Antibody pairs, reagents and methods for detecting ctni

Abstract

The application discloses an antibody pair, a reagent and a method for detecting cTnI, and relates to the field of immunodiagnosis. The antibody pair for detecting cTnI disclosed by the application comprises a first antibody and a second antibody, and the reagent and the detection method based on the antibody combination can accurately detect the presence of cTnI.

Description

Technical Field

[0001] This invention relates to the field of immunodiagnostic technology, and more specifically, to an antibody pair, reagent, and method for detecting cTnI. Background Technology

[0002] Cardiac troponin I (cTnI) typically forms a complex with troponin C, troponin T, or both simultaneously, regulating muscle contraction. Compared to other available markers of myocardial necrosis, such as creatine kinase isoenzyme MB (CK-MB), myoglobin (MYO), and lactate dehydrogenase, cTnI is a myocardial-specific and highly sensitive marker of cardiac injury due to its high tissue specificity. Clinical trials have demonstrated that cTnI levels rise within hours of myocardial infarction or the onset of myocardial symptoms, peaking approximately 8-28 hours later, and remaining at a high level for 3-10 days after the infarction. In August 2012, a joint working group of the European Society of Cardiology (ESC), the American College of Cardiology Foundation (ACCF), the American Heart Association (AHA), and the World Federation of Heart Associations (WHF) published the third edition of the globally harmonized definition of myocardial infarction, in which cardiac troponin (cTn) plays a central role and is recommended as the preferred cardiac marker.

[0003] Traditional cTnI detection methods have relatively low sensitivity, making it difficult to detect low levels of cTnI in the bloodstream. In cases of ischemic symptoms or atypical electrocardiographic changes, this can lead to delayed diagnosis or even misdiagnosis, hindering early diagnosis, risk assessment, and prognosis. With ongoing clinical research and improvements in detection methods, a new generation of highly sensitive reagents for detecting cardiac troponin I (hs-cTnI) has emerged. hs-cTnI has a lower detection limit, which helps detect previously missed minute myocardial injuries, facilitates earlier diagnosis of acute myocardial infarction, enables more appropriate screening of high-risk patients for cardiovascular events, and optimizes clinical treatment decisions and prognostic assessment. Summary of the Invention

[0004] This application provides an antibody pair that offers an important source of raw materials for the detection of cTnI and exhibits good detection performance.

[0005] To achieve the above objectives, according to one aspect of the present invention, an antibody pair for detecting cTnI is provided. The antibody pair includes a first antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:9, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:11;

[0006] The antibody pair includes a second antibody, which comprises: three heavy chain complementarity-determining regions, HCDR1, HCDR2 and HCDR3, in the heavy chain variable region shown in SEQ ID NO:21, and three light chain complementarity-determining regions, LCDR1, LCDR2 and LCDR3, in the light chain variable region shown in SEQ ID NO:23.

[0007] The complementary decision region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0008] To achieve the above objective, according to a second aspect of the present invention, an antibody pair for detecting cTnI is provided, the antibody pair comprising a first antibody having HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6.

[0009] The antibody pair includes a second antibody having HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:15, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18.

[0010] To achieve the above objectives, according to a third aspect of the present invention, an antibody pair for detecting cTnI is provided;

[0011] The antibody pair comprises a first antibody, which comprises at least one of (1)-(2):

[0012] (1) The heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11;

[0013] (2) The heavy chain shown in SEQ ID NO:10 and the light chain shown in SEQ ID NO:12;

[0014] The antibody pair comprises a second antibody, the second antibody comprising at least one of (a)-(b):

[0015] (a) The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23;

[0016] (b) The heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:24.

[0017] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody pair for detecting cTnI is provided;

[0018] The antibody pair comprises a first antibody, wherein the variable region of the heavy chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:11; or;

[0019] The antibody pair comprises a second antibody, wherein the variable region of the heavy chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:21, and the variable region of the light chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:23.

[0020] In one alternative implementation, the mutation is the addition, deletion, substitution, or modification of one or more amino acids in the framework region of the first or second antibody.

