Antibody pair, reagent and method for detecting cTnI

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of immunodiagnosis, and in particular to an antibody pair, a reagent and a method for detecting cTnI. Background Art

[0002] Cardiac troponin I (cTnI) is one of the subunits of the troponin complex. It is only expressed in myocardial tissue and has high specificity and sensitivity. When myocardial cells are damaged, cTnI appears early in the blood and lasts for a long time, which is closely related to the degree of myocardial damage and prognosis. It can be used as a specific indicator of myocardial damage and is of great significance for the diagnosis of heart disease. It is the preferred marker of myocardial damage recommended by heart disease-related organizations at home and abroad (ESC / ACCF / AHA / WHF). Sensitive troponin cTnI is widely used to assist in the diagnosis of clinical pathological myocardial damage such as acute myocardial infarction (AMI), postoperative myocardial trauma, chemotherapy cardiotoxicity, and myocardial damage related to other diseases.

[0003] The sensitivity of traditional cTnI detection methods is relatively low, and it is difficult to detect low levels of cTnI in the blood circulation. When ischemic symptoms or electrocardiographic changes are atypical, it may lead to delayed diagnosis or even misdiagnosis, which is not conducive to early diagnosis, risk assessment and prognosis of patients. With the continuous deepening of clinical application research and continuous improvement of detection methods, a new generation of high-sensitive methods for detecting cardiac troponin I (hs-cTnI) reagents have been introduced. hs-cTnI has a lower detection limit, which helps to detect minor myocardial damage that was previously easily missed, assist in the diagnosis of acute myocardial infarction at an earlier stage, and more reasonably screen patients at high risk of cardiovascular events, thereby optimizing clinical treatment decisions and prognosis assessment.

[0004] The performance of cTnI reagents is crucial for the judgment of diagnostic results. However, the complex biochemical properties of troponin make the development of highly sensitive and specific detection reagents a daunting challenge. Studies have found that cTnI is relatively unstable and susceptible to proteolytic enzymes, and the degree of influence on different parts varies. The N-terminus and C-terminus are easily degraded. Among them, the most stable region of the cTnI molecule is approximately the amino acid region of 24-110. Therefore, in order to better detect cTnI, antibodies that recognize the middle region of the molecule have become the first choice, so most of the current immunoassay systems use specific antibodies that recognize this region. Since there is no internationally agreed reference measurement procedure for cTnI, and there are many influencing factors in cTnI detection, the detection sensitivity of reagents on different platforms varies. In terms of selecting antibodies, detection reagents from different manufacturers combine antibodies with different antigen sensitivities. However, there are few types of hs-cTnI kits on the market, mainly because the sensitivity and specificity of existing antibody combinations to antigens are not high enough. Summary of the invention

[0005] The present application provides an antibody pair, which provides an important source of raw materials for the detection of cTnI and has good detection performance.

[0006] To achieve the above object, according to one aspect of the present invention, an antibody pair for detecting cTnI is provided. The antibody pair comprises a first antibody, the first antibody comprising: HCDR1, HCDR2 and

[0007] The three heavy chain complementarity determining regions of HCDR3 and the three light chain complementarity determining regions of LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO: 11;

[0008] The antibody pair includes a second antibody, wherein the second antibody comprises:

[0009] The three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and the three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:23.

[0010] The complementarity determining regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0011] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody pair for detecting cTnI, the antibody pair comprising a first antibody, the first antibody having HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6;

[0012] The antibody pair includes a second antibody having HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17

[0013] LCDR2, LCDR3 shown in SEQ ID NO:18.

[0014] In order to achieve the above object, according to a third aspect of the present invention, an antibody pair for detecting cTnI is provided;

[0015] The antibody pair comprises a first antibody, and the first antibody comprises at least one of (1)-(2):

[0016] (1) the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11;

[0017] (2) the heavy chain shown in SEQ ID NO: 10 and the light chain shown in SEQ ID NO: 12;

[0018] The antibody pair comprises a second antibody, wherein the second antibody comprises at least one of (a)-(b):

[0019] (a) the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23;

[0020] (b) The heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:24.

[0021] In order to achieve the above object, according to a fourth aspect of the present invention, an antibody pair for detecting cTnI is provided;

[0022] The antibody pair comprises a first antibody, wherein the heavy chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 11; or;

[0023] The antibody pair comprises a second antibody, wherein the heavy chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 23;

[0024] In an alternative embodiment, the mutation is the addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody.

