclonal anti-cTnl antibody specific for . In some embodiments, the invention provides for the detection of cardiac troponin I comprising an average of about 3-6 AlexaFluor647 molecules conjugated to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. provides a composition for In some embodiments, the invention provides for the detection of cardiac troponin I comprising an average of about 4-8 AlexaFluor647 molecules conjugated to an antibody such as a goat polyclonal anti-cTnl antibody specific for amino acids 27-41 of human ctnI. provides a composition for

Binding of a fluorescent moiety or a fluorescent entity that produces a fluorescent moiety to a binding partner, eg, an antibody, can be by any suitable means. Such methods are known in the art and exemplary methods are provided in the Examples. In some embodiments, after conjugation of a fluorescent moiety to a binding partner to form a label for use in the methods of the invention and prior to utilizing the label to label a protein of interest, filtration is performed. It is useful to carry out the steps. For example, an antibody-dye label can be filtered prior to use, eg, through a 0.2 micron filter or any filter suitable for removing aggregates. Other reagents used in assays of the invention can also be filtered through, for example, a 0.2 micron filter or any suitable filter. Without wishing to be bound by theory, it is believed that such filtration removes some of the antibody-dye label aggregates, for example. Such aggregates bind the protein of interest as a unit, but are prone to disintegration upon release in the elution buffer, and may give rise to false positives. That is, several labels are detected from aggregates bound only to a single protein molecule of interest. Regardless of theory, filtration was found to reduce false positives in subsequent assays and improve accuracy and precision.

IV. Sensitive Analysis of Cardiac Troponin In one aspect, the invention provides the steps of i) labeling the molecule, fragment, or complex with a label, if present, and ii) detecting the presence or absence of the label, comprising: detecting the presence of the label indicates the presence of a single molecule, fragment, or complex of cardiac Troponin in the sample; method. As used herein, a "cardiac troponin molecule" includes the entire native amino acid sequence of a particular type of cardiac troponin, including post-translationally modified forms (e.g., phosphorylated forms) as well as oxidized or chemically altered forms. Molecules that substantially comprise are included. A "fragment" of a molecule, as used herein, includes a molecule of cardiac troponin that is shorter than the entire native amino acid sequence, including modifications to the entire molecule. As used herein, a "complex" of a cardiac Troponin molecule includes a cardiac Troponin molecule or fragment associated with one or more other molecules or substances (e.g., associated with one or more other cardiac Troponin molecules). included. In some embodiments, the method comprises about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, Troponin can be detected at detection limits of less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 pg/ml. be. 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 of less than about 10 pg/ml. In some embodiments, the method is capable of detecting troponin with a detection limit of less than about 5 pg/ml. In some embodiments, the method is capable of detecting troponin with a detection limit of less than about 3 pg/ml. In some embodiments, the method is capable of detecting troponin with a detection limit of less than about 1 pg/ml. Detection limits can be determined by using suitable standards, such as the National Institute of Standards and Technology standards, eg, cTnl standards.

This method also provides a method of determining the concentration of cardiac troponin in a sample by detecting a single molecule of troponin in the sample. "Detection" of a single molecule of troponin includes detecting the molecule directly or indirectly. For indirect detection, a label corresponding to a single molecule of cardiac troponin, eg, a label bound to a single molecule of cardiac troponin, can be detected.

The type of cardiac Troponin for detection is as described herein, e.g. . In some embodiments, total cardiac troponin is detected and/or quantified. In some embodiments, total cTnT is detected. In some embodiments, total cTnl is detected and/or quantified.

A. The sample sample can be any suitable sample. Typically the sample is a biological sample, such as a biological fluid. Such fluids include, but are not limited to, exhaled breath condensate (EBC), bronchoalveolar lavage fluid (BAL), blood, serum, plasma,
Urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, gastric juice, lymphatic fluid, interstitial fluid, tissue homogenate, cell extract, saliva, sputum, feces, physiological secretions, tears, mucus, sweat, milk , semen, seminal plasma, vaginal secretions, fluids obtained from ulcers and other surface rashes, blisters, and abscesses, and tissue extracts, including biopsies of normal, malignant, and suspicious tissue, or targeted particles of interest Any other component of the body that may contain Also of interest are cell or tissue cultures, or other similar specimens such as media.

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 cardiac troponin that is desired to be measured, where the label is detectable in the instruments described herein. As is known in the art, preparation of the sample to label one or more particles can be done in homogeneous or heterogeneous formats. In some embodiments, the sample preparation is formed in a homogeneous format. In analyzer systems using homogeneous formats, unbound label is not removed from the sample. See, eg, US patent application Ser. No. 11/048,660, which is incorporated herein by reference in its entirety. In some embodiments, the particle or particles of interest are labeled by adding a labeled antibody or antibodies that bind to the particle or particles of interest.

In some embodiments, a heterogeneous assay format is used, in which steps are typically used to remove 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 format, a molecule of interest, such as a marker of a biological state, is captured using a capture binding partner, such as on a solid support. Unwanted molecules and other substances are then optionally washed away, followed by binding of detection binding partners and labels, including detectable labels (eg, fluorescent moieties). Further washing removes unbound label, which then usually, but not necessarily, releases detectable label 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 variations will be apparent to those skilled in the art.

In some embodiments, methods for detecting troponin particles employ sandwich assays with antibodies, such as monoclonal antibodies, as capture binding partners. The method involves binding troponin molecules in the sample to a capture antibody immobilized on a binding surface, and binding a label comprising a detection antibody to the troponin to form a "sandwich" complex. Labels include detectable fluorescent labels as described herein, eg, detected using the single molecule analyzers 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 to Grubb et al. and US Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by reference. Further examples specific to cardiac troponin are described in the Examples.

A capture binding partner may be attached to a solid support such as a microtiter plate or paramagnetic beads. In some embodiments, the invention provides binding partners for cardiac troponin that are attached to paramagnetic beads. Any suitable binding partner that is specific for the type of cardiac troponin it is desired to capture can be used. A binding partner may be an antibody, such as a monoclonal antibody. Antibodies are specific for free cardiac troponin (cTnl or cTnT) or complexed cardiac troponin, modified cardiac troponin, or fragments of cardiac troponin as described herein, or antibodies It can be specific for all or substantially all forms of cardiac troponin, such as cTnl or cTnT, that would be found in . The production and sources of antibodies against cardiac troponin are described elsewhere herein. Preferred antibody forms for measuring total troponin are those that are substantially unaffected by heparin, phosphorylation, oxidation, and troponin complex formation and do not cross-react with skeletal muscle troponins such as troponin-I. In some embodiments, the antibody is specific to a particular region of cardiac troponin. In some embodiments, the region includes amino acids 41-49 of human cardiac troponin I. In some embodiments, the region includes amino acids 87-91 of human cardiac troponin I. Such antibodies are well known in the art and commercially available, eg, from BiosPacific, Emeryville, Canada. Examples of capture antibodies useful in embodiments of the present invention are antibodies reactive with free cardiac troponin I (cTnl) amino acids 41-49 and cTnl complexed with other troponin components, eg, monoclonal antibodies. The antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complex formation, and preferably 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 embodiments of the present invention is an antibody, such as a monoclonal antibody, that reacts with free cardiac troponin I (cTnl) amino acids 87-91 and cTnl complexed with other troponin components. . The antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complex formation, and preferably does not cross-react with skeletal muscle troponin-I. An exemplary antibody of this type is monoclonal antibody clone number A34440228P commercially available from BiosPacific, Emeryville, Canada. It will be appreciated that antibodies identified herein as being useful as capture antibodies can also be useful as detection antibodies, and vice versa.

Binding of a binding partner, such as an antibody, to the solid support may be covalent or non-covalent. In some embodiments, the binding is non-covalent. One example of non-covalent binding that is well known in the art is the biotin-avidin/streptavidin interaction. Thus, in some embodiments, solid supports such as microtiter plates or paramagnetic beads are attached 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 covalent. Thus, in some embodiments, a solid support such as a microtiter plate or paramagnetic beads is covalently attached to a capture binding partner such as an antibody. Covalent attachment is particularly useful where the orientation of the capture antibody is optimized for capturing the molecule of interest. For example, in some embodiments, solid supports such as microtiter plates or paramagnetic microparticles can be used where binding of a binding partner such as an antibody is oriented, such as covalently oriented.

An exemplary protocol for oriented binding of antibodies 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-cold 20 mM sodium periodate in an equal volume of 0.1 M sodium acetate (pH 5.5) solution. IgG is allowed to oxidize 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 allowed to react with hydrazide-activated multiwell plates for at least 2 hours at room temperature. Unbound IgG is removed by washing the multiwell plate with borate buffered saline or another suitable buffer. Plates may be dried for storage if desired. A similar protocol can be followed for microbeads if the material of the microbeads is suitable for such binding.

In some embodiments, the solid support is a microtiter plate. In some embodiments, the solid support is a paramagnetic bead. 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 antibodies to paramagnetic beads are well known in the art. An example is given in the Examples.

A cardiac troponin of interest is contacted with a capture binding partner, such as a capture antibody immobilized on a solid support. A number of sample preparations may be used, such as preparation of serum from a blood sample or a concentration procedure prior to contacting the sample with capture antibodies. Protocols for binding proteins in immunoassays are well known in the art and are included in the Examples.

The time allowed for binding will vary depending on the conditions. It will be apparent that in some circumstances, particularly clinical settings, shorter binding times are desirable. The use of paramagnetic beads and the like can reduce the time required for binding. 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 about 12, 10, 8, 6, 4, 3, 2, or 1 hour, or about Less than 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some embodiments, the time allowed 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 allowed 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 allowed 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 allowed 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 allowed 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 allowed 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 allowed 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, after binding of the troponin particles to a capture binding partner, such as a capture antibody, particles that may be non-specifically bound, and other unwanted substances in the sample, are washed away and specifically bound. leaving substantially only the troponin particles with It will be appreciated that in other embodiments, no wash is used between sample and label additions, thereby reducing sample preparation time even further. Thus, in some embodiments, the time allowed for both the binding of the protein of interest to a capture binding partner, such as an antibody, and the binding of the label to the protein of interest is about 12, 10, 8, 6, less 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 time allowed 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 time allowed 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 time allowed 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 time allowed 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 time allowed 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 15 minutes. In some embodiments, the time allowed 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 time allowed 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 target analytes, can be derived from animal serum. Endogenous human heterophile antibodies, or human anti-animal antibodies capable of binding immunoglobulins of other species, are present in more than 10% of patient sera or plasma. These circulating heterophile antibodies can interfere with immunoassay measurements. In sandwich immunoassays, these heterophile antibodies can cross-link capture and detection (diagnostic) antibodies, thereby generating false-positive signals, or block binding of diagnostic antibodies, thereby generating false-negative signals. can be generated. In a competitive immunoassay, a heterophile antibody can bind to the assay antibody and inhibit its binding to troponin. They can also prevent or enhance the separation of antibody-troponin complexes from free troponin, particularly when anti-species antibodies are used in separation systems. Therefore, it is difficult to predict the interfering impact of these heterophile antibodies. It is therefore advantageous to block the binding of any heterophile antibodies. In some embodiments of the invention, the immunoassay comprises depleting heterophile antibodies from the sample using one or more heterophile antibody blocking agents. Methods for removing heterophilic antibodies from samples tested in immunoassays are known and include heating the sample in sodium acetate buffer (pH 5.0) at 90° C. for 15 minutes and centrifuging at 1200 g for 10 minutes. Alternatively, heterophile antibodies can be precipitated using polyethylene glycol (PEG); immunoextraction of interfering heterophile immunoglobulins from the specimen using protein A or protein G. or adding non-immune mouse IgG. Embodiments of the methods of the present invention contemplate sample preparation prior to analysis with a single molecule detector. The suitability of the method of pretreatment can be determined. Biochemicals are commercially available to minimize immunoassay interference caused by heterophile antibodies. For example, a product called MAK33, an IgG1 monoclonal antibody against h-CK-MM, is available from Boehringer Mannheim. The MAK33 Plus product contains 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 commercial source of biochemicals for neutralizing heterophile antibodies is the Immunoglobulin Inhibiting Reagent sold by Bioreclamation Inc, East Meadow, NY. This product is a preparation of immunoglobulins (IgG and IgM) from multiple species, primarily murine IgG2a, IgG2b, and IgG3 from Balb/c mice. In some embodiments, heterophile antibodies are depleted from the sample by binding an interfering antibody against protein A or G, using methods known in the art. can be immunoextracted from In some embodiments, heterophile antibodies are neutralized using one or more heterophile antibody inhibitors. The heterophile inhibitor can be selected from the group consisting of anti-isoform heterophile antibody inhibitors, anti-idiotype heterophile antibody inhibitors, and anti-anti-idiotype heterophile antibody inhibitors. In some embodiments, combinations of heterophilic antibody inhibitors may be used.

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

The label is then eluted from the protein of interest. Preferred elution buffers are effective in releasing label without creating significant background. It is also useful if the elution buffer is bacteriostatic. Elution buffers for use in the present invention include chaotropes (e.g., urea or guanidinium compounds), buffers (e.g., borate-buffered saline), protein carriers (e.g., for coating the walls of capillary tubes in detection instruments). albumin, such as human albumin, bovine albumin, or fish albumin, or IgG), and a detergent (e.g., an ionic or non-ionic detergent selected to produce relatively low background, e.g. , Tween 20, Triton X-100, or SDS)).

The elution buffer/label aliquot taken into the single molecule detector is referred to as the "processed 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 involves attaching a) a capture antibody immobilized on a binding surface to i) troponin particles in the sample, and ii) a labeled analogue of the troponin particles containing a detectable label (the detection reagent). and b) measuring the amount of label using a single particle analyzer. Another such method involves binding a) an antibody with a detectable label (detection reagent) to i) troponin particles in the sample, and ii) an analogue of troponin particles immobilized on a binding surface (capture reagent). and b) measuring the amount of label using a single particle analyzer. By "analogue of troponin" herein is meant a species that competes with troponin for binding to the capture antibody. Examples of competitive immunoassays are disclosed in US Pat. No. 4,235,601 to Deutsch et al., US Pat. No. 4,442,204 to Liotta, and US Pat. No. 5,208,535 to Buechler et al., all of which are incorporated herein by reference. there is

C. Troponin Detection and Concentration Determination After elution, the label is passed through a single molecule detector, eg, in the elution buffer. The processed sample may contain no label, a single label, or multiple labels. The number of labels corresponds to or is proportional to the number of molecules of cardiac troponin captured during the capture step (when sample dilution or fractionation is used).

