WO2025035371A1 - 检测大尺寸cTnITC的组合物、试剂盒和方法、及其应用 - Google Patents

检测大尺寸cTnITC的组合物、试剂盒和方法、及其应用 Download PDF

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WO2025035371A1
WO2025035371A1 PCT/CN2023/112994 CN2023112994W WO2025035371A1 WO 2025035371 A1 WO2025035371 A1 WO 2025035371A1 CN 2023112994 W CN2023112994 W CN 2023112994W WO 2025035371 A1 WO2025035371 A1 WO 2025035371A1
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antibodies
antibody
amino acids
group
independently selected
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French (fr)
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张轶
卡特鲁卡伊万
里巴科娃纳塔利娅
刘雨青
刘君君
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Shenzhen Mindray Bio Medical Electronics Co Ltd
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Shenzhen Mindray Bio Medical Electronics Co Ltd
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Priority to PCT/CN2023/112994 priority Critical patent/WO2025035371A1/zh
Priority to CN202380101552.9A priority patent/CN121773334A/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present application relates to the field of immunoassay, and specifically to compositions and kits for detecting large-size cTnITC (large-size cardiac troponin ternary complex), detection methods, and their applications in in vitro diagnosis of myocardial injury.
  • cTnITC large-size cardiac troponin ternary complex
  • Cardiac troponin is a specific marker of myocardial injury and can be used to detect myocardial injury after or during myocardial infarction.
  • Troponin contains three subunits, namely cardiac troponin I (cTnI), cardiac troponin T (cTnT), and troponin C (TnC).
  • the concentrations of cTnI and cTnT in serum are highly correlated with the severity of myocardial injury and are highly specific and sensitive markers of myocardial infarction recommended by the American and European Cardiology Societies.
  • cardiac troponin in the blood, including pulmonary embolism, acute or chronic heart failure, cardiac trauma, endocarditis, and myocarditis, in addition to myocardial infarction. This reduces the clinical specificity of cTn for myocardial infarction, and it is usually necessary to cooperate with other means for the diagnosis of myocardial infarction.
  • cTnITC Cardiac troponin is usually bound to actin filaments in the form of a ternary complex cTnITC.
  • cTnITC ternary complex
  • LMW-cTnITC low molecular weight ternary complex
  • cTnIC binary complex
  • free cTnT free cTnT.
  • the traditional immunological methods for detecting cardiac troponin are mainly enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography (GICA), electrochemiluminescence (ECL), chemiluminescence immunoassay (CLIA) and other technologies.
  • ELISA enzyme-linked immunosorbent assay
  • GICA colloidal gold immunochromatography
  • ECL electrochemiluminescence
  • CLI chemiluminescence immunoassay
  • chemiluminescence immunoassay technology has high sensitivity, strong specificity, wide linear range, strong operability and high degree of automation, and is the immunoassay method with the broadest clinical application prospects.
  • the existing high-sensitivity cardiac troponin detection methods are usually used to detect cTnI and cTnT, and cannot distinguish between troponin complexes and free troponin.
  • the double antibody sandwich immunoassay method of cTnITC complex is achieved by capturing antibodies to recognize binary TnIC complex epitopes and detecting antibodies to recognize TnT antigens.
  • This detection method has a low signal-to-noise ratio and low specificity, making it difficult to achieve rapid, sensitive and specific detection of cTnITC complexes.
  • the task of the present application is to provide a technical solution that can detect specific forms of cTnITC in samples with high sensitivity and speed, and to achieve specific detection through the combination of capture antibodies and detection antibodies.
  • adding antibodies that specifically bind to TnC to capture antibodies or detection antibodies is very beneficial for improving the signal-to-noise ratio of the detection system and enhancing the detection sensitivity.
  • the present application provides a composition for detecting large-sized cTnITC in a sample, comprising a first group of antibodies and a second group of antibodies, wherein:
  • the first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT;
  • the second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.
  • the present application provides a kit for detecting large-sized cTnITC in a sample, comprising a capture antibody and a detection antibody, wherein the capture antibody is selected from one of a first group of antibodies and a second group of antibodies, and the detection antibody is selected from the other of the first group of antibodies and the second group of antibodies; wherein,
  • the first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT;
  • the second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.
  • the present application provides a method for detecting large-sized cTnITC in a sample in vitro, comprising the following steps:
  • the capture antibody is selected from one group of the first group of antibodies and the second group of antibodies
  • the detection antibody is selected from the other group of the first group of antibodies and the second group of antibodies:
  • the first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT;
  • the second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.
  • the present application provides use of the composition described in any one of the first aspects in preparing a reagent for detecting large-size cTnITC.
  • the present application provides use of the composition of any one of the first aspect, the kit of any one of the second aspect, or the method of any one of the third aspect in in vitro diagnosis of myocardial injury.
  • the present application provides a method for in vitro diagnosing myocardial injury in the first aspect, comprising the step of detecting large-sized cTnITC in a sample from a subject using the composition described in any one of the first aspect, the kit described in any one of the second aspect, or the method described in any one of the third aspect.
  • FIG1 is a signal-to-noise ratio analysis of a large-size cTnITC detection kit.
  • FIG2 is a diagram showing the validation of the specificity of the large-size cTnITC detection kit using serum samples.
  • FIG. 3 shows the linearity analysis of large-size cTnITC detection kit 3.
  • FIG. 4 shows the linearity analysis of large-size cTnITC detection kit 4.
  • FIG5 is a signal-to-noise ratio analysis of large-size cTnITC detection kit 3 and detection kit 3a.
  • FIG6 is an analysis of the signal-to-noise ratio of large-size cTnITC detection kit 3 and detection kit 3a for clinical serum samples.
  • FIG. 7 is a diagram showing the validation of the specificity of the large-size cTnITC detection kit 3a using serum samples.
  • FIG8 is an analysis of the changing trends of large-size cTnITC detected by large-size cTnITC detection kit 3 and high-sensitivity cTnI and cTnT detected by existing kits.
  • FIG9 is an analysis of the concentration change rates of large-size cTnITC detected by large-size cTnITC detection kit 3 and high-sensitivity cTnI and cTnT detected by existing kits.
  • the terms "comprises,” “comprising,” or any other variation thereof are intended to encompass non-exclusive inclusion, such that a method or apparatus comprising a series of elements includes not only the elements explicitly stated, but also other elements not explicitly listed, or elements inherent to the implementation of the method or apparatus. In the absence of further limitations, an element defined by the phrase “comprising a " does not exclude the presence of other related elements in the method or apparatus comprising the element.
  • the term "at least one” means 1 or more than 1 under reasonable conditions, such as 2, 3, 4, 5 or 10, etc.
  • first and second are merely used to distinguish similar objects and do not represent a specific order for the objects. It is understood that “first” and “second” can be interchanged for a specific order or precedence where permitted. It should be understood that the objects distinguished by “first” and “second” can be interchanged where appropriate so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein.
  • the term "specific binding” refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as a reaction between an antibody and an antigen to which it is directed.
  • the binding affinity between two molecules can be described by a KD value.
  • the KD value refers to the dissociation constant obtained by the ratio of kd (dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (association rate of a specific binding molecule-target molecule interaction; also known as kon), or refers to kd/ka expressed as a molar concentration (M).
  • M molar concentration
  • an antibody that specifically binds to an antigen refers to an antibody that binds to the antigen with a KD of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less.
  • KD KD of less than about 10-5 M
  • suitable studies are, for example, binding studies, blocking and competition studies using structurally and/or functionally closely related molecules.
  • FACS fluorescence activated cell sorting
  • FACS titration flow cytometry titration
  • SPR surface plasmon resonance technology
  • ITC isothermal titration calorimetry
  • fluorescence titration or radiolabeled ligand binding assays include, for example, immunoblotting (Western Blot), ELISA (including competitive ELISA) tests, RIA tests, ECL tests and IRMA tests.
  • TnT troponin T
  • TnI troponin I
  • TnC troponin C
  • cardiac troponin refers to all troponin isoforms expressed in cardiac cells, preferably subendocardial cells. These isoforms have been fully characterized in the art, such as described in Anderson 1995, Circulation Research, vol.76, no.4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493.
  • cardiac troponin also includes variants of specific cardiac troponins, such variants having at least the same basic biological and immunological properties as the specific cardiac troponin. In particular, if they are detected by the same specificity mentioned herein, they share the same basic biological and immunological properties. It should be understood that the isoforms of troponin can be measured together (simultaneously or sequentially) or separately (i.e., other isoforms are not measured at all).