[0025] In an alternative embodiment, the framework region of the conservative variant formed by mutation of the first antibody has at least 80% identity with the framework region of the first antibody before mutation; or;

[0026] The framework region of the conservative variant formed by mutation of the second antibody has at least 80% identity with the framework region of the second antibody before mutation.

[0027] In order to achieve the above object, according to a fifth aspect of the present invention, an antibody pair for detecting cTnI is provided;

[0028] The antibody pair comprises a first antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO:11;

[0029] The antibody pair comprises a second antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:23.

[0030] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided an antibody pair for detecting cTnI, 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 a κ-type or λ-type light chain constant region;

[0031] In an alternative embodiment, the heavy chain constant region of the first antibody is SEQ ID NO: 7 or a sequence having at least 80% identity thereto, and the light chain constant region of the first antibody is SEQ ID NO: 8 or a sequence having at least 80% identity thereto;

[0032] In an alternative embodiment, the heavy chain constant region of the second antibody is SEQ ID NO: 19 or a sequence having at least 80% identity thereto, and the light chain constant region of the second antibody is SEQ ID NO: 20 or a sequence having at least 80% identity thereto;

[0033] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG1, hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM;

[0034] In an optional embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0035] In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a reagent for detecting cTnI, the reagent comprising a first group of antibodies and a second group of antibodies, the first group of antibodies comprising a first antibody in an antibody pair;

[0036] The second set of antibodies includes the second antibody in the antibody pair;

[0037] Optionally, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeling antibody;

[0038] The reagent comprises the first antibody and the second antibody of the above-mentioned 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;

[0039] In an optional embodiment, the coated antibody or labeled antibody is coupled to biotin or a biotin derivative;

[0040] In an optional embodiment, the labeling substance coupled to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0041] In an alternative embodiment, the coated antibody is coupled to a solid carrier;

[0042] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0043] In order to achieve the above object, according to an eighth aspect of the present invention, a method for detecting cTnI is provided, comprising:

[0044] a) contacting the above antibody pair or reagent with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and

[0045] b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of cTnI in the test sample.

[0046] In order to achieve the above object, according to the ninth aspect of the present invention, there is provided use of the above antibody pair or reagent in detecting cTnI or preparing a product for detecting cTnI. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 .Clinical relevance of the chemiluminescent reagent of the present invention and Abbott's hs-cTnI reagent

[0048] Figure 2 .Fluorescence detection calibration curve

[0049] Figure 3 .Full results of clinical relevance of the fluorescent detection reagent of the present invention and Abbott's hs-cTnI reagent

[0050] Figure 4 . Results of the 0-1 ng / ml interval of the clinical relevance of the fluorescent detection reagent of the present invention and the hs-cTnI reagent of Abbott DETAILED DESCRIPTION

[0051] In a first aspect, an embodiment of the present invention provides an antibody pair for detecting cTnI, the antibody pair comprising a first antibody, the first antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:11; the antibody pair comprises a second antibody, the second antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:21 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:23.

[0052] The complementarity determining regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0053] In the present invention, the term "antibody" is used in the broadest sense, and the antibody in the method of the present invention may be a whole antibody, or an antigen-binding fragment or a polymerized antibody capable of binding to cTnI.

[0054] The whole antibody may be monoclonal. Such a whole antibody is generally an antibody made by any suitable method known in the art. For example, by immunizing a mammal, usually a rabbit or a mouse, with cTnI under suitable conditions and isolating the antibody molecule from, for example, the serum of the mammal. Monoclonal antibodies may 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 optional embodiment, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody. The antigen-binding fragment of the above-mentioned antibody generally has the same binding specificity as the antibody from which it is derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the antigen-binding fragment of the above-mentioned antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, it is easy for a person skilled in the art to obtain the above-mentioned antigen-binding fragment. The antigen-binding fragment of the above-mentioned antibody can also be synthesized by recombinant genetics techniques also known to a person skilled in the art or by, for example, an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems. Polymeric antibodies refer to polymers formed by the polymerization of whole antibodies and antigen-binding fragments.

[0055] In a second aspect, an embodiment of the present invention provides an antibody pair for detecting cTnI, the antibody pair comprising a first antibody and a second antibody; the first antibody has HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; the second antibody has HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18.