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 autosampler for taking the prepared sample and optionally a recovery system for recovering the sample.

Some embodiments utilize a capillary flow system and include a capillary flow cell, a laser for illuminating an interrogation space in the capillary through which the sample to be processed passes, and a detector for detecting radiation emitted from the interrogation space. The processed sample is analyzed with a single molecule analyzer that includes a detector for and a motive source for moving the processed sample through the interrogation space. In some embodiments, the single molecule analyzer further comprises a microscope objective lens, such as a high numerical aperture microscope objective lens, that collects light emitted from the processed sample as it passes through the interrogation space. 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 motive power source is a pump for supplying pressure. In some embodiments, the present invention provides an analyzer that includes a sampling system capable of automatically collecting multiple samples that provides flow communication between a sample container and an interrogation space. provide the 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 0.01 pL and 5 pL, or between about 0.01 pL and 0.5 pL, or between about 0.02 pL and about 300 pL, or between about 0.02 pL and about 50 pL; 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 between 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 detectors used in the methods of the present invention utilize a capillary flow system, a capillary flow cell, a continuous wave for illuminating the interrogation space in the capillary through which the sample to be processed passes. A laser, a high numerical aperture microscope objective lens to collect the light emitted from the interrogation space as the sample passes through the interrogation space, and an avalanche photodiode to detect the radiation emitted from the interrogation space. A detector and a pump for providing pressure to move the processing sample through the interrogation space, where the interrogation space is between about 0.02 pL and about 50 pL. In some embodiments, the single molecule detectors used in the methods of the present invention utilize a capillary flow system, a capillary flow cell, a continuous wave for illuminating the interrogation space in the capillary through which the sample to be processed passes. a laser, a high numerical aperture microscope objective lens (where the lens has a numerical aperture of at least about 0.8) that collects light emitted from the interrogation space as the sample passes through the interrogation space; an avalanche photodiode detector for detecting radiation emitted from the interrogation space, and a pump for providing pressure to move the processing sample through the interrogation space, in which case the interrogation The gating space is between about 0.004 pL and about 100 pL. In some embodiments, the single molecule detectors used in the methods of the present invention utilize a capillary flow system, a capillary flow cell, a continuous wave for illuminating the interrogation space in the capillary through which the sample to be processed passes. a laser, a high numerical aperture microscope objective lens (where the lens has a numerical aperture of at least about 0.8) that collects light emitted from the interrogation space as the sample passes through the interrogation space; an avalanche photodiode detector for detecting radiation emitted from the interrogation space, and a pump for providing pressure to move the processing sample through the interrogation space, in which case the interrogation The gating space is between about 0.05 pL and about 10 pL. In some embodiments, the single molecule detectors used in the methods of the invention utilize a capillary flow system, a continuous wave laser for illuminating the interrogation space in the capillary flow cell, through which the sample to be processed passes. , a high numerical aperture microscope objective lens (where the lens has a numerical aperture of at least about 0.8) that collects light emitted from the interrogation space as the processing sample passes through the interrogation space; an avalanche photodiode detector for detecting radiation emitted from the interrogation 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 detectors used in the methods of the invention utilize a capillary flow system, a continuous wave laser for illuminating the interrogation space in the capillary flow cell, through which the sample to be processed passes. , a high numerical aperture microscope objective lens (where the lens has a numerical aperture of at least about 0.8) that collects light emitted from the interrogation space as the processing sample passes through the interrogation space; an avalanche photodiode detector for detecting radiation emitted from the interrogation 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 for a molecule of interest in a sample, wherein a range in sample concentration is at least about 100-fold, or 1000-fold, or 10000-fold, or 100000-fold, or 30000-fold, or 1000000-fold, or 10000000-fold, or 30000000-fold.

In some embodiments, the methods of the present invention reduce the concentration of the analyte between the first and second samples introduced to the detector by about 50%, 40%, 30%, Utilizing single-molecule detectors capable of detecting differences of less than 20%, 15%, or 10%, the volume of the first sample and said 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 less than 1 femtomole. In some embodiments, the methods of the present invention are capable of detecting less than about 50% difference in analyte concentration between the first and second samples introduced to the detector. Utilizing certain single molecule detectors, the volume of the first sample and said 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 femtomoles. In some embodiments, the methods of the present invention are capable of detecting less than about 40% difference in analyte concentration between the first and second samples introduced to the detector. Utilizing a single molecule detector, the volume of the first sample and said 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 femtomoles. In some embodiments, the methods of the present invention are capable of detecting less than about 20% difference in analyte concentration between the first and second samples introduced to the detector. Utilizing a single molecule detector, the volume of the first sample and said 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 femtomoles. In some embodiments, the methods of the present invention are capable of detecting less than about 20% difference in analyte concentration between the first and second samples introduced to the detector. Utilizing a single molecule detector, the volume of the first sample and said 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 femtomoles. In some embodiments, the methods of the present invention are capable of detecting less than about 20% difference in analyte concentration between the first and second samples introduced to the detector. Utilizing a single molecule detector, the volume of the first sample and said 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 femtomoles.

Single molecule detectors and systems are described in more detail below. Further embodiments of single molecule analyzers useful in the methods of the invention, such as detectors with more than one interrogation window, detectors utilizing electrokinetic or electrophoretic flow, etc. US patent application Ser. No. 11/048,660, the entire text of which is incorporated herein by reference.

Equipment may be cleaned between runs. To maintain capillary conditioning, i.e., to keep the surface of the capillary relatively constant from sample to sample and reduce variability, a wash buffer that maintains the salt and detergent concentration of the sample is added in part. Embodiments can be used.

A feature that contributes to the very high sensitivity of the instruments and methods of the present invention is the method of detecting and counting the label, which in some embodiments binds to the single molecule to be detected or is more sensitive. It typically corresponds to a single molecule to be detected. Briefly, a given interrogation space of the capillary is subjected to EM radiation from a laser emitting light at an excitation wavelength suitable for the fluorescent moiety used for labeling for a predetermined period of time, during which time Detecting the emitted photons effectively separates the processed sample flowing through the capillary 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; ie, the label has been detected. If the total number of photons is not at the predetermined threshold level, no detection event is registered. In some embodiments, the concentration of the processed sample is sufficiently dilute that a high percentage of detected events represent only one label that passed the window, whereas this That corresponds to a single molecule of interest in the original sample, ie some number of detection events represents more than one label in a single bin. In some embodiments, higher concentrations of labels in the processed sample, i.e. concentrations where the probability of two or more labels being detected as a single detection event, is no longer significant. Additional purification is applied to allow accurate detection.

Although other bin times may be used without departing from the scope of the invention, in some embodiments the bin times are selected in the range of about 1 microsecond to about 5 ms. In some embodiments, bin times are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, Greater than 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In some embodiments, bin times are 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 5000 microseconds. In some embodiments, the bin time is approximately 1 to 1000 microseconds. In some embodiments, the bin time is approximately 1 to 750 microseconds. In some embodiments, the bin time is approximately 1 to 500 microseconds. In some embodiments, the bin time is approximately 1 to 250 microseconds. In some embodiments, the bin time is approximately 1 to 100 microseconds. In some embodiments, the bin time is approximately 1 to 50 microseconds. In some embodiments, the bin time is approximately 1 to 40 microseconds. In some embodiments, the bin time is approximately 1 to 30 microseconds. In some embodiments, the bin time is approximately 1 to 500 microseconds. In some embodiments, the bin time is approximately 1 to 20 microseconds. In some embodiments, the bin time is approximately 1 to 10 microseconds. In some embodiments, the bin time is approximately 1 to 500 microseconds. In some embodiments, the bin time is approximately 1 to 5 microseconds. In some embodiments, the bin time is approximately 5 to 500 microseconds. In some embodiments, the bin time is approximately 5 to 250 microseconds. In some embodiments, the bin time is approximately 5 to 100 microseconds. In some embodiments, the bin time is approximately 5 to 50 microseconds. In some embodiments, the bin time is about 5-20 microseconds. In some embodiments, the bin time is about 5-10 microseconds. In some embodiments, the bin time is approximately 10 to 500 microseconds. In some embodiments, the bin time is approximately 10 to 250 microseconds. In some embodiments, the bin time is about 10-100 microseconds. In some embodiments, the bin time is about 10-50 microseconds. In some embodiments, the bin time is about 10-30 microseconds. In some embodiments, the bin time is approximately 10-20 microseconds. In some embodiments, the bin time is approximately 5 microseconds. In some embodiments, the bin time is approximately 5 microseconds. In some embodiments, the bin time is approximately 6 microseconds. In some embodiments, the bin time is approximately 7 microseconds. In some embodiments, the bin time is about 8 microseconds. In some embodiments, the bin time is approximately 9 microseconds. In some embodiments, the bin time is approximately 10 microseconds. In some embodiments, the bin time is approximately 11 microseconds. In some embodiments, the bin time is approximately 12 microseconds. In some embodiments, the bin time is approximately 13 microseconds. In some embodiments, the bin time is approximately 14 microseconds. In some embodiments, the bin time is approximately 5 microseconds. In some embodiments, the bin time is approximately 15 microseconds. In some embodiments, the bin time is approximately 16 microseconds. In some embodiments, the bin time is approximately 17 microseconds. In some embodiments, the bin time is approximately 18 microseconds. In some embodiments, the bin time is approximately 19 microseconds. In some embodiments, the bin time is approximately 20 microseconds. In some embodiments, the bin time is approximately 25 microseconds. In some embodiments, the bin time is approximately 30 microseconds. In some embodiments, the bin time is approximately 40 microseconds. In some embodiments, the bin time is approximately 50 microseconds. In some embodiments, the bin time is approximately 100 microseconds. In some embodiments, the bin time is approximately 250 microseconds. In some embodiments, the bin time is approximately 500 microseconds. In some embodiments, the bin time is approximately 750 microseconds. In some embodiments, the bin time is approximately 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, typical noise values or statistical values are chosen. In most cases the noise is expected to follow a Poisson distribution. Accordingly, in some embodiments, determining the concentration of particle-label complexes in the sample comprises determining the background noise level.

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

Given a background noise value, a threshold energy level can be assigned. As discussed above, a threshold is determined to discriminate true signal (due to label fluorescence) from background noise. Care must be taken in choosing the threshold so that the number of false positive signals from random noise is minimized while also minimizing the number of true signals that are rejected. Methods for selecting the threshold include determining a fixed value above the noise level and calculating the threshold based on the noise signal distribution. In one embodiment, the threshold is set to a fixed number of standard deviations above the background level. Assuming a Poisson distribution of noise, this method can be used to assess the number of false positive signals over the time course of the experiment. In some embodiments, the threshold level is calculated as a 4 sigma value above background noise. For example, given an average background noise level of 200 photons, the analyzer system would set a threshold level of 4√200 above the average background/noise level of 200 photons to be 256 photons. Establish. Thus, in some embodiments, determining the concentration of the label in the sample comprises establishing a threshold level above which the photon signal indicates the presence of the label. Conversely, a photon signal with an energy level not above the threshold level energy level indicates no label.

A number of bin measurements are taken to determine the concentration of the sample and each bin measurement is checked for the presence or absence of label. Typically, 60,000 or more measurements can be taken per minute (e.g., in embodiments where the bin size is 1 ms, the number of measurements is correspondingly higher for smaller bin sizes, e.g., 10 microseconds). 6,000,000 measurements per minute for a bin size of . Therefore, there is no definitive single measurement and the method provides a high margin of error. Bins determined not to contain label ("no" bins) are not taken into account, only measurements made in bins determined to contain label ("yes" bins) determine the concentration of label in the processed sample. take into account in doing so. By not taking into account measurements made in the "no" or no label bins, the signal-to-noise ratio and the accuracy of the measurements are increased. Thus, in some embodiments, determining the concentration of label in the sample comprises detecting bin measurements that reflect the presence of label.

The signal-to-noise ratio, or sensitivity of the analyzer system, can be increased by minimizing the time to detect background noise during bin measurements in which particle-label complexes are detected. For example, in a bin measurement lasting 1 millisecond where one particle-label complex is detected as it passes through the interrogation space within 250 microseconds, 750 microseconds of the 1 millisecond is background noise emission. is spent to detect 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 times are 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 portion, the flow rate, and the power of the laser. Various combinations of relevant factors that allow 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. It will be appreciated by those skilled in the art that if the bin time is reduced, the laser power directed into the interrogation space must be increased in order to keep the total energy applied to the interrogation space constant during the bin time. be understood. For example, if the bin time is reduced from 1000 microseconds to 250 microseconds, as a first approximation, the laser power should be increased by about a factor of four. These settings allow the same number of photons to be detected in 250 µs as the number of photons counted during the 1000 µs given by the previous settings, allowing samples to be processed faster with lower background and thus greater sensitivity. be able to analyze. Additionally, the flow rate may be adjusted to expedite sample processing. These numbers are illustrative only and one skilled in the art can adjust the parameters accordingly to achieve the desired results.

In some embodiments, the interrogation space encompasses the entire cross-section of the sample stream. If the interrogation space encompasses the entire cross-section of the sample stream, only the number of labels counted for a set length of time and the volume across the cross-section of the sample stream is the number of labels in the processed sample. Required to calculate 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 illuminated by the laser beam. In some embodiments, the interrogation space is adjusted by adjusting the analyzer aperture 306 (FIG. 1A) or 358 and 359 (FIG. 1B) to reduce the illumination volume imaged by the objective lens to the detector. can be defined. In embodiments in which the interrogation space is defined to be smaller than the cross-sectional area of the sample stream, the concentration of label is released by the conjugate from a standard curve generated using one or more samples of known standard concentration. can be determined by interpolating the resulting signals. In still other embodiments, the concentration of label can be determined by comparing the measured particles to an internal standard of label. In embodiments where diluted samples are analyzed, 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 encompasses the entire cross-section of the sample stream, the number of labels counted across the cross-section of the sample stream for a set length of time (bins). Only the number and volume of interrogated samples in bins are needed to calculate the sample concentration. The total number of labels contained in the "yes" bins is determined and related to the sample volume represented by the total number of bins used in the assay to determine the concentration of label in the processed sample. Thus, in one embodiment, determining the concentration of label in the processed sample comprises determining the total number of labels that detected the "yes" bin, and determining the total number of labels detected as the total volume of sample analyzed. including associating with The total sample volume analyzed is the volume of sample that passes through the capillary flow cell and across the interrogation space in a specified time interval. Alternatively, the signal emitted by the label in a number of bins can be interpolated from a standard curve generated by determining the signal emitted by the label in the same number of bins with standard samples containing known concentrations of label. Determine the concentration of the labeled complex in .