[0021] In one alternative implementation, the framework region of the conserved variant formed by the mutation of the first antibody has at least 80% identity with the framework region of the first antibody before the mutation; or;

[0022] The framework region of the conserved variant formed by the mutation of the second antibody has at least 80% identity with the framework region of the second antibody before the mutation.

[0023] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody pair for detecting cTnI is provided;

[0024] The antibody pair comprises a first antibody that binds to an epitope, the epitope being the same as the epitope bound by the antibody comprising the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11.

[0025] The antibody pair comprises a second antibody that binds to an epitope: the epitope is the same as the epitope bound to the antibody comprising the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23.

[0026] To achieve the above objectives, according to a sixth aspect of the present invention, an antibody pair for detecting cTnI is provided, wherein the heavy chain constant region of the antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and the light chain constant region is selected from κ-type or λ-type light chain constant regions.

[0027] In an optional embodiment, the heavy chain constant region of the first antibody is SEQ ID NO:7 or a sequence having at least 80% identity with it, and the light chain constant region of the first antibody is SEQ ID NO:8 or a sequence having at least 80% identity with it.

[0028] In an optional embodiment, the heavy chain constant region of the second antibody is SEQ ID NO:19 or a sequence having at least 80% identity with it, and the light chain constant region of the second antibody is SEQ ID NO:20 or a sequence having at least 80% identity with it.

[0029] In an optional implementation, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0030] In one alternative embodiment, the antibody is a polymer formed by the polymerization of antibody monomers.

[0031] To achieve the above objectives, according to a seventh aspect of the present invention, a reagent for detecting cTnI is provided, the reagent comprising a first group of antibodies and a second group of antibodies, wherein the first group of antibodies comprises a first antibody in an antibody pair;

[0032] The second group of antibodies includes the second antibody in the antibody pair;

[0033] Optionally, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeled antibody;

[0034] The reagent includes the first antibody and the second antibody in the above antibody pair; the first antibody is a coating antibody and the second antibody is a labeled antibody, or the second antibody is a coating antibody and the first antibody is a labeled antibody;

[0035] In an optional embodiment, the coated antibody or labeled antibody is conjugated with biotin or a biotin derivative;

[0036] In one optional embodiment, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0037] In one optional embodiment, the coated antibody is coupled to a solid-phase support;

[0038] In one alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0039] To achieve the above objectives, according to an eighth aspect of the present invention, a method for detecting cTnI is provided, comprising:

[0040] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody pair or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0041] b) Detect the presence of the immune complex, the presence of which indicates the presence of cTnI in the test sample.

[0042] To achieve the above objectives, according to a ninth aspect of the present invention, the use of the above-described antibody pairs or reagents in the detection of cTnI or in the preparation of products for the detection of cTnI is provided. Detailed Implementation

[0043] In a first aspect, embodiments of the present invention provide an antibody pair for detecting cTnI, the antibody pair comprising a first antibody, the first antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:11; the antibody pair comprising a second antibody, the second antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:21 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:23.

[0044] The complementary decision region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0045] In this invention, the term "antibody" is used in the broadest sense, and the antibody in the method of this invention can be a whole antibody, an antigen-binding fragment, or a polymeric antibody capable of binding to cTnI.

[0046] Whole antibodies can be monoclonal. Such whole antibodies are typically prepared by any suitable method known in the art. For example, antibody molecules are isolated from the serum of a mammal, typically a rabbit or mouse, by immunizing it with cTnI under suitable conditions, for example. Monoclonal antibodies can be obtained by hybridoma or recombinant methods. The antigen-binding fragment includes an antigen-binding site, such as a Fab or F(ab)2 fragment. In an alternative embodiment, the antigen-binding fragment is selected from any one of the F(ab')2, Fab', Fab, Fv, and scFv of the antibody. The antigen-binding fragment of the above-described antibody typically has the same binding specificity as the antibody from which it originates. As will be readily understood by those skilled in the art according to the description of the present invention, the antigen-binding fragment of the above-described antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the structure of the complete antibody disclosed in this invention, those skilled in the art can readily obtain the above-described antigen-binding fragment. The antigen-binding fragment of the above-described antibody can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, an automated peptide synthesizer, such as those sold by AppliedBioSystems. Polymerized antibodies are polymers formed by the polymerization of whole antibodies and antigen-binding fragments.