[0056] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0057] The definition methods of CDR are well known in the art, and the CDR definition methods 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., US Pept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one 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 extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the annotations in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person 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 Table 1 also belong to the scope of protection of the present disclosure.

[0058] Table 1: CDR Definition 1

[0059] 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

[0060] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with the amino acid numbering on the heavy chain represented by "H+number" and the amino acid numbering on the light chain represented by "L+number". One of ordinary skill in the art can unambiguously map the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0061] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0062] 3If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0063] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0064] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0065] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[0066] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0067] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0068] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0069] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0070] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0071] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0072] The antibodies of the present invention also include a framework region. In the present invention, the "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to the region other than CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

[0073] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0074] In some optional embodiments of the present invention, the antibody described in the present invention can also be an antibody that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the FR region with the first antibody and the second antibody in the antibody pair.

[0075] In a third aspect, an embodiment of the present invention provides an antibody pair for detecting cTnI.

[0076] 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.

[0077] In an optional embodiment, the antibody pair comprises a first antibody having a heavy chain shown in SEQ ID NO:10 and a light chain shown in SEQ ID NO:12; the antibody pair comprises a second antibody having a heavy chain shown in SEQ ID NO:22 and a light chain shown in SEQ ID NO:24.

[0078] In a fourth aspect, an embodiment of the present invention provides an antibody pair for detecting cTnI, the antibody pair comprising a first antibody, the heavy chain variable region of the first antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:9, and the light chain variable region of the first antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:11; the antibody pair comprises a second antibody, the heavy chain variable region of the second antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:21, and the light chain variable region of the second antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:23.

[0079] The conservative variant refers to a mutant in which the amino acid at the mutation site is replaced by an amino acid with the same chemical properties as the original amino acid.

[0080] The mutation is the addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody.

[0081] In a fifth aspect, an embodiment of the present invention provides an antibody pair for detecting cTnI, the antibody pair comprising a first antibody, the first antibody binding to an epitope, and the epitope is the same as the epitope bound by an antibody comprising 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, the second antibody binding to an epitope: the epitope is the same as the epitope bound by an antibody comprising a heavy chain variable region shown in SEQ ID NO:21 and a light chain variable region shown in SEQ ID NO:23.

[0082] The epitope is also called an antigen epitope (AE). The epitope determines the ability of the antigen to specifically bind to the antibody. The antibody binds to the same epitope, that is, the amino acid fragments that the antibody binds to the target antigen are consistent. Whether an antibody recognizes the same epitope as other antibodies can be confirmed by competition between the two for the epitope. Competition between antibodies can be evaluated by competitive binding assays, and the methods include: ELISA, fluorescence energy transfer assay (FRET) or fluorescence microassay technology (FMAT). The amount of the antibody bound to the antigen is indirectly related to the binding ability of the candidate competing antibody (tested antibody) that competes for the same epitope. That is, the greater the binding amount or affinity of the tested antibody to the same epitope, the lower the binding amount of the antibody to the antigen, and the greater the binding amount of the tested antibody to the antigen. Specifically, the antibody and the antibody to be evaluated that have been appropriately labeled are added to the antigen at the same time, and the antibody bound is detected by the label. By pre-labeling the antibody, the amount of the antibody bound to the antigen can be easily determined. There is no particular limitation on the label, and the labeling method corresponding to the measurement technology is selected. Specific labeling methods include: fluorescent labeling, radioactive labeling, enzyme labeling, etc.

[0083] In an optional embodiment, the antibody further comprises a constant region, wherein the heavy chain constant region 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 a κ-type or λ-type light chain constant region. The λ-type light chain constant region can be selected from λ1, λ2, λ3, and λ4 subtypes.

[0084] 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 above-mentioned constant region (SEQ ID NO: 7, 8, 19 or 20).

[0085] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG1, hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM;

[0086] In an optional embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0087] In some optional embodiments, the species of origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0088] In a seventh aspect, an embodiment of the present invention provides a reagent for detecting cTnI.

[0089] The reagents include a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody in the above antibody pair;

[0090] The second group of antibodies includes the second antibody in the above antibody pair.

[0091] In an optional embodiment, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other group is a labeling antibody.

[0092] It should be noted that the meaning of reagent in this application and the meaning of kit can be regarded as equivalent to each other.

