WO2025035370A1 - 检测总cTnITC的组合物、试剂盒和方法、及其应用 - Google Patents

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

Info

Publication number
WO2025035370A1
WO2025035370A1 PCT/CN2023/112993 CN2023112993W WO2025035370A1 WO 2025035370 A1 WO2025035370 A1 WO 2025035370A1 CN 2023112993 W CN2023112993 W CN 2023112993W WO 2025035370 A1 WO2025035370 A1 WO 2025035370A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibodies
antibody
amino acids
group
independently selected
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/112993
Other languages
English (en)
French (fr)
Inventor
刘雨青
张轶
刘君君
卡特鲁卡·伊万
别列兹尼科娃·阿纳斯塔西娅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Mindray Bio Medical Electronics Co Ltd
Original Assignee
Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Mindray Bio Medical Electronics Co Ltd filed Critical Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority to PCT/CN2023/112993 priority Critical patent/WO2025035370A1/zh
Priority to CN202380101490.1A priority patent/CN121794576A/zh
Publication of WO2025035370A1 publication Critical patent/WO2025035370A1/zh
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • 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 immunoassays, and in particular to a composition and a kit for detecting total ternary cardiac troponin complex cTnITC, a detection method, and their application in in vitro diagnosis of myocardial injury.
  • 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.
  • Cardiac troponin is usually bound to actin filaments in the form of a ternary complex cTnITC.
  • a ternary complex cTnITC When the myocardium is damaged, troponin is degraded from the myofilaments and released into the blood.
  • the full-length cTnITC In cells or in the blood circulation, the full-length cTnITC is degraded by proteases and gradually forms a low molecular weight ternary complex (LMW-cTnITC), a binary complex (cTnIC) and free cTnT.
  • 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 the epitope of the binary TnIC complex and detecting antibodies to recognize the TnT antigen.
  • This detection method has a low signal-to-noise ratio and low specificity, making it difficult to achieve rapid, sensitive and specific detection of the total content of the ternary complex cTnITC. Therefore, developing a highly sensitive and fast detection kit for detecting the total amount of troponin ternary complex cTnITC in the blood (ie, the total cTnITC content) is particularly important for the diagnosis and prognosis of cardiovascular diseases.
  • the task of the present application is to provide a technical solution that can detect the total content of cardiac troponin ternary complex cTnITC in a sample with high sensitivity and speed, and to achieve specific detection of total cTnITC through the combination of capture antibody and detection antibody.
  • adding an antibody that specifically binds to TnC to the capture antibody or the detection antibody is very beneficial for improving the signal-to-noise ratio of the detection system and enhancing the sensitivity of cTnITC detection.
  • the present application provides a composition for detecting a ternary complex of cardiac troponin 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 section of the amino acid sequence of cTnT at positions 223-287;
  • the present application provides a kit for detecting a ternary complex of cardiac troponin 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 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 a ternary complex of cardiac troponin 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 section of the amino acid sequence of cTnT at positions 223-287;
  • 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 the preparation of a reagent for detecting a ternary complex of cardiac troponin.
  • 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 diagnosing myocardial injury in vitro, comprising the step of detecting the cardiac troponin ternary complex 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 the total cTnITC detection kit.
  • FIG2 is a diagram showing the specificity of the total cTnITC detection kit validated using serum samples.
  • FIG. 3 shows the linearity analysis of total cTnITC detection kit 3.
  • FIG. 4 is a linearity analysis of total cTnITC detection kit 4.
  • FIG5 is an analysis of the signal-to-noise ratio of total cTnITC detection kit 3 and kit 3a.
  • FIG6 is an analysis of the signal-to-noise ratio of total cTnITC detection kit 3 and kit 3a for clinical serum samples.
  • FIG. 7 is a diagram showing the validation of the specificity of the total cTnITC detection kit 3a using serum samples.
  • FIG8 is an analysis of the changing trends of total cTnITC detected by the total cTnITC detection kit and high-sensitivity cTnI and cTnT detected by the existing kits.
  • FIG9 is an analysis of the concentration change rate of total cTnITC detected by the total cTnITC detection kit and high-sensitivity cTnI and cTnT detected by the existing kit.
  • 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.
  • troponin refers to a protein on myosin in myocytes that regulates calcium-mediated interaction between actin and myosin. It is present in cardiac and skeletal muscles and is expressed by troponin T. (TnT), troponin I (TnI) and troponin C (TnC) are composed of three subunits. Among them, TnT is a tropomyosin binding subunit that interacts with actin and tropomyosin; TnI is an inhibitory subunit that inhibits the ATPase activity of actomyosin; TnC is the only subunit that binds to calcium and can cause skeletal muscle or cardiac muscle contraction.
  • cardiac troponin and “cTn” refer 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, which have at least the same basic biological and immunological properties as the specific cardiac troponin.
  • troponins may be assayed together (simultaneously or sequentially) or individually (ie without assaying the other isoform 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.
  • total cardiac troponin ternary complex or “total cTnITC” are used interchangeably herein and are intended to include a complex formed by all full-length proteins or fragments of TnC, full-length proteins or fragments of cTnI and one or more segments of amino acid residues 223-287 of cTnT, and optionally 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 a light chain (LC) and a 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 Antigen binding site.
  • the distribution of amino acids in various regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. In this article, unless the context clearly indicates otherwise, when referring to the term "antibody”, it includes not only complete 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 method that can sensitively detect the total content of various forms of cardiac troponin ternary complex cTnITC in a sample and can specifically distinguish it from other existing forms of cTn, including cTnT, cTnI and cTnIC.
  • the present application provides a composition for detecting a ternary complex of cardiac troponin 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 section of the amino acid sequence of cTnT at positions 223-287;
  • 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 a ternary complex of cardiac troponin 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 section of the amino acid sequence of cTnT at positions 223-287;
  • 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 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 fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical Any substance detected by chemical means. It is particularly preferred that such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).
  • immunological detection e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent 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 detectable label 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 label 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 total cTnITC can be directly or indirectly detected.
  • 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 a ternary complex of cardiac troponin in a sample in vitro. The following steps are involved:
  • 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 section of the amino acid sequence of cTnT at positions 223-287;
  • 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 first group of antibodies further comprises one or more antibodies 3, each of the antibodies 3 being independently selected from antibodies that specifically bind to any segment of the amino acid sequence at positions 67-222 of cTnT.