  • cardiac troponin T As used herein, the terms “cardiac troponin T”, “cTnT” refer to the cardiac troponin T subunit, the amino acid sequence of which is disclosed in the UniProt database, accession number P45379.
  • cardiac troponin I As used herein, the terms “cardiac troponin I”, “cTnI” refer to the cardiac troponin I subunit, the amino acid sequence of which is disclosed in the UniProt database, accession number P19429.
  • troponin C As used herein, the terms “troponin C”, “TnC” refer to the troponin C subunit, the amino acid sequence of which is disclosed in the UniProt database, accession number P63316.
  • large-size cardiac troponin or “large-size cTnITC” are used interchangeably herein and are intended to include a complex formed by any full-length protein or fragment of TnC, full-length protein or fragment of cTnI, one or more segments of amino acid residues 223-287 of cTnT, and one or more segments of amino acid residues 1-222 of cTnT.
  • antibody has the meaning generally understood in the art and refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Antibody light chains can be classified as ⁇ (kappa) and ⁇ (lambda) light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ or ⁇ , and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively.
  • the distribution of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.
  • antibody it includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the present application is applicable to different detection systems, including but not limited to fluorescent labeling reaction systems, enzyme-linked immunosorbent assay systems, bioluminescence systems, etc.
  • One object of the present application is to provide a detection reagent that can sensitively and specifically detect large-sized cTnITC (including full-length cTnITC and moderate hydrolysis products, which still include cTnI, cTnT and TnC subunits or their fragments, and TnT fragments at positions 67-222), without recognizing further hydrolyzed ternary complexes, binary complexes or free subunits and their fragments.
  • the present application provides a composition for detecting large-sized cTnITC in a sample, comprising a first group of antibodies and a second group of antibodies, wherein:
  • the first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT;
  • the second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.
  • the second aspect of the present application provides a kit for detecting large-sized cTnITC in a sample, comprising a capture antibody and a detection antibody, wherein the capture antibody is selected from one of the first group of antibodies and the second group of antibodies, and the detection antibody is selected from the other of the first group of antibodies and the second group of antibodies; wherein,
  • the first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT;
  • the second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.
  • the capture antibody includes at least antibody 1, and the detection antibody includes at least antibody 2; or, the capture antibody includes at least antibody 2, and the detection antibody includes at least antibody 1.
  • the first group of antibodies does not include antibodies that specifically bind to any section of the amino acid sequence of positions 223-287 of cTnT.
  • the capture antibody can be coated on the surface of a solid phase carrier, such as magnetic beads, latex particles, ELISA plates, plastic beads, plastic tubes, immunochromatographic test strips, and test cards to capture the antigen in the sample, and then a detection antibody with a detectable label is used to bind to the capture antibody coated on the surface of the solid phase carrier, and the amount of the detectable label on the bound detection antibody is measured to determine the antigen content in the sample.
  • a solid phase carrier such as magnetic beads, latex particles, ELISA plates, plastic beads, plastic tubes, immunochromatographic test strips, and test cards
  • the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. It is particularly preferred that such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).
  • immunological detection e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.
  • Such labels are well known in the art, including but not limited to enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), chemiluminescent substances (e.g., acridinium ester compounds), and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels.
  • enzymes e.g., horseradish peroxidas
  • the markers covered in the present invention can be detected by methods known in the art.
  • radioactive labels can be detected using photographic film or scintillation counters
  • fluorescent markers can be detected using photodetectors to detect emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate.
  • Chemiluminescent substances are generally detected by providing an excitation liquid and/or a catalyst to the luminescent substance to detect the emitted light.
  • Biotin is generally detected by providing avidin (for example, streptavidin) modified with the above-mentioned marker to biotin and detecting the marker carried by the avidin connected to biotin.
  • the detectable marker as described above can be connected to the antibody or its antigen-binding fragment of the present invention by a linker of different lengths to reduce potential steric hindrance.
  • the detectable marker is selected from fluorescein, chemiluminescent substances (such as acridinium ester compounds), enzymes (such as horseradish peroxidase, alkaline phosphatase), radioactive isotopes, biotin, colloidal gold and magnetic particles.
  • the kit may further include a reagent for making the corresponding detectable label detectable (a substrate for the detectable label).
  • a reagent for making the corresponding detectable label detectable a substrate for the detectable label
  • the kit may also include a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase.
  • OPD o-phenylenediamine
  • TMB tetramethylbenzidine
  • ABTS ABTS
  • luminol compounds for horseradish peroxidase
  • p-NPP p-nitrophenyl phosphate
  • AMPPD alkaline phosphatase.
  • the detectable label is a chemiluminescent reagent (e.g., an
  • the kit in the present application is particularly suitable for immunoassays, such as ELISA, and in particular, double antibody sandwich chemiluminescent immunoassay.
  • the amount of large-size cTnITC can be detected directly or indirectly.
  • the amount or concentration of the detection antibody can be directly detected based on the detectable label signal of the detection antibody, and the signal is directly related to the target antigen in the sample.
  • Such signals are sometimes also referred to as intensity signals herein-for example, they can be obtained by measuring the intensity value of the specific physical or chemical properties of the detection antibody.
  • Indirect measurement includes measuring signals from secondary components (ie, not the detection antibody itself) or biological readout systems, such as measurable cell responses, ligands, markers, or enzymatic reaction products.
  • the present application provides a method for detecting large-sized cTnITC in a sample in vitro, comprising the following steps:
  • the capture antibody is selected from one group of the first group of antibodies and the second group of antibodies
  • the detection antibody is selected from the other group of the first group of antibodies and the second group of antibodies:
  • the first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT;
  • the second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.
  • the second group of antibodies further includes:
  • one or more antibodies 3 each of which is independently selected from an antibody that specifically binds to cTnIC; and/or,
  • One or more antibodies 4 each of which is independently selected from an antibody that specifically binds to any section of the amino acid sequence at positions 18-210 of cTnI.
  • the signal-to-noise ratio of the detection reagent can be significantly improved.
  • the first group of antibodies includes one or more of the antibody 1, and the second group of antibodies includes one or more of the antibody 2 and one or more of the antibody 3.
  • the first group of antibodies includes at least two antibodies 1.
  • the second group of antibodies includes at least two antibodies 2.
  • the first group of antibodies includes at least two antibodies 1; the second group of antibodies includes at least two antibodies 2 and optionally at least two antibodies 3.
  • the combination of multiple antibodies that bind to the same target protein is beneficial for the detection of large-sized cTnITC.
  • each of the antibodies 1 is independently selected from antibodies that specifically bind to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT. In some embodiments, each of the antibodies 1 is independently selected from antibodies that specifically bind to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT. An antibody that specifically binds to amino acids 119-138, 132-151, or 171-190 of cTnT. In some embodiments, each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 119-138 or 132-151 of cTnT.
  • each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 1-15, amino acids 13-22, amino acids 18-22, amino acids 18-28, amino acids 18-35, amino acids 22-31, amino acids 22-40, amino acids 23-29, amino acids 24-40, amino acids 25-40, amino acids 26-35, amino acids 34-37, amino acids 41-49, amino acids 83-89, amino acids 86-90, amino acids 87-90, amino acids 117-126, amino acids 130-145, amino acids 169-178, amino acids 186-192, amino acids 190-196, or amino acids 195-209 of cTnI.
  • each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 22-40, 41-49, or 83-89 of cTnI. In some embodiments, each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 41-49 of cTnI.
  • the capture antibody is the first group of antibodies
  • the detection antibody is the second group of antibodies. Studies have found that when the first group of antibodies are used as capture antibodies and the second group of antibodies are used as detection antibodies, the detection of large-sized cTnITC has a higher signal-to-noise ratio.
  • an exemplary scheme includes obtaining a monoclone with specific binding activity by hybridoma technology, specifically including the selection and preparation of two parent cells, cell fusion, selective culture and cloning of hybridoma cells, preparation of monoclonal antibodies, specific identification and purification, etc. Further, epitope identification and screening steps may also be included, such as using ELISA or surface plasmon resonance (SPR) and other technologies to identify antibody binding epitopes.
  • SPR surface plasmon resonance
  • cTnITC Due to the low sensitivity of existing cTnITC detection kits, its application in the diagnosis of myocardial injury is seriously affected. In clinical practice, cTnI and cTnT are also commonly selected as highly specific and highly sensitive markers of myocardial infarction. Based on the aforementioned composition, kit and detection method, the present application achieves a highly sensitive and highly specific rapid detection of large-size cTnITC, which is conducive to a deep understanding of the correlation between troponin and myocardial injury.
  • cTnITC is more sensitive to myocardial injury and can be used as a more sensitive myocardial injury marker to help diagnose the disease and judge the prognosis.