[0093] In an optional embodiment, the coated antibody or labeled antibody is coupled to biotin or a biotin derivative;

[0094] In an optional embodiment, the labeling substance coupled to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0095] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0096] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0097] In an optional embodiment, 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.

[0098] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0099] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0100] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0101] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0102] In an optional embodiment, the colloidal metal is colloidal gold.

[0103] In an alternative embodiment, the coated antibody is coupled to a solid carrier;

[0104] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0105] In an eighth aspect, an embodiment of the present invention provides a method for detecting cTnI, comprising:

[0106] a) contacting the above antibody pair or reagent with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and

[0107] b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of cTnI in the test sample.

[0108] In order to achieve the above object, according to the ninth aspect of the present invention, there is provided use of the above antibody pair or reagent in detecting cTnI or preparing a product for detecting cTnI.

[0109] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0111] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0112] Based on long-term creative research on cTnI, the applicant discovered two antibodies that can meet the requirements for cTnI detection.

[0113] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0114] Example 1 Antibody Preparation

[0115] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting cTnI monoclonal antibody was an existing hybridoma cell line, which was revived for future use.

[0116] (1) Antibody gene preparation

[0117] mRNA was extracted from the hybridoma cell line secreting cTnI monoclonal antibody, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0118] (2) Sequence analysis of cTnI antibody variable region gene

[0119] The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was approximately 320bp, and there was a 57bp leader peptide sequence in front of it; in the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was approximately 360bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0120] (3) Construction of recombinant antibody expression plasmid

[0121] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced 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; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody are designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the Light Chain gene fragment and the Heavy Chain gene fragment are amplified by PCR amplification method.

[0122] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0123] 2. Recombinant Antibody Production

[0124] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium, and use it as a cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. Use protein A affinity chromatography column to affinity purify the supernatant.

[0125] The obtained antibodies were named 30C-119 and 30C-12-21. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0126] Table 2 Antibody sequences

[0127] Antibody Name Heavy chain Light chain 30C-119 SEQ ID NO:10 SEQ ID NO:12 30C-12-21 SEQ ID NO:22 SEQ ID NO:24

[0128] Example 2 Preparation of detection reagents

[0129] 2.1 Chemiluminescent reagents

[0130] 1. Process of preparing reagents

[0131] A) Coating: Carboxyl magnetic microparticles were used as solid phase carriers. 100 mg of carboxyl magnetic microparticle suspension was taken. After magnetic separation, the supernatant was added to MES buffer (0.02 M MES, pH 6.0) for resuspending. EDC aqueous solution (EDC concentration was 10 mg / mL to 20 mg / mL) was added to activate the surface carboxyl groups of the carboxyl magnetic microparticles. Anti-cTnI monoclonal antibodies were then added and suspended at room temperature for 2 h to 10 h. After magnetic separation and removal of the supernatant, the suspension was resuspended in magnetic microparticle diluent (25 mM HEPES, 1% BSA, pH 7.2) to obtain carboxylated magnetic microparticles coated with cTnI monoclonal antibodies at a concentration of 10 mg / mL.

[0132] The mass ratio of EDC to carboxyl magnetic particles is 1:50-100.

[0133] The mass ratio of 30C-119 antibody to carboxyl magnetic particles is 1:50-100.

[0134] The particle size of the carboxyl magnetic particles is 0.05 μm to 3 μm.

[0135] B) Labeling: Take labeling buffer solution (0.02M PBS, pH 7.2) in a centrifuge tube, add 30C-12-21 antibody, and mix thoroughly; add acridinium ester solution, mix thoroughly, shake and react at room temperature in the dark for 1h to 2h, then remove impurities to obtain cTnI monoclonal antibody labeled with acridinium ester marker.

[0136] The impurity removal operation is to desalt with a centrifugal desalting column. First, the centrifugal desalting column is treated with purified water and PBS buffer (0.02M PBS, pH 7.2) respectively, and finally the cTnI monoclonal antibody solution labeled with an acridinium ester marker is added to collect the liquid in the centrifuge tube.

[0137] The molar ratio of acridinium ester to cTnI monoclonal antibody is 1:5 to 1:20.