  • the signal-to-noise ratio of the detection reagent can be significantly improved.
  • the second group of antibodies further includes:
  • one or more antibodies 4 each of which is independently selected from an antibody that specifically binds to cTnIC; and/or
  • the first group of antibodies includes one or more of the antibody 1 and one or more of the antibody 3; the second group of antibodies includes one or more of the antibody 2, and one or more of the antibody 4 and/or one or more of the antibody 5.
  • the first group of antibodies includes one antibody 1, and optionally one antibody 3; the second group of antibodies includes one antibody 2, and optionally one antibody 4 and/or one antibody 5.
  • 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 and optionally at least two antibodies 3; the second group of antibodies includes at least two antibodies 2 and optionally at least two antibodies 4 and/or at least two antibodies 5.
  • the combination of multiple antibodies that bind to the same target protein is useful for the detection of total cTnITC. Beneficial.
  • each of the antibodies 1 is independently selected from antibodies that specifically bind to amino acids 223-242 and 262-281 of cTnT. In some embodiments, the antibody 1 specifically binds to amino acids 223-242 of cTnT.
  • each of the antibodies 3 is independently selected from an antibody that specifically binds 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 3 is independently selected from an antibody that specifically binds to amino acids 119-138 or amino acids 132-151 of cTnT.
  • each of the antibodies 5 is independently selected from an antibody that specifically binds to amino acids 1-15, 13-22, 18-22, 18-28, 18-35, 22-31, 22-40, 23-29, 24-40, 25-40, 26-35, 34-37, 41-49, 83-89, 86-90, 87-90, 117-126, 130-145, 169-178, 186-192, 190-196, or 195-209 of cTnI.
  • each of the antibodies 5 is independently selected from an antibody that specifically binds to amino acids 22-40, amino acids 41-49, or amino acids 83-89 of cTnI.
  • each of the antibodies 5 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 total 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 detection kit Since the existing cTnITC detection kit has low sensitivity, 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, this application realizes the rapid detection of total cTnITC with high sensitivity and high specificity, which is conducive to a deeper understanding of the correlation between troponin and myocardial injury. Studies have shown that compared with cTnI and cTnT, total cTnITC It is more sensitive to myocardial injury and can serve 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 the preparation of a reagent for detecting a ternary complex of cardiac troponin.
  • 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 diagnosing myocardial injury in vitro, comprising the step of detecting the cardiac troponin ternary complex 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.
  • the specific detection of the ternary troponin complex cTnITC is conducive to a deeper understanding of the correlation between troponin and disease.
  • the total concentration change trend of the ternary troponin complex cTnITC is consistent with that of cTnT and cTnI, which has clinical value; the total concentration of cTnITC changes faster and is more sensitive to the patient's myocardial injury. It can be used as a more sensitive myocardial injury marker to help diagnose and prognose the disease.
  • the detection kit of the present application is used in combination with a fully automatic chemiluminescence analyzer to achieve automated, rapid and high-throughput detection of the ternary troponin complex cTnITC.
  • the detection 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 chemiluminescent immunoassay method to construct a detection kit for detecting the total ternary troponin complex cTnITC in the sample.
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • the detection antibodies are: Antibody 4: 20C6cc (specifically binds to the cTnIC complex epitope).
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • the detection antibody is: Antibody 5: 19C7cc (specifically binds to cTnI amino acid fragment 41-49).
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC); Antibody 4: 20C6cc (specific binding to the cTnIC complex epitope);
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • the detection antibodies are: Antibody 2: 7B9cc (specifically binds to TnC); Antibody 5: 19C7cc (specifically binds to cTnI amino acid fragment 41-49).
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC).
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to cTnT amino acid fragment 223-242).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC).
  • the capture antibody is: Antibody 1:155 (specifically binds to cTnT amino acid fragment 262-281).
  • the detection antibodies are: Antibody 2: 7B9cc (specifically binds to TnC); Antibody 4: Tcom8 (specifically binds to the cTnIC complex epitope).
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); Antibody 3: 406cc (specifically binds to the cTnT amino acid fragment 132-151).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC); Antibody 4: 20C6cc (specific binding to the cTnIC complex epitope);
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); Antibody 3: 300cc (specifically binds to the cTnT amino acid fragment 119-138).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC); Antibody 4: 20C6cc (specific binding to the cTnIC complex epitope);
  • the capture antibodies are: Antibody 1: 155 (specifically binds to the cTnT amino acid fragment 262-281); Antibody 3: 406cc (specifically binds to the cTnT amino acid fragment 132-151).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC); Antibody 4: 20C6cc (specific binding to the cTnIC complex epitope);
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); Antibody 3: 7G7 (specifically binds to the cTnT amino acid fragment 67-86).
  • the detection antibodies are: Antibody 2: 7B9cc (specific binding to TnC); Antibody 4: 20C6cc (specific binding to the cTnIC complex epitope);
  • the capture antibodies are: Antibody 1: 7E7 (specifically binds to the cTnT amino acid fragment 223-242); Antibody 3: 1C11cc (specifically binds to the cTnT amino acid fragment 171-190).
  • the detection antibodies are: Antibody 2: 7B9cc (specifically binds to TnC); Antibody 4: Tcom8 (specifically binds to the cTnIC complex epitope).
  • the experiment also used a kit in which the antibody 3 used in the total cTnITC detection kits 3-5 and 8-12 was replaced with the following antibodies: antibody 7F4 with a specific binding site for the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc with a specific binding site for the cTnT amino acid 145-164 fragment.
  • the experiment also used a kit in which the antibody 5 used in the total cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site for the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site for the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site for the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site for the cTnI amino acid 190-196 fragment.
  • antibody M18cc with a specific binding site for the cTnI amino acid 18-28 fragment
  • antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site for the cTnI amino acid 86-90 fragment
  • antibody M46 with a specific binding site for the cTnI amino acid 130-145 fragment
  • antibody MF4cc with a specific binding site for the
  • Total cTnITC Assay Kit includes:
  • Magnetic bead coating working solution used to achieve the capture of the ternary cTnITC complex 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 the ternary cTnITC complex antigen captured by superparamagnetic microparticles.
  • the enzyme marker working solution comprises: a detection antibody labeled with alkaline phosphatase.
  • the detection method is as follows:
  • Step 1 Add the sample, magnetic bead coating working solution, and enzyme labeling working solution to the reaction tube. After incubation, the ternary cTnITC complex in the sample binds to the antibody coated on the magnetic beads, and the antibody-alkaline phosphatase marker binds to the ternary cTnITC complex 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 the ternary cTnITC complex in the sample.
  • the amount of analyte in the sample is determined by the calibration curve.
  • the above-mentioned total cTnITC detection kit is used in conjunction with Mindray's fully automatic chemiluminescence analyzers CL2000i, CL6000i, CL8000i and other models.