[0047] In a second aspect, embodiments of the present invention provide an antibody pair for detecting cTnI, the antibody pair comprising a first antibody and a second antibody; the first antibody having HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; the second antibody having HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:15, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18.

[0048] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0049] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, with slight variations in labeling across different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0050] Table 1: CDR Definition 1

[0051] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0052] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0053] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0054] 3If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0055] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0056] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0057] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0058] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0059] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0060] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0061] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0062] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0063] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0064] The antibody of the present invention also includes a frame region. In the present invention, the "frame region" or "FR" region includes a heavy chain frame region and a light chain frame region, which refers to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR. The heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including LFR1, LFR2, LFR3 and LFR4 frame regions.

[0065] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0066] In some optional embodiments of the present invention, the antibody described in the present invention may also be an antibody having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the first antibody and the second antibody in the antibody pair in the FR region.

[0067] Thirdly, embodiments of the present invention provide an antibody pair for detecting cTnI.

[0068] The antibody pair comprises a first antibody having a heavy chain variable region shown in SEQ ID NO:9 and a light chain variable region shown in SEQ ID NO:11; the antibody pair comprises a second antibody having a heavy chain variable region shown in SEQ ID NO:21 and a light chain variable region shown in SEQ ID NO:23.

[0069] In an optional embodiment, the antibody pair comprises a first antibody having a heavy chain as shown in SEQ ID NO:10 and a light chain as shown in SEQ ID NO:12; and the antibody pair comprises a second antibody having a heavy chain as shown in SEQ ID NO:22 and a light chain as shown in SEQ ID NO:24.

[0070] Fourthly, embodiments of the present invention provide an antibody pair for detecting cTnI, the antibody pair comprising a first antibody, wherein the variable region of the heavy chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:11; the antibody pair comprising a second antibody, wherein the variable region of the heavy chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:21, and the variable region of the light chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:23.

[0071] The conserved variant refers to a mutant obtained by replacing the amino acid at the mutation site with an amino acid with the same chemical properties as the original amino acid.

[0072] The mutation is the addition, deletion, substitution, or modification of one or more amino acids in the framework region of the first or second antibody.

[0073] Fifthly, embodiments of the present invention provide an antibody pair for detecting cTnI, the antibody pair comprising a first antibody that binds to an epitope, the epitope being the same as the epitope bound by an antibody containing the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11; the antibody pair comprising a second antibody that binds to an epitope, the epitope being the same as the epitope bound by an antibody containing the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23.

[0074] The epitope, also known as an antigen epitope (AE), determines the ability of an antigen to specifically bind to an antibody. Antibodies bind to the same epitope because the amino acid fragments they bind to the target antigen are identical. Whether an antibody recognizes the same epitope as other antibodies can be confirmed by their competition for the epitope. Competition between antibodies can be evaluated using competitive binding assays, such as ELISA, fluorescence energy transfer assay (FRET), or fluorescence microassay (FMAT). The amount of antibody binding to the antigen is indirectly related to the binding ability of candidate competing antibodies (the antibody being tested) that compete for the same epitope. That is, the greater the binding amount or affinity of the antibody being tested for the same epitope, the lower the amount of antibody binding to the antigen, and the higher the amount of antibody binding to the antigen. Specifically, an appropriately labeled antibody and the antibody to be evaluated are simultaneously added to the antigen, and the binding of the antibody is detected using the labeling. By pre-labeling the antibody, the amount of antibody binding to the antigen can be easily determined. There are no particular limitations on the labeling; a labeling method appropriate to the assay technique is selected. Labeling methods include: fluorescent labeling, radioactive labeling, enzyme labeling, etc.