  • the present application provides use of the composition described in any one of the first aspects in preparing a reagent for detecting large-size cTnITC.
  • the present application provides the composition of any one of the first aspect, the test of any one of the second aspect, Use of the kit or the method described in any one of the third aspects in in vitro diagnosis of myocardial injury.
  • the present application provides a method for diagnosing myocardial injury in vitro, comprising the step of detecting large-sized cTnITC in a sample from a subject using the composition described in any one of the first aspect, the kit described in any one of the second aspect, or the method described in any one of the third aspect.
  • the sample is a body fluid or tissue sample of a subject, such as whole blood, serum, plasma (including lithium heparin plasma, EDTA plasma), urine, saliva, biological tissue or cells, preferably whole blood, serum or plasma.
  • a body fluid or tissue sample of a subject such as whole blood, serum, plasma (including lithium heparin plasma, EDTA plasma), urine, saliva, biological tissue or cells, preferably whole blood, serum or plasma.
  • This application achieves a highly sensitive and specific rapid detection of large-sized cTnITC.
  • an antibody that specifically binds to TnC as a capture antibody or a detection antibody, the signal-to-noise ratio of the system can be significantly improved, thereby increasing sensitivity.
  • composition or kit of the present application can be used in combination with a fully automatic chemiluminescence analyzer to achieve automated, rapid and high-throughput detection of large-size cTnITC.
  • the kit of the present application has high sensitivity, good specificity and good repeatability.
  • the capture antibody-detection antibody was applied to the double antibody sandwich chemiluminescence immunoassay method to construct a detection kit for detecting large-sized cTnITC in samples.
  • the capture antibodies were: Antibody 1: antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.
  • the capture antibodies were: Antibody 1: antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 4: Antibody 19C7cc that specifically binds to amino acid fragment 41-49 of cTnI.
  • the capture antibodies were: Antibody 1: antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.
  • the capture antibodies were: Antibody 1: antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 4: antibody 19C7cc that specifically binds to amino acid fragment 41-49 of cTnI.
  • the capture antibodies were: Antibody 1: antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 2: Antibody 7B9cc that specifically binds to TnC.
  • the capture antibodies were: Antibody 1: Antibody 1C11cc that specifically binds to amino acid fragment 171-190 of cTnT;
  • the detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody Tcom8 that specifically binds to the cTnIC complex epitope.
  • the capture antibodies were: Antibody 1: antibody 406cc that specifically binds to amino acid fragment 132-151 of cTnT;
  • the detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.
  • the capture antibodies were: Antibody 1: 300cc of an antibody that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.
  • the experiment also used a kit obtained by replacing the antibody 1 used in the large-size cTnITC detection kits 3-8 with the following antibodies: antibody 7F4 or 7G7 with a specific binding site in the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc with a specific binding site in the cTnT amino acid 145-164 fragment.
  • the experiment also used a kit obtained by replacing the antibody 4 used in the large-size cTnITC detection kit 4 with the following antibodies: antibody M18cc with a specific binding site in the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site in the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site in the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site in the cTnI amino acid 190-196 fragment.
  • the antibodies in this article were purchased from Haitide Biotechnology Co., Ltd.
  • the large-format cTnITC assay kit includes:
  • Magnetic bead coating working solution used to achieve the capture of large-sized cTnITC in the sample.
  • the magnetic bead coating working solution comprises: a mixture of superparamagnetic particles coated with capture antibodies.
  • Enzyme marker working solution used to detect large-sized cTnITC antigens captured by superparamagnetic microparticles.
  • the enzyme marker working solution includes: detection antibody labeled with alkaline phosphatase.
  • the detection method of large-size cTnITC is as follows:
  • Step 1 Add the sample, magnetic bead coating working solution, and enzyme marker working solution to the reaction tube. After incubation, the large-sized cTnITC in the sample binds to the antibody coated on the magnetic beads, and the antibody-alkaline phosphatase marker binds to the large-sized cTnITC in the sample. After the reaction is completed, the solid phase is placed in a magnetic field, the magnetic field attracts the magnetic beads, the substances bound to the solid phase are retained, and the unbound substances are washed away.
  • Step 2 Add the chemiluminescent substrate to the reaction tube.
  • the luminescent substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxetane, AMPPD) is decomposed by alkaline phosphatase, removing a phosphate group to generate an unstable intermediate.
  • the intermediate generates methyl benzoate anion through intramolecular electron transfer.
  • chemiluminescence is generated, and the number of photons generated in the reaction is measured by a photomultiplier tube.
  • the number of photons generated is proportional to the concentration of large-size cTnITC in the sample.
  • the amount of analyte in the sample is determined by the calibration curve.
  • the above-mentioned large-size cTnITC detection kit can be used in conjunction with Mindray's fully automatic chemiluminescence analyzers CL2000i, CL6000i, CL8000i and other models.
  • test results are shown in Figure 1, where the signal-to-noise ratio of detection kit 3 is significantly higher than that of detection kit 1 and detection kit 5, and the signal-to-noise ratio of detection kit 4 is significantly higher than that of detection kit 2 and detection kit 5, indicating that the use of antibody 2 that specifically binds to TnC together with antibody 3 or antibody 4 can significantly improve the signal-to-noise ratio; detection kits 3, 7 and 8 have a higher signal-to-noise ratio, indicating that antibody 1 with specific binding sites at cTnT amino acids 119-138 and 132-151 is significantly helpful in improving the signal-to-noise ratio.
  • the antibody 1 used in kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 with a specific binding site at the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc with a specific binding site at the cTnT amino acid 145-164 fragment, and the antibody 4 used in kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site at the cTnI amino acid 18-28 fragment, 16A11cc, 16A12cc or 8E10cc with a specific binding site at the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site at the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site at the cTnI amino acid 190-196 fragment, all of which can effectively reflect the signal differences of the samples.
  • cTnT Hytest, 8RTT5
  • cTnI Hytest, 8RT17
  • cTnIC Hytest, 8ICR3
  • cTnITC Hytest, 8ITCR
  • kits 1-8 can only recognize cTnITC antigen, but cannot recognize cTnT, cTnI and binary cTnIC.
  • the antibody 1 used in kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 with a specific binding site at the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc with a specific binding site at the cTnT amino acid 145-164 fragment
  • the antibody 4 used in kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site at the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site at the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site at the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site at the cTnI amino acid
  • LoB test results are derived from 5 blank samples, run for 4 days, and each test is repeated 4 times.
  • LoD test results are derived from 5 low-concentration samples, run for 4 days, and each test is repeated 4 times.
  • Kits 1-8 were selected to establish blank limits and detection limits. The test results are shown in Table 1.
  • the blank limit and detection limit of detection kit 3 were significantly lower than those of detection kit 1 and detection kit 5, and the blank limit and detection limit of detection kit 4 were significantly lower than those of detection kit 2 and detection kit 5, indicating that the addition of antibody 2 that specifically binds to TnC and its use together with antibody 3 or antibody 4 can significantly improve the sensitivity of the kit.
  • the antibody 1 used in the kits 3-8 is replaced with the following antibodies: antibody 7F4 or 7G7 having a specific binding site at the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc having a specific binding site at the cTnT amino acid 145-164 fragment, and the antibody 4 used in the kit 4 is replaced with the following antibodies: antibody M18cc having a specific binding site at the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc having a specific binding site at the cTnI amino acid 86-90 fragment, and antibody M46 having a specific binding site at the cTnI amino acid 130-145 fragment,
  • the antibody MF4cc whose specific binding site is at the cTnI amino acid 190-196 fragment, has lower LoB and LoD values.
  • Detection kits 3 and 4 had high signal-to-noise ratios and low limits of detection and were used for linear analysis.
  • Clinical serum samples were selected as high-concentration samples, and the high-concentration samples were diluted in a certain ratio to obtain a series of diluted samples, and the concentration range of the series of diluted samples was 0-6000 ng/L.
  • the samples were analyzed by chemiluminescent immunoassay using detection kit 3.
  • the average value of the test concentration results and the theoretical concentration were linearly fitted, and the correlation coefficient was calculated within the linear range.
  • the experimental results are shown in Figure 3.
  • the test concentration of the diluted sample is linear with the theoretical concentration.
  • the R2 value in the linear range (0-6000ng/L) is 0.9982.
  • the samples were analyzed by chemiluminescent immunoassay using the detection kit 4.
  • the average value of the test concentration results and the theoretical concentration were linearly fitted, and the correlation coefficient was calculated within the linear range.
  • the experimental results are shown in Figure 4.
  • the test concentration of the diluted sample was linear with the theoretical concentration.