  • samples containing different concentrations of antigen including two high-concentration samples (high-value sample 1, high-value sample 2) and two low-concentration samples (low-value sample 1, low-value sample 2), where the antigen is recombinant cardiac troponin ternary complex cTnITC (Hytest, 8ITCR).
  • the samples were analyzed using total cTnITC detection kits 1-12. At the same time, the blank sample signal without antigen was recorded and the signal-to-noise ratio was calculated.
  • the test results are shown in Figure 1.
  • the signal-to-noise ratio of detection kit 5 is higher than that of detection kit 6, indicating that the addition of antibody 3 that specifically binds to the amino acid fragment 67-222 of cTnT improves the signal-to-noise ratio.
  • 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 4 or antibody 5 can significantly improve the signal-to-noise ratio.
  • the signal-to-noise ratios of detection kits 3, 8 and 9 are similar, indicating that antibodies that specifically bind to different fragments of amino acids 67-222 of cTnT are selected as antibody 3, and the signal-to-noise ratios of the resulting kits are close.
  • the signal-to-noise ratio of detection kit 8 is significantly better than that of detection kit 10, indicating that the selection of an antibody that specifically binds to amino acids 223-242 of cTnT as antibody 1 has a better signal-to-noise ratio than the selection of an antibody that specifically binds to amino acids 262-281 of cTnT.
  • the signal-to-noise ratio of detection kit 3 was significantly higher than that of detection kits 11 and 12, indicating that the antibody specifically binding to cTnT positions 119-138 as antibody 3 is more advantageous than the antibody specifically binding to cTnT positions 67-86 or 171-190.
  • the antibody 3 used in the total cTnITC detection kits 3-5 and 8-12 was replaced with the following antibodies: antibody 7F4 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 7F5 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 7F6 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 7F7 whose specific binding site is the cTnT amino
  • the antibody 2F3, 1A11 or 1F11cc of the 145-164 fragment of cTnI was used, and the antibody 5 used in the total cTnITC detection kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site for the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site for the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site for the cTnI amino acid
  • cTnITC detection kits 1-9 were used for analysis by chemiluminescent immunoassay.
  • the analyzed antigens include: cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), cTnITC (Hytest, 8ITCR).
  • the experimental results are shown in Figure 2.
  • the detection kits 1-9 of the ternary troponin complex cTnITC can only recognize the cTnITC antigen, and cannot recognize cTnT, cTnI and binary cTnIC.
  • the antibody 3 used in the total cTnITC detection kits 3-5 and 8-12 is replaced with the following antibodies: antibody 7F4 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment, and the antibody 5 used in the total cTnITC detection kit 4 is replaced with the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which can effectively recognize the cTnITC antigen.
  • LoB test results were derived from 5 blank samples, run over 4 days, with each test repeated 4 times.
  • LoD test results were derived from 5 low-concentration samples, run over 4 days, with each test repeated 4 times.
  • Kits 1-9 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 5 were lower than those of detection kit 6, indicating that the addition of antibody 3 that specifically binds to cTnT amino acid fragment 119-138 improved the sensitivity.
  • 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 use of antibody 2 that specifically binds to TnC together with antibody 4 or antibody 5 significantly improved the sensitivity of the kit.
  • the antibody 3 used in the total cTnITC detection kits 3-5 and 8-12 is replaced by the following antibodies: antibody 7F4 whose specific binding site is the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc whose specific binding site is the cTnT amino acid 145-164 fragment, and the antibody 5 used in the total cTnITC detection kit 4 is replaced by the following antibodies: antibody M18cc whose specific binding site is the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc whose specific binding site is the cTnI amino acid 86-90 fragment, antibody M46 whose specific binding site is the cTnI amino acid 130-145 fragment, and antibody MF4cc whose specific binding site is the cTnI amino acid 190-196 fragment, all of which have 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.
  • the concentration range of the series samples was 0-120ng/L and 0-6000ng/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. Within the linear range, the R2 value of the low concentration range (0-120ng/L) is 0.9990, and the R2 value of the high concentration range (0-6000ng/L) is 0.9992.
  • 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. Within the linear range, the R2 value of the low concentration range (0-120ng/L) was 0.9979, and the R2 value of the high concentration range (0-6000ng/L) was 0.9988.
  • the capture antibody of detection kit 3 was exchanged with the detection antibody to construct detection kit 3a.
  • Detection Kit 3 includes:
  • Capture antibodies Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • Detection antibodies Antibody 2: 7B9cc (specifically binds to TnC); Antibody 4: 20C6cc (specifically binds to the cTnIC complex epitope).
  • Detection Kit 3a includes:
  • Antibody 2 7B9cc (specifically binds to TnC); Antibody 4: 20C6cc (specifically binds to the cTnIC complex epitope);
  • Detection antibodies Antibody 1: 7E7 (specifically binds to the amino acid fragment 223-242 of cTnT); Antibody 3: 329cc (specifically binds to the amino acid fragment 119-138 of cTnT).
  • test kits 3 and 3a to analyze the samples respectively. At the same time, record the signal of the blank sample without antigen and calculate the signal-to-noise ratio. The test results are shown in Figure 5, which shows that the signal-to-noise ratio decreases after the capture antibody and the detection antibody of test kit 3 are swapped.
  • test kits 3 and 3a Clinical serum samples of patients with cardiovascular diseases were analyzed using test kits 3 and 3a, respectively. At the same time, the signals of healthy human serum samples were recorded and the signal-to-noise ratio was calculated. The test results are shown in Figure 6.
  • the signal-to-noise ratio of test kit 3 was significantly higher than that of test kit 3a, indicating that the preferred capture antibodies and detection antibodies are: the capture antibodies are: antibody 1 (specifically binding to cTnT amino acid fragment 223-242) and antibody 3 (specifically binding to cTnT amino acid fragment 119-138), and the detection antibodies are: antibody 2 (specifically binding to TnC) and antibody 4 (specifically binding to cTnIC complex epitopes), and the signal-to-noise ratio of the kit is higher. Among them, the signal-to-noise ratio of test kit 3a is lower than that of test kit 3, and its signal difference change law in different samples is similar to that of test 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 ternary troponin complex cTnITC detection kit 3a was used for analysis by chemiluminescent immunoassay.
  • the analyzed antigens include: cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), cTnITC (Hytest, 8ITCR).
  • the experimental results are shown in Figure 7.
  • the detection kit 3a of the ternary troponin complex cTnITC can recognize the 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 overall sensitivity and specificity.
  • Total cTnITC Assay Kit 3 was used for clinical performance evaluation.
  • the results are shown in Figure 8.
  • the concentration value of total cTnITC is consistent with the change trend of the high-sensitivity cTnI and cTnT test values, indicating that the concentration value of total cTnITC has clinical value and can be used to evaluate the myocardial injury of patients.
  • the concentration change of total cTnITC was further analyzed. As shown in Figure 9, compared with cTnI and cTnT, the concentration value of total cTnITC changes faster, indicating that the concentration value change of total cTnITC is more sensitive to the myocardial injury of patients.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本申请涉及检测总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223-287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