[0075] In an optional embodiment, the antibody further includes a constant region, wherein the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; the light chain constant region is selected from the κ-type or λ-type light chain constant region. The λ-type light chain constant region may be selected from the λ1, λ2, λ3, and λ4 subtypes.

[0076] It should be noted that, in some embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant regions (SEQ ID NO: 7, 8, 19, or 20).

[0077] In an optional implementation, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0078] In one alternative embodiment, the antibody is a polymer formed by the polymerization of antibody monomers.

[0079] In some alternative implementations, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0080] Seventhly, embodiments of the present invention provide a reagent for detecting cTnI.

[0081] The reagent includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody in the antibody pair described above.

[0082] The second group of antibodies includes the second antibody in the antibody pair described above.

[0083] In one optional implementation, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeled antibody.

[0084] It should be noted that the meaning of "reagent" in this application can be considered equivalent to the meaning of "kit".

[0085] In an optional embodiment, the coated antibody or labeled antibody is conjugated with biotin or a biotin derivative;

[0086] In one optional embodiment, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0087] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar dyes, Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0088] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0089] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0090] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0091] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0092] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0093] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0094] In an optional embodiment, the colloidal metal is colloidal gold.

[0095] In one optional embodiment, the coated antibody is coupled to a solid-phase support;

[0096] In one alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0097] Eighthly, embodiments of the present invention provide a method for detecting cTnI, comprising:

[0098] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody pair or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0099] b) Detect the presence of the immune complex, the presence of which indicates the presence of cTnI in the test sample.

[0100] To achieve the above objectives, according to a ninth aspect of the present invention, the use of the above-described antibody pairs or reagents in the detection of cTnI or in the preparation of products for the detection of cTnI is provided.

[0101] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0103] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. "Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JEColigan et al., eds., 1991), each of which is explicitly incorporated herein by reference.

[0104] Based on long-term, creative research on cTnI, the applicant discovered two antibodies that can meet the requirements for cTnI detection.

[0105] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0106] Example 1 Antibody Preparation

[0107] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. The BD SMART™ RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting cTnI monoclonal antibody was an existing hybridoma cell line, which was revived and ready for use.

[0108] (1) Antibody gene preparation

[0109] mRNA was extracted from hybridoma cell lines secreting cTnI monoclonal antibodies, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After bacterial growth, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0110] (2) Sequence analysis of the cTnI antibody variable region gene

[0111] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was approximately 320 bp, with a 57 bp leader peptide sequence preceding it. Among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was approximately 360 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0112] (3) Construction of recombinant antibody expression plasmid

[0113] The pcDNA™ 3.4 TOPO® vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The LightChain gene fragment and Heavy Chain gene fragment were amplified by PCR.

[0114] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0115] 2. Recombinant antibody production

[0116] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3-5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which was then used as a cell dilution buffer. Diluents for plasmid DNA and transfection reagent were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. Cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column.

[0117] The resulting antibodies were named 35C-17 and 31C-16. The heavy chain (H) and light chain (L) sequences of the above antibodies are shown in the table below:

[0118] Table 2 Antibody Sequences

[0119] Antibody name Heavy chain Light chain 35C-17 SEQ ID NO:10 SEQ ID NO:12 31C-16 SEQ ID NO:22 SEQ ID NO:24

[0120] Example 2 Preparation of detection reagents

[0121] The kit includes: working solution of carboxymagnetic microparticles coated with monoclonal antibody, working solution of acridine ester-labeled monoclonal antibody marker, recombinant cTnI calibrator, pre-activation solution and activation solution.

[0122] 1. The process of preparing reagents

[0123] A) Coating: Using carboxyl magnetic microparticles as a solid-phase carrier, 100 mg of carboxyl magnetic microparticle suspension was taken, and after magnetic separation, the supernatant was added to MES buffer (0.02 M MES, pH 6.0) for resuspending. EDC aqueous solution (EDC concentration of 10 mg / mL~20 mg / mL) was added to activate the surface carboxyl groups of the carboxyl magnetic microparticles. Then, anti-cTnI monoclonal antibody (31C-16) was added, and the mixture was suspended at room temperature for 0.5 h~10 h. After magnetic separation to remove the supernatant, the mixture was resuspended in magnetic microparticle dilution buffer (25 mM HEPES, 1% BSA, pH 7.2) to obtain carboxylated magnetic microparticles coated with cTnI monoclonal antibody (31C-16) at a concentration of 10 mg / mL.