  • the R2 value within the linear range (0-6000ng/L) was 0.9995.
  • Example 6 Construction and testing of a detection kit for exchanging capture antibodies and detection antibodies
  • the capture antibody of detection kit 3 was exchanged with the detection antibody to construct detection kit 3a.
  • Detection Kit 3 includes:
  • the capture antibodies were: Antibody 1: antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;
  • the detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody 20C6cc that specifically binds to the epitope of the cTnIC complex.
  • Detection Kit 3a includes:
  • the capture antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody 20C6cc that specifically binds to the cTnIC complex epitope;
  • the detection antibodies are: Antibody 1: Antibody 329cc that specifically binds to the amino acid fragment 119-138 of cTnT.
  • the signal-to-noise ratio of detection kit 3 was significantly higher than that of detection kit 3a, indicating that the preferred combination of capture antibody and detection antibody is: the capture antibody is antibody 1 (specifically binding to cTnT amino acid fragment 119-138), and the detection antibody is antibody 2 (specifically binding to TnC) and antibody 3 (specifically binding to cTnIC complex epitope).
  • the detection kit with this combination of capture antibody and detection antibody has a higher signal-to-noise ratio.
  • the signal-to-noise ratio of detection kit 3a is lower than that of detection kit 3, and its signal difference variation pattern in different samples is similar. The law is similar to that of detection kit 3.
  • the specificity of the detection kit 3a was analyzed. Equal concentrations of different antigens were added to the serum of healthy people, and the detection kit 3a was used to analyze it by chemiluminescent immunoassay.
  • the analyzed antigens include: cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), cTnITC (Hytest, 8ITCR).
  • Kit 3a can recognize cTnITC antigen, but cannot recognize cTnT, cTnI and binary cTnIC.
  • Kit 3a can be used to detect clinical serum samples, and Kit 3 performs better in terms of overall sensitivity and specificity.
  • Detection kit 3 was used for clinical performance evaluation. To evaluate the clinical performance of sandwich immunoassay for detecting large-size cTnITC, serum samples from patients with myocardial injury at different time points were dynamically monitored. High-sensitivity cTnI and cTnT values in the patient's serum were also detected.
  • the concentration value of large-size cTnITC is consistent with the change trend of high-sensitivity cTnI and cTnT test values, indicating that the concentration value of large-size cTnITC has clinical value and can be used to evaluate the myocardial injury of patients. Further analysis of the concentration changes of large-size cTnITC, high-sensitivity cTnI, and cTnT was performed. As shown in Figure 9, compared with cTnI and cTnT, the concentration value of large-size cTnITC changes faster, indicating that the concentration value change of large-size cTnITC is more sensitive to the myocardial injury of patients.

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Abstract

本申请涉及检测大尺寸cTnITC的组合物、试剂盒和方法、及其应用。具体地，本申请涉及含特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体和特异性结合TnC氨基酸序列中任意一段的抗体的组合物和试剂盒、检测方法以及在体外诊断心肌损伤中的应用。

Description

检测大尺寸cTnITC的组合物、试剂盒和方法、及其应用 技术领域
本申请涉及免疫检测领域，具体涉及用于检测大尺寸cTnITC(大尺寸心肌肌钙蛋白三元复合物，large-size cardiac troponin ternary complex)的组合物和试剂盒、检测方法、以及它们在体外诊断心肌损伤中的应用。
背景技术
心肌肌钙蛋白(cTn)是心肌损伤的特异性标志物，可用于检测心肌梗死后或心肌梗死过程中的心肌损伤。肌钙蛋白包含三个亚基，分别为心肌肌钙蛋白I(cTnI)、心肌肌钙蛋白T(cTnT)、肌钙蛋白C(TnC)。cTnI和cTnT在血清中的浓度与心肌损伤的严重程度高度相关，是美国和欧洲心脏病学会推荐的心肌梗死的高特异性和高敏感型标志物。然而，导致血液中心肌肌钙蛋白浓度升高的原因有很多，除心肌梗死外，肺栓塞、急性或慢性心衰、心脏创伤、心内膜炎和心肌炎等。这降低了cTn对心肌梗死的临床特异性，通常需要配合其他手段用于心肌梗死的诊断。
心肌肌钙蛋白通常以三元复合物cTnITC的形式与肌动蛋白丝结合。在心肌损伤时，肌钙蛋白从肌丝中降解下来，释放到血液中。在细胞内或血液循环中，全长的cTnITC经过蛋白酶降解，逐渐形成低分子量的三元复合物(LMW-cTnITC)、二元复合物(cTnIC)和游离的cTnT。研究表明，肌钙蛋白在血液中的存在形式与个体的生理、病理状态相关联。检测不同形式的cTnITC复合物有助于医生更全面的掌握患者的生理、病理状态，帮助快速准确的诊断，指导患者的预后。
目前，心肌肌钙蛋白检测的传统的免疫学方法主要为酶联免疫吸附法(ELISA)、胶体金免疫层析法(GICA)、电化学发光法(ECL)、化学发光免疫分析法(CLIA)等技术。其中化学发光免疫分析技术的灵敏度高、特异性强、线性范围宽、操作性强、自动化程度高，是临床应用前景最广的免疫分析方法。基于化学发光免疫分析技术，现有的高敏心肌肌钙蛋白检测手段通常用于检测cTnI和cTnT，无法区分肌钙蛋白复合物与游离的肌钙蛋白。在已有的文献报道中，cTnITC复合物的双抗体夹心免疫分析方法通过捕获抗体识别二元TnIC复合物表位，检测抗体识别TnT抗原实现。这种检测方法的信噪比较低，特异性不强，难以实现对cTnITC复合物的快速、灵敏、特异性检测。