In some embodiments, the number of individual labels detected in a bin is related to the relative concentration of particles in the processed sample. At relatively low concentrations, such as concentrations below about 10 ⁻¹⁶ M, the number of labels is proportional to the photon signal detected in the bin. Therefore, at low concentrations of label, the photon signal is given as a digital signal. At relatively high concentrations, such as concentrations above about 10 ⁻¹⁶ M, labels and photon signals are combined with the potential for two or more labels to be counted across the interrogation space at about the same time, as one becomes significant. is lost. Thus, in some embodiments, individual particles in samples with concentrations greater than about 10 ⁻¹⁶ M are resolved by shortening the length of time of bin measurement.

Alternatively, in other embodiments, the total photon signal emitted by multiple particles in any one bin is detected. These embodiments provide a dynamic range of at least 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or greater than 8 logs of the single unit of the present invention. A single molecule detector is acceptable.

As used herein, the term "dynamic range" refers to the range of sample concentrations that can be quantified by an instrument without the need for dilution or other processing to alter the concentration of serial samples of different concentrations. , in which case the concentration is determined with an accuracy appropriate to the intended use. For example, a microtiter plate may contain a 1 femtomole concentration of sample to an analyte of interest in one well, a 10,000 femtomole concentration of sample to an analyte of interest in another well, and a Requires further processing to adjust concentration, such as dilution, by instruments with a dynamic range of at least 4 logs and a lower limit of quantitation of 1 femtomole if the wells contain 100 femtomoles of sample for the analyte of interest. It is possible to accurately quantify the concentration of all samples without Accuracy can be determined by standard methods, such as using a series of standard concentrations spanning the dynamic range and constructing a standard curve. Standard measurements that fit the resulting standard curve, ^e.g. It can be used as a measure of accuracy greater than 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

Increased dynamic range is achieved by changing the manner in which the data from the detector is analyzed and/or by using an attenuator between the detector and the interrogation space. The low-side photons of each burst above the threshold level in each detection event or bin (“event photons”) likely represent only one label because the processed sample is sufficiently diluted. In the low end range, data are analyzed to count detection events as single molecules. That is, as described above, each bin is analyzed simply as "yes" or "no" for the presence of the label. In more concentrated processed samples, the possibility of more than one label occupying a single bin becomes significant, and the number of event photons in a significant number of bins would be expected for a single label. e.g., the number of event photons in a significant number of bins doubles, triples, or more than triples the number of event photons expected for a single label. Equivalent to. For these samples, the instrument changes its method of data analysis to integrate the total number of event photons for the bins of processed samples. This total will be proportional to the total number of labels in all bins. In even more concentrated processed samples, where many labels are present in most bins, the background noise becomes an insignificant portion of the total signal obtained from each bin, and the instrument adapts the data analysis method to Change the method to count all photons (including background). An even further increase in dynamic range is achieved when the intensity of the light reaching the detector is at a concentration such that it otherwise exceeds the detector's ability to count photons accurately, i.e., saturates the detector. 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 an indication to use or not to use the attenuator if the attenuator is included in the instrument.

By using such a method, the dynamic range of the instrument can be dramatically increased. Thus, in some embodiments, the device has approximately 1000 (3 log), 10000 (4 log), 100000 (5 log), 350000 (5.5 log), 1000000 (6 log), 3500000 (6.5 log), 10000000 (7 log ), 35000000 (7.5 log), or over a dynamic range of 100000000 (8 log). In some embodiments, the instrument is capable of measuring sample concentration over a dynamic range of greater than about 100,000 (5 logs). In some embodiments, the instrument is capable of measuring sample concentration over a dynamic range of greater than about 1,000,000 (6 logs). In some embodiments, the instrument is capable of measuring sample concentration over a dynamic range of greater than about 10000000 (7 logs). 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 1000, 10000, 100000, 350000, 1000000, 3500000, 10000000, or 35000000 femtomoles. be. In some embodiments, the instrument is capable of measuring sample concentrations over a dynamic range of about 1-10 femtomoles to at least about 10,000 femtomoles. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range of about 1-10 femtomoles to at least about 100,000 femtomoles. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range of about 1-10 femtomoles to at least about 1,000,000 femtomoles. In some embodiments, the instrument is capable of measuring the concentration of a sample over a dynamic range of about 1-10 femtomoles to at least about 10,000,000 femtomoles.

In some embodiments, an analyzer or analyzer system of the invention is capable of detecting an analyte, such as a biomarker, at a detection limit of less than 1 nanomolar, or 1 picomolar, or 1 femtomole, or 1 attomole, or 1 zeptomole. It is possible. In some embodiments, the analyzer or analyzer system detects if the biomarker is present in a sample at a concentration of less than 1 nanomolar, or 1 picomolar, or 1 femtomole, or 1 attomole, or 1 zeptomole, and the size of each sample about 0.1, 1, 2 when is less than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1, 0.01, 0.001, or 0.0001 μl , less than 5, 10, 20, 30, 40, 50, 60, or 80% of the change in concentration of an analyte or multiple analytes (e.g., a biomarker or biomarkers) from one sample to another It is possible to detect In some embodiments, the analyzer or analyzer system detects less than about 20% of the first sample when the analyte is present at a concentration of less than about 1 picomolar and when each sample is less than about 50 μl in size. 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 detects less than about 20% of the first sample when the analyte is present at a concentration of less than about 100 femtomoles and when each sample is less than about 50 μl in size. 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 detects less than about 20% of the first sample when the analyte is present at a concentration of less than about 50 femtomoles and when each sample is less than about 50 μl in size. 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 detects less than about 20% of the first sample when the analyte is present at a concentration of less than about 5 femtomoles and when each sample is less than about 50 μl in size. 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 detects less than about 20% of the first sample when the analyte is present at a concentration of less than about 5 femtomoles and when each sample is less than about 5 μl in size. 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 detects less than about 20% of the first sample when the analyte is present at a concentration of less than about 1 femtomole and when each sample is less than about 5 μl in size. 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 methods of the present invention utilize highly sensitive analytical instruments such as single molecule detectors. Such single molecule detectors include the embodiments described below.

Apparatus/System In one aspect, the methods described herein utilize an analyzer system capable of detecting single particles in a sample. In one embodiment, the analyzer system is capable of single-particle detection of fluorescently labeled particles, and the analyzer system is defined within a capillary flow cell in fluid communication with a sampling system of the analyzer system. When a single particle is present in the gating space, the energy emitted by the fluorescent label excited in response to exposure by the source of electromagnetic radiation is detected. In a further embodiment of the analyzer system, a power source moves the single particles through the interrogation space of the capillary flow cell. In another embodiment of the analyzer system, an automatic 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 further embodiments, the analyzer system may include a collection system to collect at least a portion of the sample after analysis is completed.

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

In a typical embodiment, a source of electromagnetic radiation excites a fluorescent moiety attached to the label as the label passes through the interrogation space of the capillary flow cell. In some embodiments, the fluorescent labeling moiety comprises one or more fluorophores. In some embodiments, the fluorescent labeling moiety 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 an interrogation space located within the capillary flow cell. The interrogation space is typically in fluid communication with the sampling system. In some embodiments, the label is passed through the interrogation space of the capillary flow cell with a power source for advancing the label through the analyzer system. The interrogation space is positioned to receive electromagnetic radiation emitted from the radiation source. In some embodiments, the sampling system is an automated sampling system capable of sampling multiple samples without human operator intervention.

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

In a further embodiment of the analyzer system, the system further includes a sample preparation mechanism by which the sample can be partially or fully 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 includes a sample retrieval mechanism by which at least a portion, or all, or substantially all of the sample can be retrieved after analysis. In one such embodiment, the sample can be returned to the original sample. In some embodiments, samples can be returned to microtiter wells on a sample microtiter plate. Analyzer systems typically further comprise a data acquisition system for collecting and reporting the detected signals.

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

In operation, electromagnetic radiation source 301 is aligned such that its output 311 reflects off front surface 312 of mirror 302 . Lens 303 focuses light beam 311 onto a single interrogation space (an illustrative example of interrogation space 314 is shown in FIG. 2A) in capillary flow cell 313 . A microscope objective lens 305 collects light from the particles of the sample and forms an image of the light beam on aperture 306 . Aperture 306 affects the fraction of light emitted by the specimen in the interrogation space of capillary flow cell 313 that can be collected. Detector lens 307 collects the light passing through aperture 306 and focuses the light onto the active area of detector 309 after the light passes through detector filter 308 . Detector filter 308 maximizes the signal emitted by excited fluorescent moieties bound to particles while minimizing aberrant noise signals due to light scatter or ambient light. Processor 310 processes the optical signals from the particles according to the methods described herein.

In one embodiment, microscope objective 305 is a high numerical aperture microscope objective. A "high numerical aperture lens" as used herein includes lenses having a numerical aperture of 0.6 or greater. Numerical aperture is a measure of the number of highly diffracted imaging light rays captured by the objective lens. A larger numerical aperture allows more and more oblique light to enter the objective, thereby producing a higher resolved image. In addition, the brightness of the image increases at higher numerical apertures. 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 the 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 microscope objective 305 is approximately 1.25. In one embodiment using a 0.8 microscope objective lens 305, a Nikon 60X/0.8NA Achromat lens (Nikon, Inc., USA) can be used.

In some embodiments, electromagnetic radiation source 301 is a laser emitting in the visible spectrum. In all embodiments, the electromagnetic radiation source is set such that the laser wavelength is set to be of sufficient wavelength to excite the fluorescent labels attached to the particles. In some embodiments, the laser is a continuous wave laser with a wavelength of 639 nm. In other embodiments, the laser is a continuous wave laser with a wavelength of 532 nm. In other embodiments, 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 suitable wavelength to excite fluorescent moieties for use in the methods and compositions of the invention can be used without departing from the scope of the invention.

In the single particle analyzer system 300, each particle enters an excited state as it passes through the beam 311 of the electromagnetic radiation source. A detectable burst of light is emitted when 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 analyzer system 300 detects each particle as it passes through interrogation space 314. It becomes possible to detect tens of thousands of photons for . Photons emitted by the fluorescent particles are recorded by detector 309 (FIG. 1A) with a time delay indicative of the time the particle-labeling complex passes through the interrogation space. Photon intensities are recorded by detector 309 and sampling times are binned into 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 are used to determine when particles are present. Such methods involve 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. .

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

If a single molecule is to be detected at the sample concentration, the beam size and depth of focus required for single molecule detection are set, thereby defining the size of the interrogation space 314 . The interrogation space 314 is set such that, with a suitable sample concentration, only one particle is present in the interrogation space 314 during each time interval in which observations are made. It will be appreciated that the detection interrogation volume defined by the rays is typically of the "bow-tie" type rather than a perfect spherical shape. However, for purposes of definition, the "volume" of interrogation space is defined herein as the volume enclosed by a sphere of diameter equal to the diameter of the focused spot of the light beam. The focused spot of light beam 311 can have various diameters without departing from the scope of the invention. In some embodiments, the diameter of the focused spot of the light beam is from about 1 to about 5, 10, 15, or 20 microns, or from about 5 to about 10, 15, or 20 microns, or from about 10 to about 20 microns, Or about 10 to about 15 microns. In some embodiments, the diameter of the focused spot of the beam is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns. In some embodiments, the focused spot diameter of the light beam is about 5 microns. In some embodiments, the focused spot diameter of the light beam is about 10 microns. In some embodiments, the focused spot diameter of the light beam is about 12 microns. In some embodiments, the focused spot diameter of the light beam is about 13 microns. In some embodiments, the focused spot diameter of the light beam is about 14 microns. In some embodiments, the focused spot diameter of the light beam is about 15 microns. In some embodiments, the focused spot diameter of the light beam is about 16 microns. In some embodiments, the focused spot diameter of the light beam is about 17 microns. In some embodiments, the focused spot diameter of the light beam is about 18 microns. In some embodiments, the focused spot diameter of the light beam is about 19 microns. In some embodiments, the focused spot diameter of the light beam 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 fluorescent labels. An illustration incorporating 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 Ser. No. 11/048,660.

In some embodiments of analyzer system 300 , a motive force is required to move particles through capillary flow cell 313 of analyzer system 300 . In one embodiment, the motive force can be in the form of pressure. The pressure used to move the particles through the capillary flow cell can be created by a pump. In some embodiments, Scivex, Inc. HPLC pumps can be used. In some embodiments where a pump is used as the motive force, the sample can pass through the capillary flow cell at a rate of 1 μL/minute to about 20 μL/minute, or about 5 μL/minute to about 20 μL/minute. 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 particles through the analyzer system. Such methods have been previously disclosed and are incorporated by reference from prior US patent application Ser. No. 11/048,660.

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

In some embodiments, the optics of electromagnetic radiation source 301 and the optics of detector 309 are arranged in a conventional optical arrangement. In such an array, the electromagnetic radiation source and detector are aligned on different focal planes. The arrangement of the laser and detector optics of 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, electromagnetic radiation source 301 and detector 309 are aligned on the same focal plane. The confocal arrangement makes the analyzer more robust because the electromagnetic radiation source 301 and detector optics 309 do not need to be realigned when the analyzer system is moved. This arrangement also makes the analyzer easier to use, as it eliminates the need to rearrange the components of the analyzer system. Confocal arrangements of analyzer 300 (FIG. 1A) and analyzer 355 (FIG. 1B) are shown in FIGS. 3A and 3B, respectively. FIG. 3A shows that light beam 311 from electromagnetic radiation source 301 is focused by microscope objective 315 to form one interrogation space 314 (FIG. 2A) within capillary flow cell 313 . A dichroic mirror 316 that reflects the laser light but passes the fluorescent light 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 at the detector. In some embodiments, an 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 Ser. No. 11/048,660.