检测总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，具体涉及用于检测总三元心肌肌钙蛋白复合物cTnITC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。检测肌钙蛋白三元复合物cTnITC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，难以实现三元复合物cTnITC总含量的快速、灵敏、特异性检测。因此，开发一种灵敏度高、检测速度快的检测试剂盒，用于检测血液中肌钙蛋白三元复合物cTnITC的总量(也即总cTnITC的含量)，对心血管疾病的诊断与预后指导尤为重要。
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心肌肌钙蛋白三元复合物cTnITC总含量的技术方案，通过捕获抗体和检测抗体的配合，实现对总cTnITC的特异性检测，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cTnITC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心肌肌钙蛋白三元复合物的组合物，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223-287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心肌肌钙蛋白三元复合物的试剂盒，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223-287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心肌肌钙蛋白三元复合物的方法，包括以下步骤：
获得待测样品；
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，以确定待测样品中心肌肌钙蛋白三元复合物的存在和/或总量；
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223-287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心肌肌钙蛋白三元复合物的试剂中的用途。
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心肌肌钙蛋白三元复合物的步骤。
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为对总cTnITC检测试剂盒信噪比分析。
图2为用血清样本验证总cTnITC检测试剂盒的特异性。
图3为对总cTnITC检测试剂盒3的线性分析。
图4为对总cTnITC检测试剂盒4的线性分析。
图5为对总cTnITC检测试剂盒3与试剂盒3a信噪比分析。
图6为对总cTnITC检测试剂盒3与试剂盒3a对临床血清样本的检测信噪比分析。
图7为用血清样本验证总cTnITC检测试剂盒3a的特异性。
图8为采用总cTnITC检测试剂盒检测的总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为采用总cTnITC检测试剂盒检测的总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
下面将结合本发明实施例中的附图，对本发明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一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，绝不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，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，都属于本发明保护的范围。
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。
如本文中所使用的，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。
如本文中所使用的，术语“总心肌肌钙蛋白三元复合物(total cardiac troponin ternary complex)”或“总cTnITC(total cTnITC)”在本文中可互换使用，意欲包括所有TnC的全长蛋白或片段、cTnI的全长蛋白或片段和cTnT的氨基酸残基223-287中的一段或多段，以及任选的cTnT的氨基酸残基1-222中的一段或多段所形成的复合物。
如本文中所使用的，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能够灵敏地检测样品中各种形式的心肌肌钙蛋白三元复合物cTnITC的总含量，且能特异地同cTn其它存在形式，包括cTnT、cTnI及cTnIC等进行区分。
具体地，在第一个方面，本申请提供一种用于检测样品中心肌肌钙蛋白三元复合物的组合物，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223-287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心肌肌钙蛋白三元复合物的试剂盒，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223-287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，总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心肌肌钙蛋白三元复合物的方法， 包括以下步骤：
获得待测样品；
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，以确定待测样品中心肌肌钙蛋白三元复合物的存在和/或总量；
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223-287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，所述第一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cTnT第67-222位氨基酸序列中任意一段的抗体。
在此，通过将所述抗体1与特异性识别cTnT的67-222位的抗体组合，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，所述第二组抗体还包括：
一个或多个抗体4，各所述抗体4独立地选自特异性结合cTnIC的抗体；和/或
一个或多个抗体5，各所述抗体5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一个或多个所述抗体1和一个或多个所述抗体3；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4和/或一个或多个所述抗体5。
在一些实施方案中，所述第一组抗体包括一个抗体1，以及任选的一个抗体3；所述第二组抗体包括一个抗体2以及任选的一个抗体4和/或一个抗体5。
在一些实施方案中，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以及任选的至少两个抗体3；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4和/或至少两个抗体5。多个结合相同靶蛋白的抗体的组合对于总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223-242位氨基酸、第262-281位氨基酸的抗体。在一些实施方案中，所述抗体1特异性结合cTnT第223-242位氨基酸的抗体。
在一些实施方案中，各所述抗体3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3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。
在一些实施方案中，各所述抗体5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5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5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，总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不同形式的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，本申请实现了对总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，总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心肌肌钙蛋白三元复合物的试剂中的用途。
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心肌肌钙蛋白三元复合物的步骤。