发明内容
本申请的任务在于，提供一种能够灵敏度高且速度快地检测样品中特定形式的cTnITC的技术方案，通过捕获抗体和检测抗体的配合，实现特异性检测，特别地，在捕获抗体或检测抗体中加入特异性结合TnC的抗体对于提高检测体系的信噪比、提升检测灵敏度是非常有利的。
在第一个方面，本申请提供一种用于检测样品中大尺寸cTnITC的组合物，包括第一组抗体和第二组抗体，其中，
所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
在第二个方面，本申请提供一种用于检测样品中大尺寸cTnITC的试剂盒，包括捕获抗体和检测抗体，其中，所述捕获抗体选自第一组抗体和第二组抗体中的一组，所述检测抗体选自所述第一组抗体和第二组抗体中的另一组；其中，
所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
在第三个方面，本申请提供一种在体外检测样品中大尺寸cTnITC的方法，包括以下步骤：
获得检测样品；
将所述待测样品与捕获抗体接触，以形成抗体-抗原复合物；
使所述抗体-抗原复合物与结合有可检测标记的检测抗体接触，以形成抗体-抗原-抗体复合物；和
检测所述可检测标记所产生的信号，以确定待测样品中大尺寸cTnITC的存在和/或含量；
其中，所述捕获抗体选自第一组抗体和第二组抗体中的一组，所述检测抗体选自所述第一组抗体和第二组抗体中的另一组：
所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
在第四个方面，本申请提供第一方面任一项所述的组合物在制备用于检测大尺寸cTnITC的试剂中的用途。
在第五个方面，本申请提供第一方面任一项所述的组合物、第二方面任一项所述的试剂盒或第三方面任一项所述的方法在心肌损伤体外诊断中的用途。
在第六个方面，本申请提供第一方面一种在体外诊断心肌损伤的方法，其包括采用第一方面任一项所述的组合物、第二方面任一项所述的试剂盒或第三方面任一项所述的方法检测来自受试者的样品中大尺寸cTnITC的步骤。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为对大尺寸cTnITC检测试剂盒信噪比分析。
图2为用血清样本验证大尺寸cTnITC检测试剂盒的特异性。
图3为对大尺寸cTnITC检测试剂盒3的线性分析。
图4为对大尺寸cTnITC检测试剂盒4的线性分析。
图5为对大尺寸cTnITC检测试剂盒3与检测试剂盒3a的信噪比分析。
图6为对大尺寸cTnITC检测试剂盒3与检测试剂盒3a对临床血清样本的检测信噪比分析。
图7为用血清样本验证大尺寸cTnITC检测试剂盒3a的特异性。
图8为采用大尺寸cTnITC检测试剂盒3检测的大尺寸cTnITC与采用现有试剂盒检测的高敏cTnI、cTnT的变化趋势分析。
图9为采用大尺寸cTnITC检测试剂盒3检测的大尺寸cTnITC与采用现有试剂盒检测的高敏cTnI、cTnT的浓度变化率分析。
具体实施方式
下面将结合附图，对实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部。以下对实施例的描述仅仅是说明性的，绝不作为对本发明的任何限制。基于实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都在本发明的保护范围内。
在本文中，除非另有说明，否则所使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明的实施方案，下面提供相关术语的定义和解释。
如本文中所使用的，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的方法或者装置不仅包括所明确记载的要素，而且还包括没有明确列出的其他要素，或者是还包括为实施方法或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的方法或者装置中还存在另外的相关要素。
如本文中所使用的，术语“至少一个”意指在合理条件下的1个或超过1个，例如2个、3个、4个、5个或10个等。
如本文中所使用的，术语“第一”、“第二”仅仅是区别类似的对象，不代表针对对象的特定排序，可以理解地，“第一”、“第二”在允许的情况下可以互换特定的顺序或先后次序。应该理解“第一”、“第二”区分的对象在适当情况下可以互换，以使这里描述的本申请实施例能够以除了在这里图示或描述的那些以外的顺序实施。
如本文中所使用的，术语“特异性结合”是指，两分子(即结合分子与靶分子)之间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。两分子之间的结合亲和力可用KD值描述。KD值是指由kd(特定的结合分子-靶分子相互作用的解离速率；亦称为koff)与ka(特定结合分子-靶分子相互作用的缔合速率；亦称为kon)之比得到的解离常数，或者指表示为摩尔浓度(M)的kd/ka。KD值越小，两分子结合越紧密，亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合该抗原。用于分析抗体特异性的相应方法在例如以下文献中有描述：Harlow&Lane(1988)Antibodies：A Laboratory Manual，Cold Spring Harbor Laboratory Press以及Harlow& Lane(1999)Using Antibodies：A Laboratory Manual，Cold Spring Harbor Laboratory Press。适用研究的非限制性实例是，例如采用结构上和/或功能上密切相关的分子进行的结合研究、阻断和竞争研究。这些研究可采用以下方法进行，例如荧光激活细胞分选术(FACS)分析、流式细胞术滴定(FACS滴定)分析、表面等离子体共振技术(SPR，例如使用)、等温滴定量热法(ITC)、荧光滴定法或放射性标记的配体结合测定法。更多的方法包括，例如免疫印迹法(Western Blot)、ELISA(包括竞争ELISA)测试、RIA测试、ECL测试和IRMA测试。
在本文中，术语“肌钙蛋白”、“Tn”是肌细胞内肌纤蛋白上的一种调节钙介导的肌动蛋白和肌球蛋白之间相互反应的蛋白，存在于心肌和骨骼肌中，由肌钙蛋白T(TnT)、肌钙蛋白I(TnI)和肌钙蛋白C(TnC)三种亚单位组成。其中TnT是原肌球蛋白结合亚单位，与肌动蛋白及原肌球蛋白互相作用；TnI是抑制亚单位，抑制肌动球蛋白的ATP 酶活性；TnC是唯一与钙结合的亚单位，可使骨骼肌或心肌收缩。术语“心肌肌钙蛋白”、“cTn”指心脏细胞，优选心内膜下细胞，中表达的所有肌钙蛋白同种型。这些同种型在本领域中已被充分表征，例如Anderson 1995,Circulation Research,vol.76,no.4:681-686和Ferrieres 1998,Clinical Chemistry,44:487-493中所述。术语“心肌肌钙蛋白”还包括特定心肌肌钙蛋白的变体，此类变体至少具有与该特定心肌肌钙蛋白相同的基础生物学和免疫学特性。特别地，如果它们通过本文提及的相同特异性检测，则它们共享相同的基础生物学和免疫学特性。应当理解，肌钙蛋白的同种型可以被一同(同时或序贯)测定或单独(即完全不测定其它同种型)测定。
如本文中所使用的，术语“心肌肌钙蛋白T”、“cTnT”指心肌肌钙蛋白T亚单位，其氨基酸序列公开于UniProt数据库，编号P45379。
如本文中所使用的，术语“心肌肌钙蛋白I”、“cTnI”指心肌肌钙蛋白I亚单位，其氨基酸序列公开于UniProt数据库，编号P19429。
如本文中所使用的，术语“肌钙蛋白C”、“TnC”指肌钙蛋白C亚单位，其氨基酸序列公开于UniProt数据库，编号P63316。
在本文中，术语“大尺寸心肌肌钙蛋白三元复合物(large-size cardiac troponin)”或“大尺寸cTnITC(large-size cTnITC)”在本文中可互换使用，意欲包括任意的TnC的全长蛋白或片段、cTnI的全长蛋白或片段、cTnT的氨基酸残基223-287中的一段或多段和cTnT的氨基酸残基1-222中的一段或多段所形成的复合物。
在本文中，术语“抗体”具有本领域通常理解的含义，是指通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。各重链由重链可变区(VH)和重链恒定区(CH)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或 Chothia & Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
本申请适用于不同的探测体系，包括但不限于荧光标记的反应体系，酶联免疫体系，生物发光体系等。
检测试剂和方法
本申请的一个目的在于提供一种检测试剂，它能够灵敏地、特异地检测大尺寸cTnITC(包括cTnITC全长和适度水解产物，所述水解产物仍包括cTnI、cTnT和TnC亚单位或它们的片段，以及67-222位TnT片段)、而不识别进一步水解的三元复合物、二元复合物或者游离亚单位和它们的片段。
具体地，在第一个方面，本申请提供一种用于检测样品中大尺寸cTnITC的组合物，包括第一组抗体和第二组抗体，其中，
所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
在第一方面基础上，本申请第二个方面提供一种用于检测样品中大尺寸cTnITC的试剂盒，包括捕获抗体和检测抗体，其中，所述捕获抗体选自第一组抗体和第二组抗体中的一组，所述检测抗体选自所述第一组抗体和第二组抗体中的另一组；其中，