[0138] C) Use magnetic particle diluent (50mM HEPES, 1% BSA, 1% NaCl, pH 7.2) to dilute the cTnI monoclonal antibody-coated carboxylated magnetic particles to 0.3mg / mL to 0.5mg / mL magnetic particle working solution, and use acridinium ester marker diluent (50mM HEPES, 1% BSA, 1% NaCl, pH 7.2) to dilute the cTnI monoclonal antibody labeled with acridinium ester marker to 0.2μg / mL to 2μg / mL acridinium ester working solution, and store at 2 to 8°C for use. The two working solutions are combined to obtain a cTnI chemiluminescent immunoassay kit.

[0139] D) Preparation of cTnI calibrators: Use calibrator diluent (20 mM HEPES, 0.5% BSA, 1% NaCl, pH 7.2) to prepare cTnI recombinant antigen at concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, 10000 pg / mL, and 50000 pg / mL, respectively, and store at 2-8°C until use.

[0140] E) The pre-stimulation solution is a H2O2 solution with a concentration of 0.1 mol / L, and the stimulation solution is a NaOH solution with a concentration of 0.25 mol / L.

[0141] 2. Testing process

[0142] The test was performed on the Yingkai shine i2910 model fully automatic chemiluminescence immunoassay analyzer using the double antibody sandwich method, that is, the instrument sequentially added 100 μL of sample, 50 μL of magnetic particle working solution, and 50 μL of acridinium ester working solution, mixed and incubated for 10 minutes, rinsed the reaction mixture after incubation, added pre-excitation solution and excitation solution, and detected the relative luminescence intensity (RLU). The cTnI calibrator was used for calibration, and the actual sample was tested after the calibration curve was obtained, and the sample concentration was calculated according to the relative luminescence intensity of the sample.

[0143] 3. Test results and summary

[0144] A) The test data of cTnI calibrator are as follows:

[0145]

[0146] B) The actual test data of the samples to be tested are as follows:

[0147]

[0148] The coefficient of variation (CV) of the actual samples tested was <10%, indicating that the detection method has good precision.

[0149] C) The sensitivity test results are as follows:

[0150]

[0151] The calculated LoB = 0.64 ng / L, indicating that the detection method has high sensitivity.

[0152] D) The specificity test results are as follows:

[0153]

[0154] The cross-reaction rates to 1000 ng / mL cardiac troponin C, cardiac troponin T, and skeletal muscle troponin I were all less than 0.1%, indicating that the detection method has high specificity.

[0155] E) The results of clinical relevance determination are as follows:

[0156]

[0157]

[0158]

[0159]

[0160] The results are summarized as Figure 1 As shown, the correlation between this test method and Abbott's hs-cTnI reagent is R 2 =0.9744, indicating that the detection method of the present invention has good clinical correlation with Abbott's hs-cTnI reagent.

[0161] 2.2 Immunofluorescence reagents

[0162] 1. Process of preparing reagents

[0163] A) Antibody labeling: Take 100ul of fluorescent microspheres with 1% solid content and add 900ul of activation buffer and mix well. After centrifugation to remove the supernatant, add 1mL of activation buffer and mix well by ultrasonic. Then add activator, shake and mix well in the dark for 20min, centrifuge to remove the supernatant, add MES buffer with an equal volume as the microspheres, pH=6.5, and mix well by ultrasonic. After adding 0.2-0.4mg of cTnI labeled antibody (30C-12-21), shake and mix well in the dark for 3h, finally add blocking buffer for blocking, shake and mix in the dark for 45min, terminate labeling, centrifuge to remove the supernatant, re-dissolve the microspheres with microsphere preservation solution, mix well by ultrasonic, and store at 4°C before use.

[0164] B) Preparation of microsphere working solution: After the cTnI marker is finally diluted to 10-20% with the microsphere diluent, the marker is sprayed onto the glass fiber using a spray pad apparatus.

[0165] C) Prepare the dried microsphere pad: put the sprayed fluorescent pad into a 50°C oven and dry it for more than 2 hours.

[0166] D) Sample pad treatment: dilute the blocking agent to 0.4 mg / ml using sample pad diluent and spread on the glass fiber, then put in a 50°C oven to dry overnight.

[0167] E) NC membrane coating: cTnI coating antibody (30C-119) was diluted to 1.0 mg / ml using coating diluent and then coated; the membrane was placed in a 50°C oven to dry overnight.

[0168] F) Preparation of fluorescent chromatography strips: Use a strip cutter to cut the fluorescent chromatography strips into strips of required width, assemble them, and add samples for detection.