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 wavelengths 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, set between 408.9 and 686.1 nm. , whose optimum performance is set between 488 and 514.5 nm.

1. Chemiluminescent labels can be used in some embodiments of the electromagnetic radiation source analyzer system. Such embodiments may not necessarily utilize an EM radiation source for detecting particles. In another embodiment, the extrinsic label or intrinsic property of the particle is a photointeractive 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 labels and/or particles. An EM radiation source for exciting fluorescent labels is preferred.

In some embodiments, the analyzer system consists of electromagnetic radiation source 301 . Any number of radiation sources may be used in any one analyzer system 300 without departing from the scope of the invention. A number of electromagnetic radiation sources have been previously disclosed and are incorporated by reference from prior US patent application Ser. No. 11/048,660. In some embodiments, all continuous wave electromagnetic (EM) radiation sources emit electromagnetic radiation of the same wavelength. In other embodiments, different sources emit different wavelengths of EM radiation.

In one embodiment, the EM radiation sources 301, 351, 352 are continuous wave lasers producing wavelengths between 200 nm and 1000 nm. Such EM radiation sources have the advantage of being small, durable and relatively inexpensive. Furthermore, they are generally capable of producing a greater fluorescence signal 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 potentially doubling the frequency. Lasers provide continuous illumination without attached electronic or mechanical devices to interrupt the illumination, 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 beam diameter from a continuous wave laser of such 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 alternate embodiments, the exposure time to the laser beam can be about 500 μs or less. In an alternate embodiment, the exposure time can be about 100 μs or less. In an alternate embodiment, the exposure time can be about 50 μs or less. In an alternate embodiment, the exposure time can be about 10 μs or less.

LEDs are another low cost and reliable illumination source. Recent advances in high-brightness LEDs and dyes with high absorption cross-sections and high quantum yields support the applicability of LEDs for 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 pulsed wave laser. In such embodiments, the pulse size of the laser is an important factor. In such embodiments, the size, focused spot, and total energy emitted by the laser are important and must be of sufficient energy to be able to excite the fluorescent label. When using a pulsed laser, longer duration pulses may be required. In some embodiments, 2 nanosecond laser pulses can be used. In some embodiments, 5 nanosecond laser pulses can be used. In some embodiments, pulses between 2 and 5 nanoseconds can be used.

The optimal laser intensity depends on the characteristics of single-dye photobleaching and the length of time required to traverse the interrogation space (particle velocity, distance between interrogation spaces if multiple are used, and (including the size of the interrogation space). For maximum signal, it is desirable to illuminate the sample at the highest intensity that does not result in photobleaching of the high percentage dye. Preferred intensities are those at which no more than 5% of the dye fades by the time the particles cross the interrogation space.

The power of the laser is set according to the type of dye molecules that need to be stimulated and the length of time the dye molecules are stimulated and/or the speed at which the dye molecules pass through the capillary flow cell. Laser power is defined as the rate at which energy is delivered by a beam and is measured in Joules/second or Watts. It will be appreciated that the higher the power of the laser, the less time the laser may irradiate the particles, while providing a certain amount of energy to the interrogation space while the particles pass through the space. Thus, 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 about 0.1, 0.5, 1, 2, 3, 4, 5 , 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 power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is about 0.5, 1, 2, 3, 4, 5, 6, 7, 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 power and irradiation time is such that the total energy received by the interrogation space during the irradiation time is about 1 microjoule. 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 about 3 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 about 5 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 about 10 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 about 15 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 about 20 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 about 30 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 about 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 about 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 about 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 about 70 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 about 80 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 about 90 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 about 100 microjoules.

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

The time the laser illuminates 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 more. The time the laser illuminates 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 illuminates the interrogation space can be set between about 1 and 1000 microseconds. The time the laser illuminates the interrogation space can be set between about 5 and 500 microseconds. The time the laser illuminates the interrogation space can be set between about 5 and 100 microseconds. The time the laser illuminates the interrogation space can be set between about 10 and 100 microseconds. The time the laser illuminates the interrogation space can be set between about 10 and 50 microseconds. The time that the laser illuminates the interrogation space can be set between about 10 and 20 microseconds. The time the laser illuminates the interrogation space can be set between about 5 and 50 microseconds. The time that the laser illuminates the interrogation space can be set between about 1 microsecond and 100 microseconds. In some embodiments, the laser illuminates the interrogation space for about 1 microsecond. In some embodiments, the laser illuminates the interrogation space for about 5 microseconds. In some embodiments, the laser illuminates the interrogation space for about 10 microseconds. In some embodiments, the laser illuminates the interrogation space for about 25 microseconds. In some embodiments, the laser illuminates the interrogation space for about 50 microseconds. In some embodiments, the laser illuminates the interrogation space for about 100 microseconds. In some embodiments, the laser illuminates the interrogation space for about 250 microseconds. In some embodiments, the laser illuminates the interrogation space for about 500 microseconds. In some embodiments, the laser illuminates the interrogation space for about 1000 microseconds.

For example, the time that the laser illuminates the interrogation space is 1 millisecond, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds for lasers providing powers of 3 mW, 4 mW, 5 mW, or greater than 5 mW. , or 10 microseconds. In some embodiments, the label is illuminated with a laser that provides 3 mW of power and illuminates the label for about 1000 microseconds. In other embodiments, the label is illuminated for less than 1000 milliseconds with a laser providing a power of about 20 mW or less. In another embodiment, a laser power of 20 mW illuminates the label for about 250 microseconds or less. In some embodiments, a laser power of about 5 mW illuminates 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 the ray within 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 0.01 pL and 1 pL, or between about 0.01 pL and 0.5 pL, or between about 0.02 pL and about 300 pL, or between about 0.02 pL and about 50 pL, or 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 between about 0.05 pL and about 50 pL, or 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 between about 0.1 pL and about 25 pL herein has a volume defined by In some embodiments, the interrogation space has a volume between about 0.01 pL and 10 pL. In some embodiments, interrogation space 314 has a volume between about 0.01 pL and 1 pL. In some embodiments, interrogation space 314 has a volume between about 0.02 pL and about 5 pL. In some embodiments, interrogation space 314 has a volume between about 0.02 pL and about 0.5 pL. In some embodiments, interrogation space 314 has a volume between about 0.05 pL and about 0.2 pL. In some embodiments, interrogation space 314 has a volume of about 0.1 pL. Other useful interrogation space volumes are described herein. Those skilled in the art should understand that the interrogation space 314 can be selected for maximum performance of the analyzer. A very small interrogation space has been shown to minimize background noise, while a large interrogation space has the advantage that low concentration samples can be analyzed in a reasonable amount of time. In embodiments using 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 present invention, the interrogation space is large enough to detect concentrations of particles ranging from about 1000 femtomoles (fM) to about 1 zeptomole (zM). In one embodiment of the invention, the interrogation space is large enough to allow detection of particles at concentrations ranging from about 1000 fM to about 1 attomole (aM). In one embodiment of the invention, the interrogation space is large enough to allow detection of concentrations of particles ranging from about 10 fM to about 1 attomole (aM). In many cases, the large interrogation space allows detection of particles at concentrations below about 1 fM without additional preconcentrators or techniques. Those skilled in the art will appreciate that the size of the most appropriate interrogation space will depend 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 beam 311 can be adjusted to change the volume of interrogation space 314 . In another embodiment, the field of view of detector 309 can be varied. Accordingly, radiation source 301 and detector 309 can be adjusted such that single particles are irradiated and detected within interrogation space 314 . In another embodiment, the width of aperture 306 (FIG. 1A), which determines the field of view of detector 309, can vary. This configuration allows the interrogation space to be changed in near real time to fill in more or less concentrated samples, thereby ensuring the probability of having more than one particle in the interrogation space at the same time. make low. Similar changes may be made to 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 is passed through a capillary flow cell prior to 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, the depth of focus and beam diameter of the electromagnetic radiation source will limit the interrogation space in the capillary flow cell. size is determined.

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

The interrogation space is immersed 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. Additionally, the liquid may contain sieving agents such as pH adjusting agents, ionic compositions, or soluble macroparticles or polymers or gels. It is contemplated that a valve or other device may exist between the interrogation spaces to temporarily interrupt the fluid connection. Temporarily interrupted interrogation spaces are considered to be connected by liquid.

In another embodiment of the invention, the interrogation space resides within a flow cell 313 constrained by the size of the laminar flow of sample material within the diluent volume, also called sheath flow. is a single interrogation space that In these and other embodiments, the interrogation space can be defined by the sheath flow alone or in combination with the dimensions of the illumination source or the field of view of the detector. The sheath flow can be shaped in many ways, including: the sample material is the inner material in concentric laminar flow and the outer is the diluent volume; the diluent volume is on 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; A liquid volume concentrically completely surrounds the sample material; the sample material is the inner material of a discontinuous series of droplets, and a diluent volume completely surrounds each droplet of the sample material.

In some embodiments, the single molecule detectors of the invention contain only one interrogation space. In some embodiments, multiple interrogation spaces are used. A number of interrogation spaces have been previously disclosed and are incorporated by reference from US patent application Ser. No. 11/048,660. Those skilled in the art will appreciate that in some cases the analyzer will include 2, 3, 4, 5, 6, or more than 6 distinct interrogation spaces.

3. In one embodiment of the motive force analyzer system, particles are moved through the interrogation space by a motive force. In some embodiments, the motive force for moving particles is pressure. In some embodiments, pressure is supplied by a pump, pneumatic source, vacuum source, centrifuge, or combinations thereof. In some embodiments, the motive force for moving particles is an electrokinetic force. The use of electrokinetic forces as a motive force has been previously disclosed in prior applications and is incorporated by reference from US patent application Ser. No. 11/048,660.

In one embodiment, pressure can be used as a driving force to move particles through the interrogation space of the capillary flow cell. In a further embodiment, a pump provides pressure to move the sample. Suitable pumps are known in the art. In one embodiment, Scivax, Inc. Pumps manufactured for HPLC applications can be used as motive power, such as those manufactured by In other embodiments, pumps manufactured for microfluidics applications can be used when the pump is used for smaller sample volumes. These pumps are described in U.S. Pat. Nos. 5,094,594, 5,730,187, 6,033,628, and 6,533,553, which disclose devices capable of pumping liquid volumes in the nanoliter or picoliter range. ing. All materials in the pump that come into contact with the sample are preferably made of highly inert materials such as polyetheretherketone (PEEK), fused silica, or sapphire.

A motive force is required to move the sample through the capillary flow cell and push the sample through the interrogation space for analysis. A motive force is still required to force the flushing sample through the capillary flow cell after the sample has passed through. For sample retrieval, a motive force is still required to push the sample back into the sample retrieval container. Standard pumps come in a variety of sizes, and a suitable size can be selected to match anticipated sample size and flow requirements. In some embodiments, separate pumps are used for sample analysis and system flushing. The capacity 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 flush 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. These pump sizes are exemplary only and one of ordinary skill in the art will appreciate that the pump size will vary depending on the application, sample size, viscosity of the liquid in which the pump is to be used, tubing size, flow rate, temperature and techniques of the art. It will be appreciated that the selection can be made according to other factors well known in the art. In some embodiments, the pumps of the system are driven by stepper motors that are easy to control very accurately with a microprocessor.

In a preferred embodiment, the flush and analytical pumps are used in series with special check valves controlling the direction of flow. The tubing is designed so that when the analytical pump draws up the maximum amount of sample, the sample does not reach the pump itself. This is accomplished by selecting the ID and length of the tubing between the analytical pump and the analytical capillary such that the tubing volume exceeds the stroke volume of the analytical pump.

4. A detector embodiment detects light (eg, light in the ultraviolet, visible, or infrared range) emitted by a fluorescent label after exposure to electromagnetic radiation. A detector 309 (FIG. 1A) or a plurality of detectors (364, 365, FIG. 1B) captures the amplitude and duration of bursts of photons from the fluorescent moiety and further converts the amplitudes and durations of the bursts of photons into electrical signals. can be converted to Detection devices such as CCD cameras, video input module cameras, and streak cameras can be used to generate images with continuous signals. In another embodiment, devices such as bolometers, photodiodes, photodiode arrays, avalanche photodiodes, and photomultiplier tubes that produce a continuous signal can be used. Any combination of the aforementioned detectors can also be used. In one embodiment, an avalanche photodiode is used to detect photons.

Using specific optical optics between the interrogation space 314 (Fig. 2A) and its corresponding detector 309 (Fig. 1A), emission wavelength, emission intensity, burst size, burst duration, and fluorescence polarization can be determined. Several distinct characteristics of emitted electromagnetic radiation can be detected, including: In some embodiments, detector 309 is a photodiode used in reverse bias. A photodiode set in reverse bias usually has a very high resistance. This resistance drops when light of the appropriate frequency illuminates the P/N junction. Therefore, a reverse-biased 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 photodiodes can be avalanche photodiodes, which can be operated at much higher reverse biases than conventional photodiodes, thus avalancheing each photogenerated carrier. Increased by breakdown to provide internal gain within the photodiode, increasing the effective responsivity (sensitivity) of the device. The choice of photodiode is determined by the energy or emission wavelength emitted by the fluorescently labeled particles. In some embodiments, the photodiode is a silicon photodiode that detects energy in the range of 190-1100 nm, and in another embodiment the photodiode is a germanium photodiode that detects energy in the range of 800-1700 nm. a diode, in another embodiment the photodiode is an indium gallium arsenide 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 is a lead sulfide photodiode that detects energy in the range of . In some embodiments, the 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, Mass.).

In some embodiments, the detector is an avalanche photodiode detector that detects energies between 300 nm and 1700 nm. In one embodiment, silicon avalanche photodiodes can be used to detect wavelengths between 300 nm and 1100 nm. Indium gallium arsenide photodiodes can be used to detect wavelengths between 900 nm and 1700 nm. 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 specific 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 at different spectral energies when excited by an EM radiation source. In one embodiment, the analyzer system includes a first detector capable of detecting fluorescence energy in the 450-700 nm range, such as that emitted by a green dye (eg, Alexa 546), and a near-infrared dye (eg, Alexa 647 A second detector can be included that can detect fluorescence energy in the range of 620-780 nm, such as that emitted by ). detectors for detecting fluorescence energy in the 400-600 nm range, such as those emitted by blue dyes (e.g. Hoechst 33342) and in the 560-700 nm range, such as those emitted by red dyes (Alexa546 and Cy3); A detector for detecting energy can also be used.