所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
也就是说，捕获抗体至少包括抗体1，检测抗体至少包括抗体2；或者，捕获抗体至少包括抗体2，检测抗体至少包括抗体1。
在本申请实施例中，所述第一组抗体不包括特异性结合cTnT第223-287位氨基酸序列中任意一段的抗体。
在一些实施方案中，可以将捕获抗体包被于固相载体表面，例如磁珠、胶乳粒子、酶标板、塑料珠、塑料管、免疫层析试纸条、检测卡，捕获样品中的抗原，然后用带有可检测标记的检测抗体与包被于固相载体表面的捕获抗体结合，通过测定所结合的检测抗体上可检测标记的量，确定样品中抗原的含量。
所述可检测标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、化学发光物质(如吖啶酯类化合物)、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。本发明中涵盖的标记物可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测。化学发光物质(如吖啶酯类化合物)一般通过给发光物质提供激发液和/或催化剂来检测发射的光。生物素一般通过给生物素提供上述标记物修饰的亲和素(例如，链霉亲和素)及检测与生物素连接的亲和素所携带的标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所述的可检测标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。在一些实施方案中，所述可检测标记选自荧光素、化学发光物质(例如吖啶酯类化合物)、酶(例如辣根过氧化物酶、碱性磷酸酶)、放射性同位素、生物素、胶体金和磁性颗粒。
在一些实施方案中，所述试剂盒可以进一步包含用于使相应可检测标记被检测到的试剂(所述可检测标记的底物)。例如，当所述可检测标记为酶时，所述试剂盒还可以包含相应酶的显色底物，例如用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS或鲁米诺类化合物，或用于碱性磷酸酶的对硝基苯磷酸酯(p-NPP)或AMPPD。例如当所述可检测标记为化学发光试剂(例如吖啶酯类化合物)时，所述试剂盒还可以包含用于化学发光的预激发液和/或激发液。
本申请中的试剂盒特别适用于免疫测定，例如ELISA、特别是双抗体夹心化学发光免疫分析方法。
在本申请中，大尺寸cTnITC的量可直接或间接检测获得。可基于检测抗体可检测标记信号，直接检测检测抗体的量或浓度，所述信号和样品中的目标抗原直接相关。此类信号本文中有时也被称为强度信号-例如可以通过测量检测抗体的特异性物理或化学性质的强度值获得。间接测量包括测量来自于次级组分(即不是检测抗体自身)或生物学读出系统的信号，例如可测量的细胞应答、配体、标记或酶促反应产物。
在第三个方面，本申请提供一种在体外检测样品中大尺寸cTnITC的方法，包括以下步骤：
获取待测样品；
将所述待测样品与捕获抗体接触，以形成抗体-抗原复合物；
使所述抗体-抗原复合物与结合有可检测标记的检测抗体接触，以形成抗体-抗原-抗体复合物；和
检测所述可检测标记所产生的信号，以确定待测样品中大尺寸cTnITC的存在和/或含量；
其中，所述捕获抗体选自第一组抗体和第二组抗体中的一组，所述检测抗体选自所述第一组抗体和第二组抗体中的另一组：
所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
在本申请第一个方面至第三个方面中，作为一些实施方案，所述第二组抗体还包括；
一个或多个抗体3，各所述抗体3独立地选自特异性结合cTnIC的抗体；和/或，
一个或多个抗体4，各所述抗体4独立地选自特异性结合cTnI第18-210位氨基酸序列中任意一段的抗体。
在此，通过将所述抗体2与特异性识别cTnIC二元复合物的抗体或特异性识别cTnI的抗体组合，能够显著提高检测试剂信噪比。
在本申请第一个方面至第三个方面中，作为一些实施方案，在一些实施方案中，所述第一组抗体包括一个或多个所述抗体1，所述第二组抗体包括一个或多个所述抗体2以及一个或多个所述抗体3。
在本申请第一个方面至第三个方面中，作为一些实施方案，所述第一组抗体包括至少两个抗体1。在一些实施方案中，所述第二组抗体包括至少两个抗体2。在一些实施方案中，所述第一组抗体包括至少两个抗体1；所述第二组抗体包括至少两个抗体2以及任选的至少两个抗体3。多个结合相同靶蛋白的抗体的组合对于大尺寸cTnITC的检测是有益的。
本申请对抗体的结合表位进行了筛选。在一些实施方案中，各所述抗体1独立地选自特异性结合cTnT第67-86位氨基酸、第119-138位氨基酸、第132-151位氨基酸、第145-164位氨基酸或第171-190位氨基酸的抗体。在一些实施方案中，各所述抗体1独立地选自特 异性结合cTnT第119-138位氨基酸、第132-151位氨基酸或第171-190位氨基酸的抗体。在一些实施方案中，各所述抗体1独立地选自特异性结合cTnT第119-138位氨基酸或第132-151位氨基酸的抗体。
对于抗体4的结合表位，在一些实施方案中，各所述抗体4独立地选自特异性结合cTnI第1-15位氨基酸、第13-22位氨基酸、第18-22位氨基酸、第18-28位氨基酸、第18-35位氨基酸、第22-31位氨基酸、第22-40位氨基酸、第23-29位氨基酸、第24-40位氨基酸、第25-40位氨基酸、第26-35位氨基酸、第34-37位氨基酸、第41-49位氨基酸、第83-89位氨基酸、第86-90位氨基酸、第87-90位氨基酸、第117-126位氨基酸、第130-145位氨基酸、第169-178位氨基酸、第186-192位氨基酸、第190-196位氨基酸或第195-209位氨基酸的抗体。在一些实施方案中，各所述抗体4独立地选自特异性结合cTnI第22-40位氨基酸、第41-49位氨基酸或第83-89位氨基酸的抗体。在一些实施方案中，各所述抗体4独立地选自特异性结合cTnI第41-49位氨基酸的抗体。
在一些实施方案中，所述捕获抗体为所述第一组抗体，所述检测抗体为所述第二组抗体。研究发现，当第一组抗体作为捕获抗体、第二组抗体作为检测抗体时，大尺寸cTnITC的检测具有更高的信噪比。
在本申请中，可以选择市售可得的针对特定表位的抗体，也可通过本领域一般技术手段获得本申请中对大尺寸cTnITC特定表位具有特异性结合活性的抗体。一个示例性的方案包括通过杂交瘤技术获得具有特异性结合活性的单克隆，具体包括两种亲本细胞的选择和制备、细胞融合、杂交瘤细胞的选择性培养和克隆化、单克隆抗体的制备、特异性鉴定及纯化等步骤。进一步，还可包括表位鉴定以及筛选的步骤，例如利用ELISA或表面等离子共振(SPR)等技术鉴定抗体结合表位。
应用
由于现有cTnITC检测试剂盒灵敏度较低，严重影响其在心肌损伤诊断中的应用。临床上也通常选择cTnI和cTnT作为心肌梗死的高特异性和高敏感型标志物。基于前述组合物、试剂盒和检测方法，本申请实现了对大尺寸cTnITC的高敏感、高特异性的快速检测，有利于深入理解肌钙蛋白与心肌损伤的相关性。研究表明，相较于cTnI和cTnT，大尺寸cTnITC对于心肌损伤情况更为敏感，可以作为更敏感的心肌损伤标志物，帮助疾病诊断以及预后判断。
在第四个方面，本申请提供第一方面任一项所述的组合物在制备用于检测大尺寸cTnITC的试剂中的用途。
在第五个方面，本申请提供第一方面任一项所述的组合物、第二方面任一项所述的试 剂盒或第三方面任一项所述的方法在心肌损伤体外诊断中的用途。
在第六个方面，本申请提供一种在体外诊断心肌损伤的方法，其包括采用第一方面任一项所述的组合物、第二方面任一项所述的试剂盒或第三方面任一项所述的方法检测来自受试者的样品中大尺寸cTnITC的步骤。
在一些实施方案中，所述样品为受试者体液或组织样品，例如全血、血清、血浆(包括肝素锂血浆、EDTA血浆)、尿液、唾液、生物组织或细胞，优选全血、血清或血浆。
有益效果
1)本申请实现了对大尺寸cTnITC的高敏感、高特异性的快速检测。通过选择特异性结合TnC的抗体作为捕获抗体或检测抗体，能够显著提高体系信噪比，提升灵敏度。
2)由于肌钙蛋白在血液中的存在形式与个体的生理、病理状态相关联，大尺寸cTnITC的特异性检测有利于深入理解肌钙蛋白与疾病的相关性。实验结果显示，大尺寸cTnITC的变化趋势与cTnT、cTnI一致，具有临床价值；并且，大尺寸cTnITC的浓度变化率更快，对患者的心肌损伤情况更为敏感，可作为更敏感的心肌损伤标志物，帮助疾病的诊断和预后判断。
3)本申请的组合物或试剂盒能够结合全自动化学发光仪使用，可以实现对大尺寸cTnITC的自动化、快速和高通量检测。
4)本申请的试剂盒灵敏度高，特异性好，重复性好。
实施例
实施例1 检测方法及检测试剂盒的构建
将捕获抗体-检测抗体应用于双抗体夹心化学发光免疫分析方法，构建检测试剂盒，用于检测样本中的大尺寸cTnITC。
(1)大尺寸cTnITC检测试剂盒1
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc；
检测抗体为：抗体3：特异性结合cTnIC复合物表位的抗体20C6cc。
(2)大尺寸cTnITC检测试剂盒2
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc；
检测抗体为：抗体4：特异性结合cTnI氨基酸片段41-49的抗体19C7cc。
(3)大尺寸cTnITC检测试剂盒3
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体3：特异性结合cTnIC复合物表位的抗体20C6cc。
(4)大尺寸cTnITC检测试剂盒4