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)实现对总cTnITC的高敏感、高特异性的快速检测。本申请使用了特异性结合TnC的抗体作为捕获抗体或检测抗体，通过一系列实验表明，TnC抗体的使用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，三元肌钙蛋白复合物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。三元肌钙蛋白复合物cTnITC的总浓度变化趋势与cTnT、cTnI一致，具有临床价值；cTnITC的总浓度的变化率更快，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)本申请的检测试剂盒结合全自动化学发光仪使用，可以实现三元肌钙蛋白复合物cTnITC检测的自动化、快速和高通量。
4)本申请的检测试剂盒灵敏度高，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，用于检测样本中的总三元肌钙蛋白复合物cTnITC。
具体地，构建下述试剂盒用于后面的实验：
1.总cTnITC检测试剂盒1：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
检测抗体为：抗体4：20C6cc(特异性结合cTnIC复合物表位)。
2.总cTnITC检测试剂盒2：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
检测抗体为：抗体5：19C7cc(特异性结合cTnI氨基酸片段41-49)。
3.总cTnITC检测试剂盒3：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)；
4.总cTnITC检测试剂盒4：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体5：19C7cc(特异性结合cTnI氨基酸片段41-49)。
5.总cTnITC检测试剂盒5：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)。
6.总cTnITC检测试剂盒6：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)。
7.总cTnITC检测试剂盒7：
捕获抗体为：抗体1：155(特异性结合cTnT氨基酸片段262-281)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：Tcom8(特异性结合cTnIC复合物表位)。
8.总cTnITC检测试剂盒8：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：406cc(特异性结合cTnT氨基酸片段132-151片段)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)；
9.总cTnITC检测试剂盒9：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：300cc(特异性结合cTnT氨基酸片段119-138片段)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)；
10.总cTnITC检测试剂盒10：
捕获抗体为：抗体1：155(特异性结合cTnT氨基酸片段262-281)；抗体3：406cc(特异性结合cTnT氨基酸片段132-151片段)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)；
11.总cTnITC检测试剂盒11：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：7G7(特异性结合cTnT氨基酸片段67-86片段)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)；
12.总cTnITC检测试剂盒12：
捕获抗体为：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：1C11cc(特异性结合cTnT氨基酸片段171-190片段)。
检测抗体为：抗体2：7B9cc(特异性结合TnC)；抗体4：Tcom8(特异性结合cTnIC复合物表位)。
实验中也使用了将所述总cTnITC检测试剂盒3-5、8-12中所使用的抗体3替换为下述抗体的试剂盒：特异性结合位点为cTnT氨基酸67-86片段的抗体7F4、特异性结合位点为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总cTnITC检测试剂盒4中所使用的抗体5替换为下述抗体的试剂盒：特异性结合位点为cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点为cTnI氨基酸86-90片段的抗体16A11cc、16A12cc或8E10cc、特异性结合位点为cTnI氨基酸130-145片段的抗体M46、特异性结合位点为cTnI氨基酸190-196片段的抗体MF4cc。
总cTnITC检测试剂盒包括：
A.磁珠包被物工作液，用于实现对样本中三元cTnITC复合物的捕获。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三元cTnITC复合物抗原的检测。 所述酶标记物工作液包括：碱性磷酸酶标记的检测抗体。
检测方法如下：
第一步：将样本与磁珠包被物工作液、酶标记工作液添加到反应管中，经过孵育，样本中的三元cTnITC复合物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三元cTnITC复合物结合。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三元cTnITC复合物的浓度成正比。样本内分析物的量由校准曲线来确定。
上述总cTnITC检测试剂盒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 总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1、高值样本2)和两个低浓度样本(低值样本1、低值样本2)，其中抗原为重组心肌肌钙蛋白三元复合物cTnITC(Hytest，8ITCR)。使用总cTnITC检测试剂盒1-12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5的信噪比高于检测试剂盒6，表明特异性结合cTnT氨基酸片段67-222的抗体3的加入提升了信噪比。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4或抗体5共同使用能够显著提升信噪比。检测试剂盒3、8和9信噪比相似，表明选择特异性结合cTnT第67-222位氨基酸不同片段的抗体作为抗体3，所得试剂盒信噪比接近。检测试剂盒8的信噪比显著优于检测试剂盒10，表明选择特异性结合cTnT第223-242位氨基酸的抗体作为抗体1比选择特异性结合cTnT第262-281位氨基酸的抗体具有更好的信噪比。检测试剂盒3的信噪比显著高于检测试剂盒11和12，表明特异性结合cTnT第119-138的抗体作为抗体3相较于特异性结合cTnT第67-86位或第171-190位的抗体是更有优势的。
此外，将所述总cTnITC检测试剂盒3-5、8-12中所使用的抗体3替换为下述抗体：特异性结合位点为cTnT氨基酸67-86片段的抗体7F4、特异性结合位点为cTnT氨基酸 145-164片段的抗体2F3、1A11或1F11cc，将所述总cTnITC检测试剂盒4中所使用的抗体5替换为下述抗体：特异性结合位点为cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点为cTnI氨基酸86-90片段的抗体16A11cc、16A12cc或8E10cc、特异性结合位点为cTnI氨基酸130-145片段的抗体M46、特异性结合位点为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。本文抗体购自海肽生物科技有限公司。
实施例3 总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，使用总cTnITC检测试剂盒1-9分别通过化学发光免疫分析方法进行分析。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。三元肌钙蛋白复合物cTnITC的检测试剂盒1-9均仅能识别cTnITC抗原，不能识别cTnT、cTnI及二元cTnIC。
将所述总cTnITC检测试剂盒3-5、8-12中所使用的抗体3替换为下述抗体：特异性结合位点为cTnT氨基酸67-86片段的抗体7F4、特异性结合位点为cTnT氨基酸145-164片段的抗体2F3、1A11或1F11cc，将所述总cTnITC检测试剂盒4中所使用的抗体5替换为下述抗体：特异性结合位点为cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点为cTnI氨基酸86-90片段的抗体16A11cc、16A12cc或8E10cc、特异性结合位点为cTnI氨基酸130-145片段的抗体M46、特异性结合位点为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 总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1-9建立空白限和检出限，测试结果见表1。
表1：试剂盒的空白限(LoB)与检出限(LoD)