[0124] The mass ratio of EDC to carboxyl magnetic microparticles is 1:50~100.

[0125] The mass ratio of anti-cTnI monoclonal antibody (31C-16) to carboxyl magnetic microparticles is 1:50~100.

[0126] The particle size of the carboxyl magnetic microparticles ranges from 0.05 μm to 3 μm.

[0127] B) Labeling: Take the labeling buffer solution (0.02M PBS, pH 7.2) into a centrifuge tube, add the anti-cTnI monoclonal antibody (35C-17), and mix thoroughly; add the acridinium ester solution, mix thoroughly, and react with shaking at room temperature in the dark for 0.5h~2h to remove impurities, and obtain the acridinium ester-labeled cTnI monoclonal antibody (35C-17).

[0128] The purification process involved centrifugal desalting. The centrifugal desalting column was first treated with purified water and PBS buffer (0.02M PBS, pH 7.2), and finally, a cTnI monoclonal antibody solution labeled with acridinium ester was added. The liquid in the centrifuge tube was then collected.

[0129] The molar ratio of acridine ester to cTnI monoclonal antibody is 1:5 to 1:20.

[0130] C) Dilute the carboxylated magnetic microparticles coated with cTnI monoclonal antibody to a working solution of 0.1 mg / mL to 0.5 mg / mL using magnetic microparticle dilution buffer (50 mM HEPES, 1% BSA, 1% NaCl, pH 7.2). Dilute the acridine ester-labeled cTnI monoclonal antibody to a working solution of 0.1 μg / mL to 2 μg / mL using acridine ester labeling dilution buffer (50 mM HEPES, 1% BSA, 1% NaCl, pH 7.2). Store at 2–8°C for later use. Combining the two working solutions yields the cTnI chemiluminescent immunoassay kit.

[0131] D) Preparation of cTnI calibrators: The cTnI recombinant antigen was prepared to concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, 10000 pg / mL, and 50000 pg / mL using calibrator dilution buffer (20 mM HEPES, 0.5% BSA, 1% NaCl, pH 7.2), and stored at 2–8 °C for later use.

[0132] E) The pre-activation solution is a 0.1 mol / L H2O2 solution, and the activation solution is a 0.25 mol / L NaOH solution.

[0133] Example 3: Evaluation of Reagent Detection Performance

[0134] The assay was performed on a Shine i2910 fully automated chemiluminescence immunoassay analyzer using a double-antibody sandwich method. Specifically, 100 μL of sample, 50 μL of magnetic microparticle working solution, and 50 μL of acridinium ester working solution were added sequentially to the instrument. After mixing and incubation for 10 minutes, the reaction mixture was rinsed, and pre-excitation and excitation solutions were added. The relative luminescence intensity (RLU) was then measured. Calibration was performed using cTnI calibrators, and after obtaining the calibration curve, actual samples were tested. The sample concentration was calculated based on the relative luminescence intensity of the samples.

[0135] 3. Test Results and Summary

[0136] A) The test data for the cTnI calibrator are as follows:

[0137]

[0138] B) The actual test data for the sample is as follows:

[0139] Actual cTnI concentration in the sample to be tested (ng / L) Sample 1 Sample 2 Sample 3 Sample 4 Test 1 20 30 101 502 Test 2 20 28 107 506 Test 3 23 32 106 513 Test 4 19 29 104 500 Test 5 18 28 104 520 Test 6 23 29 103 506 Test 7 23 30 110 508 Test 8 19 27 105 499 Test 9 21 31 101 488 Test 10 22 29 99 515 average value 21 29 104 506 SD 1.9 1.5 3.2 9.2 CV (%) 8.9% 5.2% 3.1% 1.8%

[0140] The coefficient of variation (CV) of the actual test samples was less than 10%, indicating that the detection method has good precision.