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体4：特异性结合cTnI氨基酸片段41-49的抗体19C7cc。
(5)大尺寸cTnITC检测试剂盒5
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc。
(6)大尺寸cTnITC检测试剂盒6
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段171-190的抗体1C11cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体3：特异性结合cTnIC复合物表位的抗体Tcom8。
(7)大尺寸cTnITC检测试剂盒7
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段132-151的抗体406cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体3：特异性结合cTnIC复合物表位的抗体20C6cc。
(8)大尺寸cTnITC检测试剂盒8
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体300cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体3：特异性结合cTnIC复合物表位的抗体20C6cc。
实验中也使用了将所述大尺寸cTnITC检测试剂盒3-8中所使用的抗体1替换为下述抗体得到的试剂盒：特异性结合位点在cTnT氨基酸67-86片段的抗体7F4或7G7，特异性结合位点在cTnT氨基酸145-164片段的抗体2F3、1A11或1F11cc。实验中也使用了将所述大尺寸cTnITC检测试剂盒4中所使用的抗体4替换为下述抗体得到的试剂盒：特异性结合位点在cTnI氨基酸18-28片段的抗体M18cc，特异性结合位点在cTnI氨基酸86-90片段的抗体16A11cc、16A12cc或8E10cc，特异性结合位点在cTnI氨基酸130-145片段的抗体M46，特异性结合位点在cTnI氨基酸190-196片段的抗体MF4cc。本文抗体购自海肽生物科技有限公司。
大尺寸cTnITC检测试剂盒包括：
A.磁珠包被物工作液，用于实现对样本中大尺寸cTnITC的捕获。所述磁珠包被物工作液包括：包被着捕获抗体的超顺磁微粒混合物。
B.酶标记物工作液，用于实现对超顺磁微粒捕获的大尺寸cTnITC抗原的检测。所 述酶标记物工作液包括：碱性磷酸酶标记的检测抗体。
大尺寸cTnITC的检测方法如下：
第一步：将样本与磁珠包被物工作液、酶标记物工作液添加到反应管中，经过孵育，样本中的大尺寸cTnITC与包被在磁珠上的抗体结合，同时抗体-碱性磷酸酶标记物与样本中大尺寸cTnITC结合。反应完成后，固相置于一个磁场内，磁场吸住磁珠，结合在固相上的物质被保留，洗去未结合的物质。
第二步：将化学发光底物添加到反应管内，发光底物(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷，AMPPD)被碱性磷酸酶所分解，脱去一个磷酸基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光，再通过光电倍增管对反应中所产生的光子数进行测量。所产生光子数与样本内大尺寸cTnITC的浓度成正比。样本内分析物的量由校准曲线来确定。
上述大尺寸cTnITC检测试剂盒可以在迈瑞全自动化学发光仪CL2000i、CL6000i、CL8000i等机型上配套使用。
实施例2 大尺寸cTnITC检测试剂盒信噪比分析
配制含有不同浓度抗原的样本，包括两个高浓度样本(高值样本)和两个低浓度样本(低值样本)，其中抗原为重组心肌肌钙蛋白三元复合物(Hytest，8ITCR)。使用检测试剂盒1-8分别对样本进行分析。同时记录不含抗原的空白样本信号，计算信噪比。
测试结果见图1，其中检测试剂盒3的信噪比显著高于检测试剂盒1和检测试剂盒5，检测试剂盒4的信噪比显著高于检测试剂盒2和检测试剂盒5，表明特异性结合TnC的抗体2与抗体3或抗体4共同使用能够显著提升信噪比；检测试剂盒3、7和8具有较高信噪比，表明特异性结合位点在cTnT氨基酸119-138位和132-151位的抗体1对于信噪比提升有明显帮助。
此外，将试剂盒3-8中所使用的抗体1替换为下述抗体：特异性结合位点在cTnT氨基酸67-86片段的抗体7F4或7G7、特异性结合位点在cTnT氨基酸145-164片段的抗体2F3、1A11或1F11cc，将试剂盒4中所使用的抗体4替换为下述抗体：特异性结合位点在cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点在cTnI氨基酸86-90片段16A11cc、16A12cc或8E10cc，特异性结合位点在cTnI氨基酸130-145片段的抗体M46，特异性结合位点在cTnI氨基酸190-196片段的抗体MF4cc，均能有效反应样本的信号差异。
实施例3 大尺寸cTnITC检测试剂盒特异性分析
将等浓度的不同抗原添加至健康人的血清中，并使用检测试剂盒1-8分别通过化学发光免疫分析方法对其进行分析。分析的抗原包括：cTnT(Hytest，8RTT5)、cTnI(Hytest，8RT17)、cTnIC(Hytest，8ICR3)、cTnITC(Hytest，8ITCR)。
实验结果见图2，检测试剂盒1-8均仅能识别cTnITC抗原，但不能识别cTnT、cTnI及二元cTnIC。此外，将试剂盒3-8中所使用的抗体1替换为下述抗体：特异性结合位点在cTnT氨基酸67-86片段的抗体7F4或7G7、特异性结合位点在cTnT氨基酸145-164片段的抗体2F3、1A11或1F11cc，将试剂盒4中所使用的抗体4替换为下述抗体：特异性结合位点在cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点在cTnI氨基酸86-90片段16A11cc、16A12cc或8E10cc，特异性结合位点在cTnI氨基酸130-145片段的抗体M46，特异性结合位点在cTnI氨基酸190-196片段的抗体MF4cc，均能有效识别cTnITC抗原。
实施例4 大尺寸cTnITC检测试剂盒空白限与检出限的建立
根据临床和实验室标准协会(CLSI)的建议(EP-17A2 Protocols for Determination of Limits of Detection and Limits of Quantitation)建立空白限(LoB)与检出限(LoD)。
LoB测试结果来源于5个空白样本，运行4天，每次测试重复4次。通用公式为LoB＝平均值+1.65*SD。
LoD测试结果来源于5个低浓度样本，运行4天，每次测试重复4次。通用公式为LoD＝LoB+1.65*SD。
选择试剂盒1-8建立空白限和检出限，测试结果见表1。
表1：检测试剂盒的空白限(LoB)与检出限(LoD)
其中检测试剂盒3的空白限与检出限显著低于检测试剂盒1和检测试剂盒5，其中检测试剂盒4的空白限与检出限显著低于检测试剂盒2和检测试剂盒5，表明特异性结合TnC的抗体2的加入并与抗体3或抗体4共同使用能够显著提升试剂盒的灵敏度。
此外，将试剂盒3-8中所使用的抗体1替换为下述抗体：特异性结合位点在cTnT氨基酸67-86片段的抗体7F4或7G7、特异性结合位点在cTnT氨基酸145-164片段的抗体2F3、1A11或1F11cc，将试剂盒4中所使用的抗体4替换为下述抗体：特异性结合位点在cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点在cTnI氨基酸86-90片段16A11cc、16A12cc或8E10cc，特异性结合位点在cTnI氨基酸130-145片段的抗体M46， 特异性结合位点在cTnI氨基酸190-196片段的抗体MF4cc，均具有较低的LoB和LoD值。
实施例5 大尺寸cTnITC检测试剂盒的线性分析
检测试剂盒3和试剂盒4的信噪比高、检出限低，被用于线性分析。
选取临床血清样本为高浓度样本，将高浓度样本按一定比例稀释，得到一系列稀释样本，系列稀释样本的浓度范围为0-6000ng/L。
使用检测试剂盒3通过化学发光免疫分析方法对样本进行分析。将测试浓度结果平均值和理论浓度进行线性拟合，并计算在线性范围内相关系数。
实验结果见图3，稀释样本的测试浓度与理论浓度呈线性。线性范围内(0-6000ng/L)的R2值为0.9982。
使用检测试剂盒4通过化学发光免疫分析方法对样本进行分析。将测试浓度结果平均值和理论浓度进行线性拟合，并计算在线性范围内相关系数。实验结果见图4，稀释样本的测试浓度与理论浓度呈线性。线性范围内(0-6000ng/L)的R2值为0.9995。
实施例6 交换捕获抗体与检测抗体的检测试剂盒构建与测试
将检测试剂盒3的捕获抗体与检测抗体进行交换，构建检测试剂盒3a。
检测试剂盒3包括：
捕获抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc；
检测抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体3：特异性结合cTnIC复合物表位的抗体20C6cc。
检测试剂盒3a包括：
捕获抗体为：抗体2：特异性结合TnC的抗体7B9cc；和抗体3：特异性结合cTnIC复合物表位的抗体20C6cc；
检测抗体为：抗体1：特异性结合cTnT氨基酸片段119-138的抗体329cc。
配制含有不同浓度抗原的样本，包括三个高浓度样本和三个低浓度样本，其中抗原为重组心肌肌钙蛋白三元复合物(Hytest，8ITCR)。使用检测试剂盒3、3a分别对样本进行分析。同时记录不含抗原的空白样本信号，计算信噪比。测试结果见图5。
