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Highly sensitive system and methods for analysis of troponin

a troponin and sensitive technology, applied in the field of high-sensitivity system and methods for analysis of troponin, can solve problems such as cardiac toxicity

Inactive Publication Date: 2010-10-07
RGT UNIV OF CALIFORNIA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]In some embodiments the invention includes a composition for the detection of a troponin isoform including a binding partner to the troponin isoform attached to a fluorescent moiety, where the fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, where the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and where the total energy directed at the spot by the laser is no more than about 3 microJoules. In some embodiments of the compositions of the invention, the binding partner comprises an antibody to the troponin isoform. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the troponin isoform is a cardiac isoform. In some embodiments, the cardiac isoform is selected from the group consisting of cTnI and cTnT. In some embodiments, the cardiac isoform is cTnI. In some embodiments, the antibody is specific to a specific region 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 that comprises at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance group. In some embodiments, the fluorescent moiety includes a dye that can be AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. In some embodiments, the fluorescent moiety comprises AlexaFluor 647.
[0009]In some embodiments the invention involves a composition comprising a set of standards for the determination of a concentration of a cardiac troponin, where at least one of the standards is at a concentration of cardiac troponin less than about 10 pg / ml.
[0010]In some embodiments the invention involves a kit containing a composition including an antibody to cardiac troponin attached to a fluorescent dye moiety, where the moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, where the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and where the total energy directed at the spot by the laser is no more than about 3 microJoules, where 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 kits of the invention, the kits further include instructions. In some embodiments of the kits of the invention, the kits further include a composition containing a capture antibody for the cardiac troponin I attached to a solid support. In some embodiments, the solid support comprises a microtiter plate or paramagnetic microparticles. In some embodiments of the kits of the invention, the kits further include a component selected from the group consisting of wash buffer, assay buffer, elution buffer, and calibrator diluent. In some embodiments of the kits of the invention, further include a standard for the cardiac troponin.

Problems solved by technology

In some embodiments, the condition is cardiac toxicity.

Method used

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  • Highly sensitive system and methods for analysis of troponin
  • Highly sensitive system and methods for analysis of troponin
  • Highly sensitive system and methods for analysis of troponin

Examples

Experimental program
Comparison scheme
Effect test

example 1

Sandwich Assays for Biomarkers: Cardiac Troponin I (cTnI)

[0252]The 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 a mouse monoclonal capture antibody and a goat polyconal detection antibody. Ten microliters of sample were required. The working range of the assay is 0-900 pg / ml with a typical analytical limit of detection of 1-3 pg / ml. The assay required about four hours of bench time to complete.

[0253]Materials: the following materials were used in the procedure described below: Assay plate: 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, overnight at room temperature); blocked with 5% sucrose, 1% BSA in PBS, and stored at 4° C. For the standard curve, Human cardiac Troponin I (BiosPacific Cat # J34000352) was used. The diluent for the standard ...

example 2

Sandwich Bead-based Assays for TnI

[0267]The assays described above use the same microliter plate format where the plastic surface is used to immobilize target molecules. The single particle analyzer system also is compatible with assays done in solution using microparticles or beads to achieve separation of bound from unbound entities.

[0268]Materials: MyOne Streptavidin C1 microparticles (MPs) are obtained from Dynal (650.01-03, 10 mg / ml stock). Buffers use in the assay include: 10× borate buffer saline Triton Buffer (BBST) (1.0 M borate, 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); and elution buffer (BBS with 4 M urea, 0.02% Triton X-100, and 0.001% BSA, stored at 2-8C). Antibodies used in the sandwich bead-based assay include: Bio-Ab (A34650228P (BiosPacific) with 1-2 ...

example 3

Concentration Range for cTnI in a Population of Normal Non-Diseased Subjects

[0276]A reference range or normal range for cTnI concentrations in human serum was established using serum samples from 88 apparently healthy subjects (non-diseased). A sandwich immunoassay as described in Example 1 was performed and the number of signals or events as described above were counted using the single particle analyzer system of the invention. The concentration of serum troponin I was determined by correlating the signals detected by the analyzer with the standard curve as described above. All assays were perfumed in quadruplicate.

[0277]In accordance with recommendations by the current European and American Cardiology Societies (ESC / ACC) troponin assays should quantify accurately the 99th percentile of the normal range with an assay imprecision (CV) of less than 10% in order to distinguish reliably between patients with ACS and patients without ischemic heart disease, and risk stratification for ...

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Abstract

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

Description

CROSS-REFERENCE[0001]This application also claims priority under 35 USC §119 to U.S. Provisional Application No. 60 / 789,304, filed Apr. 4, 2006, U.S. Provisional Application No. 60 / 793,664, filed Apr. 19, 2006, U.S. Provisional Application No. 60 / 808,662, filed May 26, 2006, U.S. Provisional Application No. 60 / 861,498, filed Nov. 28, 2006, and U.S. Provisional Application No. 60 / 872,986, filed Dec. 4, 2006, all of which are incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION[0002]Each year in the United States, some six million people present to emergency departments with chest pain. Although only 15% to 20% of these patients are ultimately diagnosed with an acute coronary syndrome (ACS), about half are admitted for evaluation. Conversely, 2% of patients with ACS are mistakenly discharged. As patients with ACS have a relatively high risk of major adverse cardiovascular events in the short term, there is a clear need for accurate objective tools by which to...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/543G01N33/68G01N33/53C12Q1/02C12Q1/48C12Q1/32C12Q1/52C07K16/00G01N31/00B01L3/00
CPCG01N21/6428G01N33/582Y10T436/105831G01N2800/32G01N33/6887G01N2800/324G01N2201/1247G01N2201/12761G01N15/1459G01N15/06G01N2015/1006G01N2015/1486G01N2800/52G01N15/075G01N33/577
Inventor GOIX, PHILIPPE J.PUSKAS, ROBERTTODD, JOHNLIVINGSTON, RICHARD A.HELD, DOUGLAS
Owner RGT UNIV OF CALIFORNIA
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