其中检测试剂盒5的空白限与检出限低于检测试剂盒6，表明特异性结合cTnT氨基酸片段119-138的抗体3的加入提升了灵敏度。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，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与抗体4或抗体5共同使用显著提升了试剂盒的灵敏度。
除以上试剂盒外，将所述总cTnITC检测试剂盒3-5、8-12中所使用的抗体3替换为下述抗体：特异性结合位点为cTnT氨基酸67-86片段的抗体7F4、特异性结合位点为cTnT氨基酸145-164片段的抗体2F3、1A11或1F11cc，将所述总cTnITC检测试剂盒4中所使用的抗体5替换为下述抗体：特异性结合位点为cTnI氨基酸18-28片段的抗体M18cc、特异性结合位点为cTnI氨基酸86-90片段的抗体16A11cc、16A12cc或8E10cc、特异性结合位点为cTnI氨基酸130-145片段的抗体M46、特异性结合位点为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 总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。
选取浓度不同的两个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，系列样本的浓度范围为0-120ng/L和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，低浓度范围(0-120ng/L)的R2值为0.9990，高浓度范围(0-6000ng/L)的R2值为0.9992。
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，低浓度范围(0-120ng/L)的R2值为0.9979，高浓度范围(0-6000ng/L)的R2值为0.9988。
实施例6 交换捕获抗体与检测抗体的试剂盒构建与测试
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：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
检测抗体：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)。
检测试剂盒3a包括：
捕获抗体：抗体2：7B9cc(特异性结合TnC)；抗体4：20C6cc(特异性结合cTnIC复合物表位)；
检测抗体：抗体1：7E7(特异性结合cTnT氨基酸片段223-242)；抗体3：329cc(特异性结合cTnT氨基酸片段119-138)。
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，说明检测试剂盒3捕获抗体和检测抗体对调后，信噪比降低。
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为：捕获抗体为：抗体1(特异性结合cTnT氨基酸片段223-242)和抗体3(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4(特异性结合cTnIC复合物表位)，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的近似。
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，使用三元肌钙蛋白复合物cTnITC检测试剂盒3a通过化学发光免疫分析方法进行分析。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。三元肌钙蛋白复合物cTnITC的检测试剂盒3a能识别cTnITC抗原，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在整体灵敏度和特异性上的表现更优选。
实施例7 总cTnITC特异性免疫分析的临床性能评估
总cTnITC检测试剂盒3被用于临床性能评估。
为评估三元肌钙蛋白复合物cTnITC夹心免疫测定法的临床性能，对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总cTnITC的浓度值。
结果如图8所示，总cTnITC的浓度值与高敏cTnI、cTnT测试值的变化趋势一致，表明总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。进一步对总cTnITC的浓度变化情况进行分析，如图9所示，相比于cTnI和cTnT，总cTnITC的浓度值变化速率更快，表明总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 (21)