Systems 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 type 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 more than two detectors can be used without departing from the scope of the invention.

One skilled in the art can arrange one or more detectors in each interrogation space with one or more interrogation spaces defined in the flow cell, and that each detector It should be understood that it can be arranged to detect any of the enumerated characteristics of emitted electromagnetic radiation. For example, the use of multiple detectors for multiple interrogation spaces has been previously disclosed in an earlier application and is hereby incorporated by reference from US patent application Ser. No. 11/048,660. Once the particles have been labeled to render them detectable (or where the particles have unique characteristics that render them detectable), any suitable mechanism of detection known in the art (e.g., CCD cameras, video input module cameras, streak cameras, bolometers, photodiodes, photodiode arrays, avalanche photodiodes, and photomultipliers producing continuous signals, and combinations thereof) are outside the scope of this invention. can be used without Different characteristics of electromagnetic radiation can be detected, including emission wavelength, emission intensity, burst size, burst duration, fluorescence 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. An included sampling system is capable of automatically taking multiple samples and providing fluid communication between the sample container and the first interrogation space.

In some embodiments, the analyzer system of the invention includes a sampling system for introducing an aliquot of the sample into the single particle analyzer for analysis. Any mechanism that can introduce a sample can be used. The sample can be drawn up using any vacuum suction created by a pump, or pressure applied to the sample to force the liquid into the tube, or any other mechanism that acts to introduce the sample into the sampling tube. Generally, but not necessarily, the sampling system introduces a known sample volume of the sample into the single particle analyzer, and in some embodiments the presence or absence of one or more particles is detected. It is not critical to know the sample size exactly. In preferred embodiments, 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 will , 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500, or 2000 μl of sample for analysis. In some embodiments, the sampling system has a , 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 of sample for analysis. In some embodiments, the sampling system provides between about 5 μl and 200 μl, or between about 5 μl and about 100 μl, or between about 5 μl and 50 μl of sample for analysis. In some embodiments, the sampling system provides between about 10 μl and 200 μl, or between about 10 μl and 100 μl, or between about 10 μl and 50 μl of sample for analysis. In some embodiments, the sampling system provides between about 0.5 μl and about 50 μl of sample for analysis.

In some embodiments, the sampling system provides a sample size that can 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.

Sample volume accuracy and inter-sample volume precision of the sampling system are currently required for analysis. In some embodiments, the precision of the collection volume is determined by the pump used and is typically about 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0, . Represented by a CV less than 5, 0.1, 0.05, or 0.01%. In some embodiments, the sample-to-sample precision 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 less than 0.01%. In some embodiments, the intra-assay precision 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 intra-assay precision of the sampling system exhibits a CV of less than about 5%. In some embodiments, the inter-assay precision of the sampling system is represented by a CV of less than about 10, 5, or 1%. In some embodiments, the inter-assay precision of the sampling system exhibits a CV of less than about 5%.

In some embodiments, the sampling system is advantageous in that sample carryover is low and no additional washing steps are required between samples. Thus, in some embodiments, sample carryover is about 1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or less than 0.001%. In some embodiments, sample carryover is less than about 0.02%. In some embodiments, sample carryover is less than about 0.01%.

In some embodiments, the sampling device provides a sample loop. In these embodiments, multiple samples are drawn into the tube in succession, 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 buffer "plugs" are used, the plugs can be recovered by ejecting the buffer plugs 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 includes a 96-well plate positioner and mechanisms for immersing sample tubes in and out of the wells, e.g., mechanisms for imparting motion along the X, Y, and Z axes. include. In some embodiments, the sampling system provides multiple sampling tubes from which samples can be stored and retrieved when testing is initiated. In some embodiments, all samples from multiple tubes are analyzed with one detector. In other embodiments, multiple single molecule detectors may be connected to the sample tube. The samples can be prepared by a process that includes operations performed on the samples in the wells of the plate prior to sampling by the sampling system, or the samples can be prepared within the analyzer system, or some combination of both. can be prepared.

D. SAMPLE PREPARATION SYSTEM Sample preparation includes the steps that require the preparation of crude samples for analysis. These steps include, by way of example, separation steps such as centrifugation, filtration, distillation, chromatography; concentration, cell lysis, pH change, buffer addition, diluent addition, reagent addition, heating or cooling, labeling binding of label, cross-linking by irradiation, separation of unbound label, inactivation and/or removal of interfering compounds, and any other necessary to prepare the sample for analysis by a single particle analyzer. One or more steps of the process can be involved. In some embodiments, blood is processed to separate plasma or serum. Additional labeling, removal of unbound label, and/or dilution steps can also be performed on the serum or plasma sample.

In some embodiments, the analyzer system includes 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 first be centrifuged. A sample preparation system may then partially process the sample within the analyzer. Processing within the analyzer includes labeling the sample, mixing the sample with buffers, and other processing steps that will be known to those of skill in the art. In some embodiments, the blood sample is processed outside the analyzer system to provide a sample of serum or plasma, which is introduced into the analyzer system and further processed by the sample preparation system to produce particles or particles of interest. The particles are labeled and optionally unbound label is removed. In other embodiments, sample preparation can include immunodepletion of the sample to remove particles that are not of interest or that can interfere with analysis of the sample. In still other embodiments, the sample can be depleted of particles that can interfere with analysis of the sample. For example, sample preparation can include depletion of heterophile antibodies known to interfere with immunoassays using non-human antibodies to directly or indirectly detect particles of interest. . Similarly, other proteins that interfere with the measurement of particles of interest can be removed from the sample using antibodies that recognize the interfering proteins.

In some embodiments, samples can be subjected to solid phase extraction prior to assay and analysis. For example, a serum sample to be assayed for cAMP can first be subjected to solid phase extraction using a c18 column to which it binds. Other proteins such as proteases, lipases, and phosphatases are washed from the column and cAMP is eluted, essentially free of proteins that can degrade cAMP or interfere with the measurement of cAMP. Solid phase extraction can be used to remove the basic matrix of the sample, which can reduce the sensitivity of the assay. In still other embodiments, the particles of interest present in the sample can be concentrated by drying or lyophilizing the sample, solubilizing the particles to a volume smaller than the original volume of the sample. For example, a sample of inspired concentration (EBC) can be dried and resuspended in a small volume of a suitable buffer solution to enhance 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, inspired breath concentrate (EBC), biopsy sample, forensic sample, bioterrorism sample, etc. Provide a sample preparation system that provides a complete preparation of a sample to be analyzed with 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 initial sample is a blood sample that is further processed by the analyzer system. In some embodiments, the sample is a serum or plasma sample that is further processed by an analyzer system. A serum or plasma sample can be further processed, for example, by contacting with a label that binds to the particle or particles of interest, and then using the sample with or without removal of unbound label. can be done.

In some embodiments, sample preparation is performed on one or more microtiter plates, such as 96-well plates, either external to the analytical system or in the sample preparation component of the analytical system. Reservoirs, such as reagents and buffers, can be in intermittent fluid communication with the wells of the plate by tubes or other suitable structures, as is well known in the art. Samples can be prepared separately in 96-well plates or tubes. The steps of sample isolation, label binding, and label separation, if desired, can be performed on one plate. In some embodiments, the prepared particles are then released from the plate and the sample transferred to a tube for sampling into the 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 is described with respect to a 96-well plate, it will be appreciated that any vessel suitable for sample preparation to contain one or more samples may be used. For example, 384-well or 1536-well standard microtiter plates can be used. More generally, in some embodiments, the sample preparation system has a , or more than 10,000 samples can be held and prepared. In some embodiments, multiple samples can be taken for analysis on multiple analyzer systems. Thus, in some embodiments, two samples, or more than about 2, 3, 4, 5, 7, 10, 15, 20, 50, or 100 samples are taken from the sample preparation system and multiple sample analyzers are Run in parallel on the system.

Microfluidic systems are useful for sample preparation as part of an analyzer system, particularly for samples suspected of containing particles in sufficiently high concentrations that small amounts of sample are required for detection. can be used. The principles and techniques of microfluidic manipulation are known in the art. U.S. Pat. Nos. 5480614, 5716825, 5603351, 5858195, 5863801, 5955028, 5989402, 6041515, 6071478, 6355420, 6495104, 6386219, 9660660 Nos. 6802342, 6749734, 6623613, 6554744, 6361671, 6143152, 6132580, 5274240, 6689323, 6783992, 6537437, 6599436, See No. 6811668 and published PCT patent application WO9955461 (A1). Samples can be prepared serially or in parallel for use in single or multiple analyzer systems.

The sample preferably contains a buffer. The buffer may be mixed with the sample outside the analyzer system or provided by the sample preparation mechanism. Although any suitable buffer can be used, preferred buffers have a low background fluorescence, are inert to the detectably labeled particles, are able to maintain the working pH, and are dynamic In embodiments where is electrokinetic, it has an ionic strength suitable for electrophoresis. Buffer concentrations 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 confers solubility, function and detectability of the molecule of interest. For applications with pumping, phosphate, glycine, acetate, citrate, acidulate, carbonate/bicarbonate, imidazole, triethanolamine, glycine, amide, borate, MES , Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, Preferably the buffer is selected from the group consisting of HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS and CABS. Buffers include Gly-Gly, bicine, tricine, 2-morpholineethanesulfonic acid (
MES), 4-morpholinepropanesulfonic acid (MOPS), and 2-amino-2-methyl-1-propanol hydrochloride (AMP). A useful buffer is 2 mM Tris/borate at 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 prior applications and are hereby incorporated by reference from US patent application Ser. No. 11/048,660.

E. Sample Recovery One highly useful feature of embodiments of the analyzers and analytical systems of the present invention is the ability to analyze samples without consuming them. This can be particularly important when sample material is limited. Withdrawing the sample also makes it possible to perform other analyzes or re-analyze 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 forensic use, drug screening, and clinical diagnostic applications, will be appreciated by those skilled in the art. it is obvious.

Accordingly, in some embodiments, the analyzer system of the invention further provides a sample collection system for collecting the sample after analysis. In these embodiments, the system includes mechanisms and methods for drawing the sample into the analyzer, analyzing it, and then returning it to the sample holder, eg, sample tube, by the same route. The sample remains uncontaminated as it is not broken and does not enter any of the valves or other tubes. Additionally, all materials in the sample path are highly inert, such as PEEK, fused silica, or sapphire, so there is little contamination from the sample path. The use of stepper motor controlled pumps (particularly analytical pumps) allows precise control of the volumes drawn up and drawn back. This allows complete or almost complete recovery of the sample with little if any dilution with the flush buffer. Thus, in some embodiments, about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99% Greater than .9% of the samples are recovered after analysis. In some embodiments, the collected sample is undiluted. In some embodiments, the recovered sample is about 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, 1.05-fold, 1.01-fold, 1-fold .005-fold, or diluted less than 1.001-fold.

Any mechanism for transporting the liquid sample from the sample vessel to the analyzer can be used to collect and/or retrieve the sample. In some embodiments, the inlet end of the analytical capillary is a short length of tubing (e.g., a PEEK tube). When flushing to remove the previous sample cleanly from the device, place this tube over a waste container to catch the flushed waste. When drawing a sample, place the tube into the sample well or test tube. Typically, the sample is rapidly drawn in and then slowly pushed out while observing the particles within the sample. Alternatively, in some embodiments, the sample may be slowly withdrawn during at least a portion of the draw cycle and the sample analyzed during the slow draw. After this, the sample can be quickly returned and quickly flushed. In some embodiments, samples can be analyzed in both inward (pull) and outward (pull) cycles, which improves counting statistics and confirming results, such as small dilute samples. be. If it is desired to save the sample, it can be pushed back into the same original sample well or another. If sample storage is not desired, place tube on waste container.

VI. Methods Using Sensitive Analysis of Cardiac Troponin The methods of the present invention allow the measurement of cardiac troponin levels at concentrations much lower than those previously measured. Although cardiac troponin is an accepted marker for myocardial damage, current analytical methods find their usefulness due to the fact that detection is possible only after substantial damage to the myocardium has occurred due to the lack of sensitivity of current methods. Gender is limited. The European Union Society of Cardiology/American College of Cardiology for the Redefinition of Myocardial Infarction defines increased concentrations of cardiac troponin as a measure above the 99th percentile distribution of cardiac troponin concentrations in the reference group, an extremely low threshold. It is recommended to define A total imprecision (CV) at this <10% decision limit is recommended. however,
The analytical imprecision obtained by currently available immunoassays for cardiac troponin is not uniform, mainly in the low concentration range. Furthermore, currently available assays lack sufficient sensitivity to detect troponin levels in non-clinical (normal) subjects, and true baseline or normal population-defined troponin levels have not been defined. The analyzer system of the present invention was shown to be able to consistently detect cTnI levels at concentrations of 10 pg/ml or less with a total imprecision of less than 10% (see Examples). Accordingly, the present invention provides diagnostic, prognostic, or therapeutic methods based on sensitive detection of cardiac troponin in an individual.

In some embodiments, the invention provides a method for i) determining a cardiac Troponin concentration in a sample obtained from an individual or determining a cardiac Troponin concentration in a series of samples from an individual, wherein: determining a concentration in a cardiac troponin assay that has a detection limit for cardiac troponin of about 50, 40, 30, 10, 5, 4, 3, 2, or less than 1 pg/ml, such as less than about 20 pg/ml; ) determining a diagnosis, prognosis, or treatment in said individual based on the concentration in the sample or series of samples; I will provide a. Methods for determining the concentration of cardiac troponin include any suitable method having the requisite sensitivity, such as the methods described herein. In some embodiments, the method utilizes a method of determining the concentration of cardiac troponin in a sample comprising detecting a single molecule of troponin or a complex or fragment thereof.