[0141] C) The results of the blank limit determination are as follows:

[0142]

[0143] The calculated LoB value of 0.52 ng / L indicates that the detection method has high sensitivity.

[0144] D) The specificity test results are as follows:

[0145] Cross-reactants Blank Sample 1000 ng / mL cardiac troponin C (cTnC) 1000 ng / mL cardiac troponin T (cTnT) 1000 ng / mL skeletal muscle troponin I (sTnI) Test 1 0 5 312 412 Test 2 0 6 320 410 Test 3 0 6 317 415 Average value (ng / L) 0 6 316 412 Cross-reactivity / 0.00% 0.03% 0.04%

[0146] The cross-reactivity rates for cardiac troponin C, cardiac troponin T, and skeletal muscle troponin I at 1000 ng / mL were all less than 0.1%, indicating that the detection method has high specificity.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody pair for detecting cTnI, characterized in that, The antibody pair consists of a first antibody against cTnI and a second antibody against cTnI. The first antibody contains three heavy chain complementarity-determining regions, identical to HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:9, and three light chain complementarity-determining regions, identical to LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:

11. The second antibody comprises: three heavy chain complementarity-determining regions identical to HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:21, and three light chain complementarity-determining regions identical to LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:

23. The complementary decision region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody pair for detecting cTnI, characterized in that, The antibody pair consists of a first antibody against cTnI and a second antibody against cTnI. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antibody are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively.

3. An antibody pair for detecting cTnI, characterized in that, The antibody pair consists of a first antibody against cTnI and a second antibody against cTnI, wherein the first antibody comprises at least one of (1)-(2): (1) The heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11; (2) The heavy chain shown in SEQ ID NO:10 and the light chain shown in SEQ ID NO:12; The second antibody comprises at least one of (a)-(b): (a) The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23; (b) The heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:

24.

4. The antibody pair for detecting cTnI according to any one of claims 1-3, characterized in that, The heavy chain constant regions of the first and second antibodies are selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; the light chain constant regions are selected from κ-type or λ-type light chain constant regions.

5. The antibody pair for detecting cTnI according to claim 4, characterized in that, The heavy chain constant region of the first antibody is SEQ ID NO:7 or a sequence having at least 80% identity with it, and the light chain constant region of the first antibody is SEQ ID NO:8 or a sequence having at least 80% identity with it. The heavy chain constant region of the second antibody is SEQ ID NO:19 or a sequence having at least 80% identity with it, and the light chain constant region of the second antibody is SEQ ID NO:20 or a sequence having at least 80% identity with it.

6. The antibody pair for detecting cTnI according to claim 4, characterized in that, The heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

7. A reagent for detecting cTnI, characterized in that, The reagent includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies is the first antibody as described in any one of claims 1-6; and the second group of antibodies is the second antibody as described in any one of claims 1-6.

8. The reagent according to claim 7, characterized in that, One of the first group of antibodies and the second group of antibodies is a coating antibody, and the other group is a labeled antibody.

9. The reagent according to claim 8, characterized in that, The coated antibody or labeled antibody is conjugated with biotin.

10. The reagent according to claim 8, characterized in that, The labeled antibody is coupled with a fluorescent dye, enzyme, radioisotope, chemiluminescent label, electrochemiluminescent label, or nanoparticle label.

11. The reagent according to claim 10, characterized in that, The nanoparticle-based markers are selected from colloidal gold, fluorescent microspheres, and latex.

12. The reagent according to claim 8, characterized in that, The coated antibody is coupled to a solid-phase carrier.

13. The reagent according to claim 12, characterized in that, The solid support is selected from microspheres, plates, and membranes.

14. Use of the antibody pair according to any one of claims 1-6 or the reagent according to any one of claims 7-12 in the preparation of a product for detecting cTnI.

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