使用检测试剂盒3、3a分别对患有心血管疾病的患者的临床血清样本进行分析。同时记录健康人血清样本的信号，计算信噪比。测试结果见图6。其中检测试剂盒3的信噪比显著高于检测试剂盒3a，表明优选的捕获抗体和检测抗体的组合为：捕获抗体为抗体1(特异性结合cTnT氨基酸片段119-138)，检测抗体为抗体2(特异性结合TnC)和抗体3(特异性结合cTnIC复合物表位)，具有该捕获抗体和检测抗体的组合的检测试剂盒的信噪比更高。其中检测试剂盒3a信噪比较检测试剂盒3低，其在不同样本中的信号差异变化规 律与检测试剂盒3近似。
对检测试剂盒3a的特异性进行分析。将等浓度的不同抗原添加至健康人的血清中，使用检测试剂盒3a通过化学发光免疫分析方法对其进行分析。分析的抗原包括：cTnT(Hytest，8RTT5)、cTnI(Hytest，8RT17)、cTnIC(Hytest，8ICR3)、cTnITC(Hytest，8ITCR)。实验结果见图7。剂盒3a能识别cTnITC抗原，但不能识别cTnT、cTnI及二元cTnIC。
综合看，试剂盒3a可以用于检测临床血清样本，试剂盒3在考虑整体灵敏度和特异性上的表现更优。
实施例7 大尺寸cTnITC特异性免疫分析的临床性能评估
检测试剂盒3被用于临床性能评估。为评估使用夹心免疫测定法检测大尺寸cTnITC的临床性能，对心肌损伤患者不同时间点的血清样本进行动态监测。同时检测患者血清中的高敏cTnI和cTnT值。
选择有心肌损伤的患者入组，患者年龄大于或等于18岁。这些患者被诊断为患有心肌梗死、心衰，或经历了心脏手术治疗，表现出高敏cTnI或高敏cTnT测试值升高。患者入组后，连续3天采集患者的肝素锂血浆样本，使用迈瑞化学发光仪及配套试剂测试样本中的大尺寸cTnITC的浓度值。
结果如图8所示，大尺寸cTnITC的浓度值与高敏cTnI、cTnT测试值的变化趋势一致，表明大尺寸cTnITC的浓度值具有临床价值，可以应用于评估患者心肌损伤情况。进一步对大尺寸cTnITC、高敏cTnI、cTnT的浓度变化情况进行分析，如图9所示，相比于cTnI和cTnT，大尺寸cTnITC的浓度值变化速率更快，表明大尺寸cTnITC的浓度值变化对患者的心肌损伤情况更为敏感。
除本文中描述的那些外，根据前述描述，本发明的各种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (20)

  1. 一种用于检测样品中大尺寸cTnITC的组合物，包括第一组抗体和第二组抗体，其中，
    所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT(心肌肌钙蛋白T)第67-222位氨基酸序列中任意一段的抗体；
    所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC(肌钙蛋白C)氨基酸序列中任意一段的抗体。
  2. 权利要求1所述的组合物，其中所述第一组抗体不包括特异性结合cTnT第223-287位氨基酸序列中任意一段的抗体。
  3. 权利要求1或2所述的组合物，其中所述第二组抗体还包括：
    一个或多个抗体3，各所述抗体3独立地选自特异性结合cTnIC(心肌肌钙蛋白IC二元复合物)的抗体；和/或，
    一个或多个抗体4，各所述抗体4独立地选自特异性结合cTnI(心肌肌钙蛋白I)第18-210位氨基酸序列中任意一段的抗体。
  4. 权利要求1-3任一项所述的组合物，其中所述第一组抗体包括一个或多个所述抗体1，所述第二组抗体包括一个或多个所述抗体2以及一个或多个所述抗体3。
  5. 权利要求1-4任一项所述的组合物，其中各所述抗体1独立地选自特异性结合cTnT第67-86位氨基酸、第119-138位氨基酸、第132-151位氨基酸、第145-164位氨基酸或第171-190位氨基酸的抗体；
    优选地，各所述抗体1独立地选自特异性结合cTnT第119-138位氨基酸、第132-151位氨基酸或第171-190位氨基酸的抗体。
  6. 权利要求3-5任一项所述的组合物，其中各所述抗体4独立地选自特异性结合cTnI第1-15位氨基酸、第13-22位氨基酸、第18-22位氨基酸、第18-28位氨基酸、第18-35位氨基酸、第22-31位氨基酸、第22-40位氨基酸、第23-29位氨基酸、第24-40位氨基酸、第25-40位氨基酸、第26-35位氨基酸、第34-37位氨基酸、第41-49位氨基酸、第83-89位氨基酸、第86-90位氨基酸、第87-90位氨基酸、第117-126位氨基酸、第130-145位氨 基酸、第169-178位氨基酸、第186-192位氨基酸、第190-196位氨基酸或第195-209位氨基酸的抗体；
    优选地，各所述抗体4独立地选自特异性结合cTnI第22-40位氨基酸、第41-49位氨基酸或第83-89位氨基酸的抗体；
    更优选地，各所述抗体4独立地选自特异性结合cTnI第41-49位氨基酸的抗体。
  7. 一种用于检测样品中大尺寸cTnITC的试剂盒，包括捕获抗体和检测抗体，其中，所述捕获抗体选自第一组抗体和第二组抗体中的一组，所述检测抗体选自所述第一组抗体和第二组抗体中的另一组；其中，
    所述第一组抗体包括一个或多个所述抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
    所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
  8. 权利要求7所述的试剂盒，其中所述第一组抗体不包括特异性结合cTnT第223-287位氨基酸序列中任意一段的抗体。
  9. 权利要求7或8所述的试剂盒，其中所述第二组抗体还包括：
    一个或多个抗体3，各所述抗体3独立地选自特异性结合cTnIC的抗体；和/或，
    一个或多个抗体4，各所述抗体4独立地选自特异性结合cTnI第18-210位氨基酸序列中任意一段的抗体。
  10. 权利要求7-9任一项所述的试剂盒，其中所述第一组抗体包括一个或多个所述抗体1，所述第二组抗体包括一个或多个所述抗体2以及一个或多个所述抗体3。
  11. 权利要求7-11任一项所述的试剂盒，其中所述捕获抗体为所述第一组抗体，所述检测抗体为所述第二组抗体。
  12. 权利要求7-11任一项所述的试剂盒，其中各所述抗体1独立地选自特异性结合cTnT第67-86位氨基酸、第119-138位氨基酸、第132-151位氨基酸、第145-164位氨基酸或第171-190位氨基酸的抗体；优选地，各所述抗体1独立地选自特异性结合cTnT第119-138位氨基酸、第132-151位氨基酸或第171-190位氨基酸的抗体；和/或
    各所述抗体4独立地选自特异性结合cTnI第1-15位氨基酸、第13-22位氨基酸、第 18-22位氨基酸、第18-28位氨基酸、第18-35位氨基酸、第22-31位氨基酸、第22-40位氨基酸、第23-29位氨基酸、第24-40位氨基酸、第25-40位氨基酸、第26-35位氨基酸、第34-37位氨基酸、第41-49位氨基酸、第83-89位氨基酸、第86-90位氨基酸、第87-90位氨基酸、第117-126位氨基酸、第130-145位氨基酸、第169-178位氨基酸、第186-192位氨基酸、第190-196位氨基酸或第195-209位氨基酸的抗体；优选地，各所述抗体4独立地选自特异性结合cTnI第22-40位氨基酸、第41-49位氨基酸或第83-89位氨基酸的抗体；更优选地，各所述抗体4独立地选自特异性结合cTnI第41-49位氨基酸的抗体。
  13. 一种在体外检测样品中大尺寸cTnITC的方法，包括以下步骤：
    获取待测样品；
    将所述待测样品与捕获抗体接触，以形成抗体-抗原复合物；
    使所述抗体-抗原复合物与结合有可检测标记的检测抗体接触，以形成抗体-抗原-抗体复合物；和
    检测所述可检测标记所产生的信号，以确定待测样品中大尺寸cTnITC的存在和/或含量；
    其中，所述捕获抗体选自第一组抗体和第二组抗体中的一组，所述检测抗体选自所述第一组抗体和第二组抗体中的另一组：
    所述第一组抗体包括一个或多个抗体1，各所述抗体1独立地选自特异性结合cTnT第67-222位氨基酸序列中任意一段的抗体；
    所述第二组抗体包括一个或多个抗体2，各所述抗体2独立地选自特异性结合TnC氨基酸序列中任意一段的抗体。
  14. 权利要求13所述的方法，其中所述第一组抗体不包括特异性结合cTnT第223-287位氨基酸序列中任意一段的抗体。
  15. 权利要求13或14所述的方法，其中所述第二组抗体还包括：
    一个或多个抗体3，各所述抗体3独立地选自特异性结合cTnIC的抗体；和/或，
    一个或多个抗体4，各所述抗体4独立地选自特异性结合cTnI第18-210位氨基酸序列中任意一段的抗体。
  16. 权利要求15所述的方法，其中所述第一组抗体包括一个或多个所述抗体1，所述 第二组抗体包括一个或多个所述抗体2，以及一个或多个所述抗体3。
  17. 权利要求13-16任一项所述的方法，其中所述捕获抗体为所述第一组抗体，所述检测抗体为所述第二组抗体。
  18. 权利要求1-6任一项所述的组合物在制备用于检测大尺寸cTnITC的试剂中的用途。
  19. 权利要求1-6任一项所述的组合物、权利要求7-12任一项所述的试剂盒或权利要求13-17任一项所述的方法在心肌损伤体外诊断中的用途。
  20. 一种在体外诊断心肌损伤的方法，其包括采用权利要求1-6任一项所述的组合物、权利要求7-12任一项所述的试剂盒或权利要求13-17任一项所述的方法检测来自受试者的样品中大尺寸cTnITC的步骤。
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