In some embodiments, a sample (e.g., blood sample, serum sample, or plasma sample) obtained from an apparently healthy population is analyzed for cardiac troponin (e.g., cardiac troponin I), and 80%, 90% of the population %, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% below that level (concentration) determines the threshold concentration of troponin . This value is a threshold. In some embodiments, the threshold is set at the 99th percentile. In some embodiments, the assay is performed using a method that has a detection level of cardiac 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 invention comprises the step of comparing a concentration value of cardiac Troponin in a sample obtained from an individual to a normal value or range of normal values for cardiac Troponin, comprising: detecting cardiac Troponin in said sample; determining a normal value or range of normal values in a cardiac troponin assay having a limit of less than about 50, 40, 30, 10, 5, 4, 3, 2 or 1 pg/ml, such as less than about 20 pg/ml; ) determining a diagnosis, prognosis, or treatment regimen in said individual based on the comparison.

In some embodiments, the cardiac troponin is cardiac troponin I or cardiac troponin T. In some embodiments, the cardiac troponin is cardiac troponin T. In some embodiments, the cardiac troponin is cardiac troponin-I. In this method, all troponins, eg, all cTnI, or cTnT, or all cTnI+cTnT, described herein can be used in diagnosis, prognosis, or determination of treatment regimens. In some embodiments, the method can use the concentration of cardiac troponin free, complex, or fragment or a comparison (e.g., ratio) thereof to determine a diagnosis, prognosis, or treatment method. .

A. A sample sample or series of samples can be any suitable sample; in some embodiments, the sample is blood, serum, or plasma. In some embodiments, the sample or series of samples are serum samples. An 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, minutes, hours, days, weeks, months, or years. For acute clinical settings, serial samples are typically taken over hours and days, with the samples separated by at most hours. Sample intervals can be multiple months or multiple years when individuals are followed for longer periods. A diagnostic, prognostic, or therapeutic method can be determined from a single sample or a series of one or more samples, or from changes in a series of samples, e.g. while 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 hours, days, weeks, months, or years. A given individual's rate of change may in some cases be a relative rather than an absolute value. In acute settings, very rapid rates of change, eg, "spikes," may indicate an impending, ongoing, or recent cardiac event. In other settings, elevated values over days, weeks, months, or years in an individual are indicative of ongoing and worsening heart damage, such as cardiac conditions (e.g., cardiac hypertrophy or congestive heart failure). ) or non-cardiac conditions (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 cardiac troponin levels between samples taken prior to and during the study can provide diagnostic or prognostic information, e.g., for coronary artery disease or other myocardial-related diseases. I can show you the possibilities. Other comparisons can also be made, such as comparing any sample to normal or threshold levels or determining the rate of change in cardiac troponin concentration in a sample, all of which are useful for heart and cardiovascular health and as described herein. It can provide useful information regarding the other pathologies described.

In some embodiments, at least one sample is taken at or near a time when the individual exhibits one or more symptoms indicative of a condition that may involve heart damage to a health care professional. The settings in which an individual may come to a health care provider include, but are not limited to, emergencies such as ambulance, emergency management, clinical management, centralized management, monitoring units, inpatients, outpatients, doctor's offices, clinics, and ambulances. Response environments, and health screening environments are included. In some embodiments, one or more samples w are taken from individuals and assayed for cardiac troponin locally, ie, in an environment at or near the sampled environment. For example, an individual presenting to a hospital can have one or more samples collected that are assayed for cardiac troponin within the hospital. For example, an individual presenting to a hospital can have one or more samples collected that are assayed for cardiac troponin within the hospital. In some embodiments, one or more samples are obtained from the individual and assayed for cardiac troponin in a 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 consistent with 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. Determining a diagnostic, prognostic, or therapeutic method. In some embodiments, step ii) comprises comparing said concentration or series of concentrations to a normal value for said concentrations, comparing said concentration or series of concentrations to a predetermined comparing to a threshold level; comparing said concentration or series of concentrations to a baseline value; or determining a rate of change in concentration for said series of concentrations.

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

Normal values, threshold values, 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 from case and control populations where the case population indicates a biological state for which a diagnostic, prognostic, or therapeutic method is desired, and the control population does not. can be determined by In some embodiments, long-term studies can be performed. For example, a case population may be a subset of a control population that exhibits a biological condition over time. It will be appreciated that data from multiple studies can be used to determine an agreed value or range of values for normal or prognostic or diagnostic levels.

In developing a diagnostic or prognostic test, data can be obtained from a group of subjects for one or more potential markers. Preferably, the subject group is divided into at least two sets, the first and second sets each having approximately equal numbers of subjects. The first set includes subjects identified as having the disease or, more generally, having the first condition. For example, this first set of patients may be patients with recent onset of disease or patients with a particular type of disease, such as AMI. Confirmation of the disease state can be done by more rigorous and/or costly tests such as MRI or CT. Hereinafter, this first set of subjects will be referred to as "patients." Subjects in the second set are simply subjects that do not fall into the first set. This second set of subjects may be "non-diseased," which are normal subjects. Alternatively, this second set of subjects can be selected to exhibit a symptom or group of symptoms similar to or similar to those exhibited by the "diseased" subjects. As yet another alternative, this second set may present them at a different time than the onset of the disease. Data for the same set of markers are preferably available for each patient. This marker set may include all candidate markers that may be suspected of being relevant for detection of a particular disease or condition. No actual known relevance is required. Embodiments of the compositions, methods, and systems described herein can be used to determine which of the candidate markers are most relevant for diagnosing a disease or condition. The levels of each marker in the two sets of subjects may be distributed over a wide range, eg as a Gaussian distribution. However, distribution fitting is not required.

1. Acute Myocardial Infarction The methods of the present invention are particularly useful for diagnosis, prognosis, and/or treatment selection in patients with suspected acute myocardial infarction (AMI). Single or serial cardiac troponin measurements in patients with suspected AMI provide increasing prognostic information that indicates appropriate early therapeutic intervention to improve prognosis and minimize the risk of adverse consequences.

Accordingly, the present invention assays a sample obtained from an individual, such as a blood sample, a plasma sample, and/or a serum sample, for cardiac troponin, such as cTnI, to about 50, 40, 30, 20, 15, 10, 9, Cardiac Troponin concentration in the sample with a detection limit of less than 8, 7, 6, 5, 4, 3, 2, or 1 pg/ml, such as less than about 20 pg/ml, where the cardiac Troponin concentration in the sample is indicative of AMI (predicts whether or not AMI) provides a method of diagnosing, predicting, and/or preventing or treating AMI in an individual. Cardiac troponin may be cTnI or cTnT, total troponin or indicative of a particular form (e.g., free, complex, or fragment); some embodiments As described herein, ratios of one or more forms of troponin are used. In some embodiments, total cTnI in a sample or series of samples is measured. In some embodiments, total cTnT in a sample or series of samples is measured. In some embodiments, total cTnI+cTnT in a sample or series of samples is measured. In some embodiments, cardiac troponin levels are determined at or near the time the individual presents symptoms indicative of AMI 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 a spike in cardiac troponin concentration in the sample provides a basis for, predicts, or provides a prognosis for AMI. In some embodiments, a spike 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 baseline A basis is indicated, predicted, or provided. In some embodiments, if available, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 in a single sample , or cardiac troponin levels greater than 50 pg/ml indicate, predict, or provide a basis for the prognosis of AMI. In some embodiments, about 1 to 10, or about 5 to 15, or about 10 to 50, about 10 to 200, about 10 to 100, or about 10 to 40, or about 15 to 50, or about 15 to A cardiac troponin level 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 provides the basis for the prognosis of AMI. , predicted or provided.

In some embodiments, diagnosis or prognosis comprises stratifying individuals based on cardiac troponin concentration in a sample or series of samples. Such stratification is based on cardiac troponin concentrations in a single sample, the presence and/or magnitude of spikes from baseline in a series of samples, the ratio of different forms of cardiac troponin, the proportion of different forms of cardiac troponin, Absolute value, percent change in concentration of cardiac troponin in a series of samples, or percent change in concentration of one or more forms of cardiac troponin, change in ratio of different forms of cardiac troponin over time in a series of samples, samples or It can be based on any other information based at least in part on the cardiac troponin concentration in the series of samples. Stratification can be based on values obtained from populations of normal and diseased subjects described herein. Appropriate treatment can also be determined based on individual stratification.

In some embodiments, the concentration of cardiac troponin is determined in combination with one or more other markers, e.g., a marker of myocardial ischemia, a marker of myocardial infarction, or a marker of stroke, and the concentration of each marker is diagnostic, It is considered in prognosis, or in determining treatment methods. As will be apparent to those skilled in the art, other clinical indicators such as EKG, symptoms, history, etc. are also commonly considered. Appropriate diagnostic, prognostic, or therapeutic algorithms can be constructed based on such marker combinations and clinical indicators in combination with troponin levels.

Markers useful in combination with cardiac troponin in the methods of the present invention include, 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 acids, cardiac fatty acid binding protein (H-FABP), ischemia-modified albumin, myosin light chain 1, myosin light chain 2 included. Markers of inflammation and plaque instability useful in combination with cardiac troponin in the methods of the invention include, but are not limited to, C-reactive protein, white blood cell count, soluble CD40 ligand, myeloperoxidase, monocyte chemoattractant protein. 1, whole blood choline, and pregnancy-associated plasma protein A. Other markers of inflammation can also be detected, including IL-8, IL-1β, IL6, IL10, TNF, and IL-12p70, as well as other cytokines or marker combinations apparent to those skilled in the art.

In some embodiments, the cardiac troponin, e.g., cTnI, is creatine kinase (CK) and its myocardial fraction CK myocardium, e.g., in the same sample or in samples 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 acids, heart fatty acid binding protein (H-FABP), deficient It is measured with a marker selected from the group consisting of blood modified albumin, myosin light chain 1, myosin light chain 2. In some embodiments, cardiac troponin, eg, cTnI, is measured along with CK-MB, eg, in the same sample, or in samples from the same individual taken at or near the same time point.

In some embodiments, cardiac troponin alone or in combination with other markers or clinical signs, measured as described herein, is used to determine reinfarction. In some embodiments, cardiac troponin, alone or in combination with other markers or clinical indications, measured as described herein, is an infarct characteristic, e.g., size, or duration from infarction. used to determine In the latter case, proteolytically generated troponin fragments in the blood can be compared to total troponin; the higher the fraction of fragments, the longer the infarction.

2. Conditions Other than AMI The methods of the invention also include diagnostic, prognostic, and therapeutic methods based on cardiac troponin levels in samples useful for conditions other than AMI.

Many pathologies involve possible or actual heart damage, and measuring cardiac troponin at the levels described herein allows for early detection and intervention of such damage. Knowledge of cardiac troponin concentrations measured by this method and compositions of the invention are useful in the diagnosis, prognosis, and treatment determination of such conditions. Conditions include percutaneous coronary intervention, cardiac surgery, heart failure, acute rheumatic fever, amyloidosis, cardiac trauma (including contusion, ablation, pacing, firing, cardioversion, catheterization and cardiac surgery), recanalization. Flow injury, cardiotoxicity due to cancer therapy, congestive heart failure, end-stage renal failure, type II glycogen storage disease (Pompe disease), heart transplantation, dyshemoglobinopathies due to transfusion hemosiderosis, hypertension, including gestational hypertension, low blood pressure, often with arrhythmias Included are blood pressure, hypothyroidism, myocarditis, pericarditis, postoperative non-cardiac surgery, pulmonary embolism, and sepsis.

In these embodiments, troponin levels can be determined concurrently with levels of markers specific for non-cardiac disease or other symptoms or clinical manifestations of disease; To do so, concentrations and/or information of markers of other symptoms or clinical signs are combined with cardiac troponin concentration information determined as described herein. For example, in embodiments of the invention, in addition to determining cardiac troponin concentration, one or more of the polypeptides described above or other protein markers useful in the diagnosis, prognosis, or discrimination of disease are determined. can be used. In some embodiments, a group of markers for disease is provided, the group comprising cardiac troponin levels described herein and at least one other marker for disease. A group can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more individual markers, including one or more cardiac troponins, eg, total cTnI. Analysis of single markers or subsets of markers can be performed by one skilled in the art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not limited to, emergency, emergency management, crisis management, centralized control, monitoring units, inpatient, outpatient, doctor's office, clinic, and health screening environments. Furthermore, one skilled in the art can use single markers or subsets of markers in combination with diagnostic threshold adjustment in each of the circumstances described above to optimize clinical sensitivity or specificity.

a. Cardiotoxicity The compositions and methods of the present invention are particularly useful for determining and monitoring cardiotoxicity resulting from therapy, eg, cardiotoxicity of drug therapy. Thus, for example, the present invention provides for i) determining cardiac Troponin concentration in a sample from an individual or determining cardiac Troponin concentration in a series of samples, wherein the individual is undergoing treatment or after at least one sample is obtained from the individual at least one sample, wherein the limit of detection of cardiac troponin in said sample is less than about 50, 40, 30, 10, 5, 4, 3, 2 or 1 pg/ml, such as less than about 20 pg/ml and ii) assessing the degree of cardiotoxicity of the treatment based on said one or more concentrations, by measuring cardiac troponin in the individual. A method for assessing cardiotoxicity of a therapy is provided. In some embodiments, the treatment is drug therapy. In some embodiments, the treatment is non-drug therapy. Methods for determining cardiac troponin concentration include any suitable method having the required sensitivity, such as those described herein. In some embodiments, the method utilizes a method of determining cardiac troponin concentration in a sample comprising detecting a single molecule of troponin, or a complex or fragment thereof.

Methods for measuring cardiotoxicity that utilize cross-reactive antibodies described herein, i.e., antibodies that react with troponin from at least two species, such as human and another species such as rat, dog, mouse, or monkey. is particularly useful. Such antibodies can be used in animal tests of drug toxicity, and the individual whose toxicity is to be evaluated can be, for example, mammals such as rats, mice, dogs, monkeys, or other animals used in such tests. be an animal. Toxicity in different species can be directly compared if the antibody used in the assay is the same antibody, thus reducing variability.

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

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

In one embodiment, the business methods herein detect a single molecule of one or more markers at a detection level of less than about 50, 20, 10, 5, or 1 pg/ml. establishing a range of concentrations of said one or more markers in a biological sample obtained from a first population by measuring concentrations of said one or more markers in and commercializing one or more of the markers established in the above steps, eg, in a diagnostic product. Diagnostic products herein include one or more antibodies that specifically bind to a cardiac troponin marker and emit at least about 200 photons when stimulated by a laser that emits at the excitation wavelength of the fluorescent moiety. The laser is focused to a spot of about 5 microns or greater in diameter containing the fluorescent moiety, and the total energy directed to the spot by the laser is about 3 microjoules or less.