[0169] 2. Testing process

[0170] Add the sample to the sample diluent (recommended sample: sample diluent ratio = 1:1), mix thoroughly and then load the sample (e.g.

[0171] 75uL of sample was loaded on a 3.5mm wide strip), the test card was allowed to react for 15min, and the test card was immediately inserted into the instrument for reading.

[0172] 3. Test results and summary

[0173] A) Standard curve:

[0174] Encapsulation 30C-119 mark 30C-12-21 Calibrators T / C value 0 0.006 0.01 0.010 0.04 0.018 0.079 0.023 0.12 0.026 0.287 0.042 0.59 0.110 1.758 0.200 4.162 0.430 10.635 1.122 21.803 2.100 50.002 4.501

[0175] The standard curve is as follows Figure 2 As shown, the overall gradient of the coated (30C-119) + labeled (30C-12-21) test recombinant antigen is better and more sensitive, which can meet the needs of clinical testing.

[0176] B) Clinical relevance:

[0177]

[0178]

[0179]

[0180]

[0181] The full results of Abbott clinical relevance are as follows Figure 3 In summary, R 2 =0.978, the results in the range of 0-1ng / ml are as follows Figure 4 Summarize,

[0182] R 2 =0.944, the overall correlation is good.

[0183] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0184] The partial amino acid sequences involved in this application are as follows:

[0185]

[0186]

Claims

1. An antibody pair for detecting cTnI, characterized in that: The antibody pair comprises a first antibody, the first antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:11; The antibody pair includes a second antibody, which comprises: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:21 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:

23.

2. The antibody pair according to claim 1, wherein the complementarity determining regions are defined by any one of the systems of Kabat, Chothia, IMGT, AbM or Contact or a combination of multiple systems.

3. An antibody pair for detecting cTnI, characterized in that: The antibody pair comprises a first antibody, wherein the first antibody has HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; The antibody pair includes a second antibody having HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:

18.

4. An antibody pair for detecting cTnI, characterized in that: The antibody pair comprises a first antibody, and 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 antibody pair comprises a second antibody, wherein 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.

5. The antibody pair for detecting cTnI according to claim 4, characterized in that: The antibody pair comprises a first antibody, wherein the heavy chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO:9, and the light chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO:11; The antibody pair comprises a second antibody, the heavy chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 23; or; Optionally, the mutation is the addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody; The framework region of the conservative variant formed by mutation of the first antibody has at least 80% identity with the framework region of the first antibody before mutation; or; The framework region of the conservative variant formed by mutation of the second antibody has at least 80% identity with the framework region of the second antibody before mutation.

6. An antibody pair for detecting cTnI, characterized in that: The antibody pair comprises a first antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO:11; The antibody pair comprises a second antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:21 and a light chain variable region set forth in SEQ ID NO:

23.

7. The antibody pair for detecting cTnI according to any one of claims 1 to 6, characterized in that: 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; the light chain constant region is selected from a κ-type or λ-type light chain constant region; Optionally, the heavy chain constant region of the first antibody is SEQ ID NO: 7 or a sequence having at least 80% identity thereto, and the light chain constant region of the first antibody is SEQ ID NO: 8 or a sequence having at least 80% identity thereto; Optionally, the heavy chain constant region of the second antibody is SEQ ID NO: 19 or a sequence having at least 80% identity thereto, and the light chain constant region of the second antibody is SEQ ID NO: 20 or a sequence having at least 80% identity thereto; Optionally, the heavy chain constant region includes CH1 of IgG1, a hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

8. A reagent for detecting cTnI, characterized in that: The reagents include a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody according to any one of claims 1 to 7; The second group of antibodies comprises the second antibody of any one of claims 1-7; Optionally, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeling antibody; Optionally, the coated antibody or labeled antibody is coupled to biotin or a biotin derivative; Optionally, the label to which the labeled antibody is coupled is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels; Optionally, the coated antibody is coupled to a solid carrier; Optionally, the solid support is selected from microspheres, plates and membranes.

9. A method for detecting cTnI, characterized in that: include: a) contacting the antibody pair according to any one of claims 1 to 7 or the reagent according to claim 8 with a sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of cTnI in the test sample.

10. Use of the antibody pair according to any one of claims 1 to 7 or the reagent according to claim 8 in detecting cTnI or preparing a product for detecting cTnI.

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