  1. 一种用于检测样品中心肌肌钙蛋白三元复合物的组合物，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223-287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cTnT第67-222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4，各所述抗体4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5，各所述抗体5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。
  4. 权利要求3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和一个或多个所述抗体3；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4和/或一个或多个所述抗体5。
  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223-242位氨基酸或第262-281位氨基酸的抗体；优选特异性结合cTnT第223-242位氨基酸的抗体。
  6. 权利要求2-5任一项所述的组合物，其中各所述抗体3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3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。
  7. 权利要求3-6任一项所述的组合物，其中各所述抗体5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5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5独立地选自特异性结合cTnI第41-49位氨基酸的抗体。
  8. 一种用于检测样品中心肌肌钙蛋白三元复合物的试剂盒，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223-287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。
  9. 权利要求8所述的试剂盒，其中所述第一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cTnT第67-222位氨基酸序列中任意一段的抗体。
  10. 权利要求8或9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4，各所述抗体4独立地选自特异性结合cTnIC的抗体；和/或
    一个或多个抗体5，各所述抗体5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。
  11. 权利要求8-10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和一个或多个所述抗体3；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4和/或一个或多个所述抗体5。
  12. 权利要求8-11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。
  13. 权利要求8-12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223-242位氨基酸或第262-281位氨基酸的抗体，优选特异性结合cTnT第223-242位氨基酸的抗体；和/或
    各所述抗体3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3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；和/或
    各所述抗体5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5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5独立地选自特异性结合cTnI第41-49位氨基酸的抗体。
  14. 一种在体外检测样品中心肌肌钙蛋白三元复合物的方法，包括以下步骤：
    获得待测样品；
    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，以确定待测样品中心肌肌钙蛋白三元复合物的存在和/或总量；
    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223-287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。
  15. 权利要求14所述的方法，其中所述第一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cTnT第67-222位氨基酸序列中任意一段的抗体。
  16. 权利要求14或15所述的方法，其中所述第二组抗体还包括：
    一个或多个抗体4，各所述抗体4独立地选自特异性结合cTnIC的抗体；和/或
    一个或多个抗体5，各所述抗体5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。
  17. 权利要求14-16任一项所述的方法，其中所述第一组抗体包括一个或多个所述抗体1和一个或多个所述抗体3；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4和/或一个或多个所述抗体5。
  18. 权利要求14-17任一项所述的方法，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。
  19. 权利要求1-7任一项所述的组合物在制备用于检测总心肌肌钙蛋白三元复合物的试剂中的用途。
  20. 权利要求1-7任一项所述的组合物、权利要求8-13任一项所述的试剂盒或权利要求14-18任一项所述的方法在心肌损伤体外诊断中的用途。
  21. 一种在体外诊断心肌损伤的方法，其包括采用权利要求1-7任一项所述的组合物、权利要8-13任一项所述的试剂盒或权利要求14-18任一项所述的方法检测来自受试者的样品中总心肌肌钙蛋白三元复合物的步骤。
PCT/CN2023/112993 2023-08-14 2023-08-14 检测总cTnITC的组合物、试剂盒和方法、及其应用 Pending WO2025035370A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2023/112993 WO2025035370A1 (zh) 2023-08-14 2023-08-14 检测总cTnITC的组合物、试剂盒和方法、及其应用
CN202380101490.1A CN121794576A (zh) 2023-08-14 2023-08-14 检测总cTnITC的组合物、试剂盒和方法、及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/112993 WO2025035370A1 (zh) 2023-08-14 2023-08-14 检测总cTnITC的组合物、试剂盒和方法、及其应用