In one embodiment, the business methods herein measure 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 establishing a concentration range of said cardiac troponin marker in a biological sample obtained from a first population by; establishing a range of normal values for cardiac troponin markers using the system including providing diagnostic services to determine whether one has an AMI condition. Diagnostic services herein may be provided by a CLIA-approved laboratory licensed under the business, or by the business itself. Diagnostic services herein may be provided directly to a healthcare provider, health insurance company, or patient. Thus, the business methods herein may generate revenue from, for example, diagnostic services or diagnostic products.

The business methods herein also contemplate providing diagnostic services to, for example, health care providers, health insurance companies, or patients. Businesses herein may provide diagnostic services by outsourcing to service laboratories or establishing service laboratories (under Clinical Laboratory Improvement Amendment (CLIA) or other regulatory approval). Such service laboratories can then perform the methods disclosed herein to ascertain whether a cardiac Troponin marker is present in the sample.

VII. Compositions The present invention provides compositions useful for the detection and quantification of cardiac troponins. The composition is labeled with a label suitable for detection by the methods of the invention, wherein one or both of the binding partners of cardiac troponin are labeled with a label suitable for detection by the methods of the invention. A binding partner pair, in some embodiments, a solid support to which a capture binding partner is bound, having a detection binding partner.

A representative embodiment includes a composition for detecting cardiac troponin comprising a binding partner of cardiac troponin conjugated to a fluorescent moiety, wherein the fluorescent moiety is at least Capable of emitting about 200 photons, the laser is focused to a spot about 5 microns or greater in diameter containing the fluorescent portion, and the total energy directed by the laser to the spot is about 3 microjoules or less. In some embodiments, the binding partner comprises an antibody against cardiac 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-reacting antibody, e.g., cardiac troponin from at least two species, e.g., at least two species selected from the group consisting of human, monkey, dog and mouse. is an antibody. In some embodiments, the antibody cross-reacts with cardiac troponins from all of humans, monkeys, dogs and mice. In some embodiments, the cardiac troponin is selected from the group consisting of cTnI and cTnT. In some embodiments, the cardiac troponin is cTnI. In some embodiments, the cardiac troponin is cTnT. The antibody may be specific for a particular region of the troponin molecule, eg, a region including amino acids 27-41 of cardiac troponin I. Fluorescent moieties can contain one or more molecules containing at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of the indolium ring is a chemically reactive group or a conjugated agent group. contains Labeling compositions can include fluorescent moieties that include one or more dye molecules selected from the group consisting of AlexaFluor 488, 532, 647, 700, or 750. Labeling compositions can include fluorescent moieties that include one or more dye molecules selected from the group consisting of AlexaFluor 488, 532, 700, or 750. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor488. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor555. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor610. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor647. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor680. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor700. Labeling compositions can include fluorescent moieties that include one or more dye molecules that are AlexaFluor750e.

In some embodiments, the invention provides a composition comprising a set of standards for determining cardiac troponin concentration, at least one of the standards being about 20, 15, 10, 5, 4 , cardiac troponin concentrations less than 3, 2, or 1 pg/ml. In some embodiments, the invention provides compositions comprising a set of standards for determining cardiac Troponin concentration, at least one of the standards having a cardiac Troponin concentration of less than about 20 pg/ml. be. In some embodiments, the invention provides compositions comprising a series of standards for determining cardiac Troponin concentration, at least one of the standards having a cardiac Troponin concentration of less than about 10 pg/ml. . In some embodiments, the invention provides a composition comprising a set of standards for determining cardiac Troponin concentration, at least one of the standards having a cardiac Troponin concentration of less than about 5 pg/ml. be. In some embodiments, the invention provides a composition comprising a set of standards for determining cardiac Troponin concentration, at least one of the standards having a cardiac Troponin concentration of less than about 1 pg/ml. be.

Other compositions of the invention are as described herein.

VIII. Kits The present invention further provides kits. Kits of the invention comprise one or more compositions useful for the sensitive detection of cardiac troponin as described herein in suitable packaging. In some embodiments, kits of the invention provide a label, eg, a binding partner such as an antibody specific for cardiac troponin, wherein the binding partner is attached to a fluorescent moiety. In some embodiments, kits of the invention provide binding partner pairs, e.g., antibody pairs that are specific for cardiac troponin, wherein at least one of the binding partners is directed to cardiac troponin described herein. is a sign. In some embodiments, binding partners, eg, antibodies, are provided in separate containers. In some embodiments, binding partners, eg, antibodies, are provided within 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 fluorescent moieties described herein.

Binding partners, eg, antibodies, solid supports, and fluorescent labels for kit components can be any suitable components, such as those described herein.

Kits may also contain reagents useful in the methods of the invention, e.g., buffers and other reagents used in binding reactions, detergents, buffers or other reagents for preconditioning equipment for performing the assay, equipment. An elution buffer or other reagents can be included to move the sample through.

The kit may include one or more standards, e.g., highly purified standards, e.g., recombinant human cTnI or human cTnT, or various fragments, conjugates thereof, etc., for use in assays of the invention. can include standards for The kit can further include instructions for use.

EXAMPLES The following examples are provided for illustrative purposes and are not intended to limit the disclosure of the remainder.

Unless otherwise specified, sample treatments in the examples were analyzed in the 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) with a 639 nm wavelength continuous wave gallium arsenite diode laser (Blue Sky Research, Milpitas, Calif.). Flow rate = 5 microliters/min through a 100 micron square ID and 300 micron square OD fused silica capillary; non-confocal arrangement of lenses (see, e.g., Figure 1A); 0.8 aperture focusing lens (Olympus) a silicon avalanche photodiode detector (Perkin Elmer, Waltham, MA);

Biomarker Sandwich Assay: Cardiac Troponin I (cTnI)
Assay: The purpose of this assay was to detect the presence of cardiac troponin I (cTNI) in human serum. The assay format was a two-step sandwich immunoassay based on mouse monoclonal capture antibody and goat polyclonal detection antibody. A 10 microliter sample was required. The working range of the assay is 0-900 pg/ml, with detection of 1-3 pg/ml being a typical analytical limit. Completion of the assay required approximately 4 hours of bench time.

Materials: The following materials were used in the following procedure: Assay Plates: Nunc Maxisorp, Product 464718, 384-well, clear, passively coated with monoclonal antibody BiosPacific A34440228P, Lot# A0316 (5 μg/ml in 0.05 M sodium carbonate). , pH 9.6, room temperature overnight); blocked with 5% sucrose, 1% BSA in PBS, stored at 4°C. Human cardiac troponin (BiosPacific catalog number J34000352) was used for the standard curve. Diluents for standard concentrations were human serum immunodepleted of endogenous cTNI, aliquoted and stored at -20°C. Standard dilutions were performed in 96-well, conical, polypropylene (Nunc product number 249944). The following buffers and solutions were used: (a) assay buffer: 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 A polyclonal antibody was affinity purified against peptide 3 (BiosPacific G129C), labeled with the fluorochrome AlexaFluor647 and stored at 4° C.; detection antibody diluent: 50% assay buffer, 50% passive blocking solution. Washing Buffer: Borate Buffered 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, the detection antibody G-129-C was prepared by dissolving 100 µg of the detection antibody G-129-C in 400 µL of coupling buffer (0.1 M NaHCO ₃ ). C was conjugated to AlexaFluor647. The antibody solution was then concentrated to 50 μl by transferring it into a YM-30 filter and subjecting the solution and filter to centrifugation. The YM-30 filters and antibody were then washed three times by adding 400 μl of coupling buffer. 50
Antibody was recovered by adding 1.1 to the filter, inverting the filter, and centrifuging at 5,000 xg for 1 minute. The resulting antibody solution was 1-2 μg/μl. The AlexaFluor 647 NHS ester was reconstituted by adding 20 μl of DMSO to one vial of AlexaFluor 647 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 AlexaFluor647 solution and mixed. The solution was ultrafiltered through YM-30 to remove low molecular weight components. The volume of retentate containing 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 an Ultrafree 0.22 centrifugation unit and spun at 12,000×g for 2 minutes. The filtrate containing conjugated antibody was collected and used for the assay.

Procedure: Preparation and Analysis of cTnI Standards and Samples:
A standard curve was generated as follows: Working standards were prepared by serially diluting cTnI stocks into standard dilutions or to reach cTnI concentrations ranging from 1.2 pg/ml to 4.3 μg/ml. was prepared (0-900 pg/ml).

10 μl of passive blocking solution and 10 μl of standards or samples were added to each well. Standards were run in quadruplicate. Plates were sealed with Axyseal sealing film, centrifuged at 3000 RPM for 1 minute and incubated at 25° C. for 2 hours with shaking. Plates were washed 5 times and centrifuged on paper towels in the inverted position until the rotor reached 3000 RPM. A 1 nM working dilution of detection antibody was prepared and 20 μl of detection antibody was added to each well. Plates were sealed, centrifuged, and the assay was incubated for 1 hour at 25° C. with shaking. 30 μl of elution buffer was added per well, the plate was 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.

For analysis, 20 μl per well were captured at 40 μl/min and 5 μl were analyzed at 5 μl/min. Data were analyzed based on a 4 sigma threshold. The raw signal versus standard concentration was plotted. A linear fit was performed to the low concentration range and a non-linear fit was performed to the full standard curve. The limit of detection (LoD) was calculated as LOD = (3 x zero standard deviation)/linear fit slope. Sample concentration was determined from an equation (linear or non-linear) appropriate to the sample signal.

An aliquot was pumped into the analyzer. Individually labeled antibodies were measured during capillary flow by setting the interrogation volume such that the emission of only one fluorescent label was detected in a defined space after laser excitation. Although each signal represents a digital event, this configuration allows for extremely high analytical sensitivity. The total fluorescence signal 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/sample. The limit of detection of the cTnI assay of the present invention was determined by the mean+3SD method.

Results: Data for a typical cTnI standard curve measured in quadruplicate using the assay protocol are shown in Table 3.

The sensitivity of the analyzer system was tested in 15 runs and was routinely found to detect sub-femtomole/l (fM) levels of calibrator, as shown in Table 4. Precision 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 of detection (LoD) was determined by 15 consecutive assays. LoD is the mean of 0 standards + 3 SD (n=4) intra-assay measurements. The average LoD was 1.7 pg/ml (range 0.4-2.8 pg/ml).

Sample recovery was determined by immunodepleting cTnI and analyzing samples of serum spiked with known amounts of cTnI. Table 5 shows data on sample collection by the system analyzed over 3 days.

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

These data demonstrate that the analyzer system of the present invention enables the performance of highly sensitive laser-guided immunoassays for sub-femtomole concentrations of cTnI.

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

Materials: MyOne Streptavidin C1 microparticles (MPs) are obtained from Dynal (650.01-03, 10 mg/ml stock). Buffers used in the assay included 10× borate-buffered saline Triton buffer (BBST) (1.0 M boric acid, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3). ); assay buffer (2 mg/ml normal goat IgG, 2 mg/ml normal mouse IgG, and 0.2 mg/ml MAB-33-IgG-polymer in 0.1 M Tris, pH 8.1; 0.025 M EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.1% NaN3, stored at 4° C.); of urea, 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 containing 1-2 biotins per IgG (BiosPacific)) and Det-Ab (G-129-C (BiosPacific) conjugated to A647). , 2-4 fluorophores per IgG). The standard is recombinant human cardiac troponin I (BiosPacific, catalog number J34120352). Calibrator diluent is BSA in TBS wEDTA at 30 mg/ml.

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

Preparation of standards and samples: Dilute standards with calibrator diluents to generate an appropriate standard curve (usually 200 pg/ml to 0.1 pg/ml). Frozen serum and plasma samples should be centrifuged at 13K rpm for 10 minutes at room temperature. Carefully remove the clarified serum/plasma without possible pellets or floaters and place in a fresh tube. Pipet 50 μl of each standard or sample into the appropriate wells.

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

Washing and Detection: Place the plate on the magnet and remove the supernatant after confirming that all MPs have been captured by the magnet. After removing the plate from the magnet, add 250 μl of wash buffer. The plate is then placed on a magnet and the supernatant is removed after confirming that all MPs have been captured by the magnet. 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 the JitterBug, 5 for 30 minutes at room temperature.

Wash and Elute: Place plate on magnet and wash 3 times with wash buffer. After confirming that all MPs have been captured by the magnet, remove the supernatant and add 250 μl of wash buffer. After washing, samples are transferred into new 96-well plates. A new plate is then placed on the magnet and the supernatant is removed after ensuring that all MPs have been captured by the magnet. After removing the plate from the magnet, add 250 μl of wash buffer. The plate is then placed on a magnet and the supernatant is removed after confirming that all MPs have been captured by the magnet. 20 μl of elution buffer is then added and the mixture is incubated on the JitterBug, 5 for 30 minutes at room temperature.

Filter off MPs and transfer to 384-well plate: Standards and samples are transferred into a 384-well filter plate placed on top of the 384-well assay plate. The plates are then centrifuged at 3000 rpm at room temperature using a plate rotor. Remove the filter plate and add the appropriate calibrator. The plate is coated and ready to run on the SMD.

SMD: Pump an aliquot into the analyzer. After laser excitation, individually labeled antibodies are measured during capillary flow by setting the interrogation volume such that the emission of only one fluorescent molecule in a defined space is detected. Although each signal represents a digital event, this configuration allows for extremely high analytical sensitivity. The total fluorescence signal 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/sample. The limit of detection of the cTnI assay of the present invention was determined by the mean+3SD method.

Concentration Ranges of cTnI in Normal Undiseased Subject Population A reference or normal range of cTnI concentrations in human serum was established using serum samples from 88 apparently healthy subjects (non-diseased). A sandwich assay as described in Example 1 was performed to count the number of signals or events described above using the single particle analyzer system of the present invention. Serum troponin I concentrations were determined by correlating the signal detected by the analyzer with the standard curve described above. All assays were performed in quadruplicate.

Following current recommendations by the European American College of Cardiology (ESC/ACC), the Troponin assay is less than 10% assay to reliably discriminate between ACS patients and those without ischemic heart disease. The 99th percentile of the normal range must be accurately quantified by assay imprecision (CV) and stratified for risk of adverse cardiac events. The assay showed that the biological threshold (cut-off concentration) for TnI was a TnI concentration of 7 pg/ml, established at the 99th percentile with a corresponding CV of 10% (Figure 5). At the 10% CV level, the precision profile points to TnI concentrations of 4 pg/ml and 12 pg/ml.

Furthermore, the assay correlates well with Troponin I standard measurements provided by the National Institute of Standards and Technology (Figure 6).

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

Detection of early release of TnI into the blood circulation of patients suffering from acute myocardial infarction (AMI) Test 1: 47 samples serially from 18 patients presenting with chest pain in the emergency department (ED) Obtained. All of these patients had non-ST elevation ECGs and were diagnosed with AMI. Concentrations of cTnI in initial samples from all 18 patients were determined by commercial assays at entry to the emergency room to be <350 pg/ml (10% cutpoint), 12 were <100 pg/ml (10% cutpoint). 99%) 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 those obtained using a commercially available assay.

Blood was drawn for the first time when the patient presented with chest pain (Sample 1) and subsequently at 4-8 hour intervals (Sample 2 at 12 hours; Sample 3 at 16 hours; Sample 4 at 24 hours; Sample 5 at hour; sample 6 at 36 hours; sample 7 at 42 hours; and sample 8) at 48 hours were taken. Sera were analyzed by the method of the present invention and current commercial methods and the results obtained are shown in FIG. The analyzer of the present invention detected TnI at the time the patient presented with chest pain (Sample 1), whereas the commercially available assay detected cTnI only at a much later time point (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 invention, indicating that sample 3 is positive for TnI and the occurrence of a cardiac event. was shown to suggest The biological threshold of commercial assays is between 111 and 333 pg/ml. Sample 3 would therefore not be considered to indicate a positive cardiac event.

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

Test 2: Fifty additional serum samples that tested negative with the commercial assay were tested using the analyzer and assay of the present invention. The results are shown in FIG. Of the 50 samples, 36 were within 99%, determined to be within the normal range established by the assay of the present invention. However, when tested, the remaining 14 samples determined to be within the commercial "normal" or non-disease range were above the biological thresholds established by the present invention.

Thus, the highly sensitive cTnI assay of the present invention allows detection of myocardial injury in patients when serum levels of cTnI are below threshold according to commercially available techniques. Use of the highly sensitive and accurate cTnI assays of the present invention allows detection of AMI sooner than with existing cTnI assays, thereby facilitating appropriate diagnosis and early medical intervention to improve outcomes. Opportunity is provided.

Claims (89)

A method for determining the presence or absence of a single molecule of troponin or a fragment or complex thereof in a sample, comprising:
i) labeling said molecule, fragment or complex with a label, if present;
ii) detecting the presence or absence of said label, wherein detecting the presence of said label indicates the presence of said single molecule, fragment or complex of troponin in said sample. 2. The method of claim 1, wherein said troponin is a cardiac isoform of troponin. 3. The method of claim 2, wherein said troponin is selected from the group consisting of cardiac troponin I (cTnI) and cardiac troponin C (cTnC). 4. The method of claim 3, wherein said troponin is cTnI. 2. The method of claim 1, wherein said single molecule of troponin is detectable with a detection limit of less than about 50 pg/ml in said detecting step. 2. The method of claim 1, wherein said troponin is detectable at a detection level of less than about 20 pg/ml. 2. The method of claim 1, wherein said label comprises a fluorescent moiety. said fluorescent moiety is capable of emitting at least about 200 photons when stimulated by a laser emitting at said moiety's excitation wavelength, said laser being focused to a spot of about 5 microns or greater in diameter containing said fluorescent moiety; 2. The method of claim 1, wherein the total energy directed at the spot by the laser is less than or equal to about 3 microjoules. 2. The method of claim 1, wherein said fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of said indolium ring comprises a chemically reactive group or a conjugated group. 2. The method of claim 1, wherein the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, or AlexaFluor700. 2. The method of claim 1, wherein said fluorescent moiety comprises AlexaFluor647. 2. The method of claim 1, wherein said label further comprises a binding partner of said troponin molecule, fragment or complex. 13. The method of claim 12, wherein said binding partner comprises an antibody specific for said troponin molecule, fragment or complex. 14. The method of claim 13, wherein said antibody is specific for a particular region of the troponin molecule. 15. The method of claim 14, wherein said antibody is specific for a region of cardiac troponin I comprising amino acids 27-41. 14. The method of claim 13, wherein said antibody comprises a polyclonal antibody. 14. The method of claim 13, wherein said antibody is a monoclonal antibody. 2. The method of claim 1, further comprising capturing said troponin or troponin complex on a solid support. 19. The method of claim 18, wherein said solid support is selected from the group consisting of microtiter plates and paramagnetic beads. 19. The method of claim 18, wherein said solid support comprises a capture partner specific for said troponin or troponin complex bound to said solid support. 21. The method of claim 20, wherein said binding of said capture partner to said solid support is non-covalent. 21. The method of claim 20, wherein said binding of said capture partner to said solid support is covalent. 23. The method of claim 22, wherein said covalent attachment of said capture partner is such that said capture partner binds to said solid support in a specific orientation. 24. The method of claim 23, wherein said specific orientation serves to maximize specific binding of said Troponin or Troponin complex to said capture partner. 21. The method of claim 20, wherein said capture partner comprises an antibody. 26. The method of claim 25, wherein said antibody is a monoclonal antibody. 26. The method of claim 25, wherein said antibody is specific for amino acids 87-91 of cardiac troponin I. 26. The method of claim 25, wherein said antibody is specific for amino acids 41-49 of cardiac troponin I. 2. The method of claim 1, wherein said sample is a blood, serum, or plasma sample. 30. The method of claim 29, wherein said sample is a serum sample. 2. The method of claim 1, wherein said label comprises a fluorescent moiety and step ii) comprises passing said label through a single molecule detector. the single molecule detector comprising:
a) a source of electromagnetic radiation for stimulating said fluorescent moiety;
b) a capillary flow cell for passing said fluorescent moiety;
c) a motive force source for moving said fluorescent moiety within said capillary flow cell;
d) an interrogation space defined within said capillary flow cell for receiving electromagnetic radiation emitted from said electromagnetic radiation source;
e) an electromagnetic radiation detector operably connected to said interrogation space for measuring the electromagnetic properties of said stimulated fluorescent moiety; and f) positioned between said interrogation space and said detector. 32. 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 regimen in an individual, comprising:
i) determining the concentration of cardiac Troponin in a sample obtained from said individual or determining the concentration of cardiac Troponin in a series of samples, wherein the detection limit of said cardiac Troponin in said sample is about 50 pg/ determining said concentration with a cardiac troponin assay that is less than ml;
ii) determining a diagnostic, prognostic, or therapeutic method in said individual based on said concentration in said sample or said concentration in said series of samples. step ii) comparing said concentration or series of concentrations to a normal value for said concentration; comparing said concentration or series of concentrations to a predetermined threshold level; comparing said concentration or series of concentrations to a baseline value; and determining a rate of change in concentration for said series of concentrations. wherein step ii) comprises comparing said troponin concentration in said sample to a predetermined threshold concentration to determine a diagnosis, prognosis, or treatment if the sample concentration is above the threshold level. Item 35. The method of Item 34. 36. The method of claim 35, wherein said threshold concentration is determined by determining the 99th percentile concentration of troponin in a normal population and setting said threshold concentration at said 99th percentile concentration. 34. The method of claim 33, wherein at least one sample is taken during or after the cardiac stress test. 34. The method of claim 33, wherein said cardiac troponin is selected from the group consisting of cardiac troponin I and cardiac troponin T. 34. The method of claim 33, wherein said cardiac troponin is cardiac troponin I. 39. The method of claim 38, wherein the cardiac troponin concentration is a total cardiac troponin concentration. 41. The method of claim 40, wherein the concentration of cardiac troponin is a concentration of cardiac troponin complex, cardiac troponin fragment, phosphorylated cardiac troponin, oxidized cardiac troponin, or a combination thereof. 42. The method of claim 41, wherein the concentration of cardiac troponin is compared to total cardiac troponin. 34. The method of claim 33, wherein the diagnosis, prognosis or treatment is of myocardial infarction. 44. The method of claim 43, wherein the diagnostic, prognostic, or therapeutic method comprises risk stratification for myocardial infarction risk level. 44. The method of claim 43, wherein said concentration or series of concentrations is determined at or near the time when said individual presents to a medical practitioner one or more symptoms indicative of myocardial ischemia or infarction or the possibility thereof. described method. 46. The method of claim 45, wherein said 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. 34. The method of claim 33, wherein said concentration is determined by a method comprising detecting a single molecule of troponin or a complex or fragment thereof. 48. The method of claim 47, comprising labeling said troponin or troponin complex with a label comprising a fluorescent moiety. said fluorescent moiety is capable of emitting at least about 200 photons when stimulated by a laser emitting at the excitation wavelength of said moiety, said laser focused to a 5 micron diameter spot containing said fluorescent moiety; 34. The method of claim 33, wherein the total energy directed by the laser to the spot is less than or equal to about 3 microjoules. 34. The method of claim 33, wherein said fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of said indolium ring comprises a chemically reactive or conjugated group. 34. The method of claim 33, wherein the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, or AlexaFluor700. 52. The method of claim 51, wherein said fluorescent moiety comprises AlexaFluor647. 34. The method of claim 33, wherein said label further comprises a binding partner of said troponin. 54. The method of claim 53, wherein said binding partner comprises an antibody specific for said troponin. 55. The method of claim 54, wherein said antibody comprises a polyclonal antibody. 34. The method of claim 33, further comprising capturing said troponin or troponin complex on a solid support. 57. The method of claim 56, wherein said solid support is selected from the group consisting of microtiter plates and paramagnetic beads. 57. The method of claim 56, wherein said solid support comprises a capture partner specific for said troponin or troponin complex bound to said solid support. Step i) further comprises assessing another indicator of said individual, and step ii) is based on assessing said another indicator of said troponin concentration and said non-troponin marker in said sample, or said series of 34. The method of claim 33, comprising determining a diagnostic, prognostic, or therapeutic method in said individual based on said concentration in a sample. 60. The method of claim 59, wherein said another indication is a clinical indication of myocardial ischemia or infarction. 61. The method of claim 60, wherein said another indicator is the concentration of one or more non-troponin markers in said sample or said series of samples. 60. The method of claim 59, wherein the one or more markers are a marker of cardiac ischemia or a marker of inflammation and a marker of plaque instability. the one or more markers of cardiac ischemia are 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 acids, heart-type fatty acid binding protein (H-FABP), ischemia-modified albumin, myosin light chain 1, myosin light chain 2. Method. 64. The method of claim 63, wherein said one or more markers comprises one or more specific markers of myocardial injury. 34. The method of claim 33, wherein the diagnosis, prognosis or treatment is for a condition other than myocardial infarction. 66. The method of claim 65, wherein said condition is cardiotoxicity. 67. The method of claim 66, wherein said cardiotoxicity is associated with drug administration to said individual. The condition is acute rheumatic fever, amyloidosis, cardiac trauma (including contusion, ablation, pacing, firing, cardioversion, catheterization and cardiac surgery), reperfusion injury, congestive heart failure, end stage renal failure , glycogen storage disease type II (Pompe disease), heart transplantation, dyshemoglobinopathy due to transfusion hemosiderosis, hypertension, including gestational hypertension, hypotension, often with arrhythmia, hypothyroidism, myocarditis, pericarditis, 66. The method of claim 65, selected from the group consisting of postoperative non-cardiac surgery, pulmonary embolism, and sepsis. 1. A composition for the detection of a troponin isoform comprising a binding partner of a troponin isoform attached to a fluorescent moiety, said fluorescent moiety being at least about 200 when stimulated by a laser emitting at said moiety's excitation wavelength. photons, wherein said laser is focused to a spot of about 5 microns or greater in diameter containing a fluorescent moiety, and wherein the total energy directed by said laser to said spot is about 3 microjoules or less. 70. The composition of claim 69, wherein said binding partner comprises an antibody against said troponin isoform. 71. The composition of claim 70, wherein said antibody is a polyclonal antibody. 71. The composition of claim 70, wherein said antibody is a monoclonal antibody. 70. The composition of claim 69, wherein said troponin isoform is a myocardial isoform. 74. The composition of claim 73, wherein said myocardial isoform is selected from the group consisting of cTnI and cTnT. 75. The composition of claim 74, wherein said myocardial isoform is cTnl. 76. The composition of claim 75, wherein said antibody is specific for a particular region of the troponin molecule. 77. The composition of claim 76, wherein said antibody is specific for a region of cardiac troponin I comprising amino acids 27-41. 70. The composition of claim 69, wherein said fluorescent moiety comprises a molecule comprising at least one substituted indolium ring system, wherein the substituent on the 3-position carbon of said indolium ring comprises a chemically reactive or conjugated group. 70. The composition of claim 69, wherein said fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor488, AlexaFluor532, AlexaFluor647, AlexaFluor680, or AlexaFluor700. 80. The composition of claim 79, wherein said fluorescent moiety comprises AlexaFluor647. A composition comprising a set of standards for determination of cardiac troponin concentration, wherein at least one standard has a cardiac troponin concentration of less than about 10 pg/ml. A kit comprising a composition comprising an antibody to cardiac troponin conjugated to a fluorochrome moiety, said fluorochrome moiety emitting at least about 200 photons when stimulated by a laser emitting at the excitation wavelength of said moiety. wherein the laser is focused to a spot of about 5 microns or greater in diameter containing the fluorescent moiety, and the total energy directed by the laser to the spot is about 3 microjoules or less; Packaged in a kit. 83. The kit of claim 82, wherein said cardiac troponin is cardiac troponin I or cardiac troponin T. 83. The kit of claim 82, wherein said cardiac troponin is cardiac troponin I. 83. The kit of claim 82, further comprising instructions. 83. The kit of claim 82, further comprising a composition comprising a capture antibody for said cardiac troponin I bound to a solid support. 87. The kit of claim 86, wherein said solid support comprises a microtiter plate or paramagnetic microparticles. 83. The kit of claim 82, further comprising components selected from the group consisting of wash buffers, assay buffers, elution buffers, and calibrator diluents. 83. The kit of claim 82, further comprising a standard of said cardiac troponin.

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