Publications (1)

Publication Number Publication Date
WO2025035370A1 true WO2025035370A1 (zh) 2025-02-20

Family

ID=94631919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/112993 Pending WO2025035370A1 (zh) 2023-08-14 2023-08-14 检测总cTnITC的组合物、试剂盒和方法、及其应用

Country Status (2)

Country Link
CN (1) CN121794576A (zh)
WO (1) WO2025035370A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5795725A (en) * 1995-04-18 1998-08-18 Biosite Diagnostics Incorporated Methods for the assay of troponin I and T and selection of antibodies for use in immunoassays
US6156521A (en) * 1997-12-19 2000-12-05 Biosite Diagnostics, Inc. Methods for the recovery and measurement of troponin complexes
US6174686B1 (en) * 1995-04-18 2001-01-16 Biosite Diagnostics, Inc. Methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays
US20040219604A1 (en) * 2003-04-30 2004-11-04 Susann Eriksson Immunoassay
US20050164317A1 (en) * 1995-04-18 2005-07-28 Biosite, Inc. Novel methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays
US20090233268A1 (en) * 2008-03-05 2009-09-17 Axela Inc. Detection of biomarkers and biomarker complexes
CN114994304A (zh) * 2022-05-18 2022-09-02 厦门宝太生物科技股份有限公司 一种cTnI干式均相化学发光检测试剂盒

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5795725A (en) * 1995-04-18 1998-08-18 Biosite Diagnostics Incorporated Methods for the assay of troponin I and T and selection of antibodies for use in immunoassays
US6174686B1 (en) * 1995-04-18 2001-01-16 Biosite Diagnostics, Inc. Methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays
US20050164317A1 (en) * 1995-04-18 2005-07-28 Biosite, Inc. Novel methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays
US6156521A (en) * 1997-12-19 2000-12-05 Biosite Diagnostics, Inc. Methods for the recovery and measurement of troponin complexes
US20040219604A1 (en) * 2003-04-30 2004-11-04 Susann Eriksson Immunoassay
US20090233268A1 (en) * 2008-03-05 2009-09-17 Axela Inc. Detection of biomarkers and biomarker complexes
CN114994304A (zh) * 2022-05-18 2022-09-02 厦门宝太生物科技股份有限公司 一种cTnI干式均相化学发光检测试剂盒

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"UniProt", Database accession no. P63316
ANDERSON, CIRCULATION RESEARCH, vol. 76, no. 4, 1995, pages 681 - 686
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
FERRIERES, CLINICAL CHEMISTRY, vol. 44, 1998, pages 487 - 493
HARLOWLANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS
HARLOWLANE: "Using Antibodies: A Laboratory Manual", 1999, COLD SPRING HARBOR LABORATORY PRESS

Also Published As

Publication number Publication date
CN121794576A (zh) 2026-04-03

Similar Documents

Publication Publication Date Title
JP6452718B2 (ja) 食物アレルギーを検出するためのペプチド、試薬及び方法
CN104374919B (zh) 糖蛋白的测定方法、试剂及糖链标记物
JP5425063B2 (ja) ＩｇＡ腎症の検査方法及び検出キット
WO2012115221A1 (ja) 心筋トロポニンの測定法
US20200333337A1 (en) Method for re-using test probe and reagents in an immunoassay
TW201643429A (zh) 前列腺抗原標準品及其用途
US20250130237A1 (en) Point of Care Assays
KR101495225B1 (ko) Ast 양의 측정을 통한 간 질환 진단, 예후 또는 모니터링 키트 및 방법
US9835621B2 (en) Process for detection of alzheimer's disease from a serum sample
WO2025035370A1 (zh) 检测总cTnITC的组合物、试剂盒和方法、及其应用
WO2025035371A1 (zh) 检测大尺寸cTnITC的组合物、试剂盒和方法、及其应用
JP7315965B2 (ja) ウィルス性肝癌の検出方法
JP7597580B2 (ja) 免疫アッセイにおける干渉の低減
WO2017170847A1 (ja) 糖尿病の罹患を測定する方法
AU2024283890A1 (en) Method and system for the diagnosis of inflammatory bowel disease (ibd)
WO2023068249A1 (ja) Ｉ型コラーゲン架橋ｎ－テロペプチドの測定試薬、その調製方法、及びそれを用いた免疫測定方法
OA19228A (en) Point of care assays
JPH04326062A (ja) ＩｇＡ測定用試薬

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23948805

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023948805

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE