WO2023061388A1 - 半乳糖凝集素-3的免疫测定 - Google Patents

半乳糖凝集素-3的免疫测定 Download PDF

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WO2023061388A1
WO2023061388A1 PCT/CN2022/124770 CN2022124770W WO2023061388A1 WO 2023061388 A1 WO2023061388 A1 WO 2023061388A1 CN 2022124770 W CN2022124770 W CN 2022124770W WO 2023061388 A1 WO2023061388 A1 WO 2023061388A1
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antibody
seq
antigen
binding portion
heart failure
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PCT/CN2022/124770
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English (en)
French (fr)
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林潮喜
张君
何丹
温志刚
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深圳市睿盟创新生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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 generally relates to the field of biological detection, and specifically, the present application provides antibodies or antigen-binding parts thereof and antibody combination products for binding to human galectin-3.
  • the present application also provides a kit for detecting the content of galectin-3 in a sample from a human subject and use thereof.
  • Galectin-3 (Gal-3) is a galactose-binding protein with a special amino acid sequence, widely present in animals from low to high. Galectins are a large family of proteins. Existing studies have confirmed 15 members of this protein family. According to their structural differences and the number of sugar recognition domains conserved in their polypeptide chains, they are classified as Divided into three groups: (1) prototype galectins (Galectin-1, 2, 5, 7, 10, 11, 13, 14 and 15), which contain a single CRD; (2) tandem repeat galectins (Galectin-4, 6, 8, 9, and 12), which connect two CRDs by a junction region; (3) chimeric galectin (Galectin-3), which contains a CRD and is connected to an extended N-terminal domain of the non-lectin.
  • Galectin-3 is one of the most studied members of the galectin family.
  • the human Galectin-3 gene is located on chromosome 14q21-22, with a total length of about 17kb, containing 251 amino acid residues, a molecular weight of about 29.31KD, and 6 exons and 5 introns.
  • Human Galectin-3 has three structural domains: (1) 1-12 amino acid NH2-(amino) terminal, which determines cell localization, and is also necessary for the highly conserved ser6 residue to participate in the anti-apoptotic activity of Galectin-3; ( 2) Collagen repeat sequence rich in glycine, tyrosine and proline, which functions as the substrate of matrix metalloproteinases (Matrix Metalloproteinases, MMPs); The -(carboxy) terminal can interact with extracellular matrix and glycoproteins.
  • MMPs matrix metalloproteinases
  • Galectin-3 is widely distributed in tumor cells, epithelial cells, fibroblasts, macrophages and other inflammatory cells, and is also expressed in a small amount in the heart, kidney, liver, brain, and pancreas. Galectin-3 is expressed in the cytoplasm, nucleus and It is expressed on the cell surface, mainly in the cytoplasm. The biological function of Galectin-3 is related to intracellular localization. It is mainly located in the cytoplasm when the cell is in a resting state, and it is mainly distributed in the nucleus when it is in a proliferating state. The secretion of Galectin-3 does not depend on the endoplasmic reticulum and Golgi apparatus. It is transferred to the outside of the cell through a non-classical mechanism similar to cell exocytosis. Soluble Galectin-3 can enter the systemic circulation.
  • Galectin-3 is the substrate of MMPs. After MMP-2 or MMP-9 decomposes Galectin-3, a 22kU sugar recognition domain and a 9kU amino acid residue fragment are generated. Galectin-3 is more likely to play a role in the process of tumor metastasis and invasion. It has been reported in the literature that in the study of breast cancer, the decomposed Galectin-3 activates PKC or induces GTPase, up-regulates the pFAK pathway, enhances the chemotaxis of vascular endothelial cells, and promotes the migration and chemotaxis of vascular endothelial cells, thereby Promote the formation of new blood vessels.
  • Galectin-3 participates in many physiological and pathological processes in vivo, including cell growth, cell adhesion, inflammatory response, immune regulation, apoptosis, and tumor transformation and metastasis. Expression is significantly increased in many cancers such as melanoma, lung cancer, breast cancer, colorectal cancer, head and neck cancer and non-Hodgkin's lymphoma. Galectin-3 can participate in the formation of fibrosis in kidney, lung, liver and other organs. Galectin-3 is closely related to rheumatic immune diseases and bronchial asthma. Galectin-3 is a pro-inflammatory signal factor, expressed in a variety of inflammatory factors, involved in macrophage phagocytosis, cell adhesion, etc.
  • Heart failure is the most researched disease related to Galectin-3.
  • Heart failure is a serious manifestation or late stage of various heart diseases. The incidence rate is increasing, and the mortality rate and rehospitalization rate remain high , is one of the major cardiovascular diseases.
  • Heart failure is a complex clinical syndrome involving various systems of the body, and its pathophysiological mechanism mainly involves hemodynamic disturbance and abnormal activation of the neuroendocrine system.
  • Galectin-3 (Gal-3) promotes myocardial fibrosis and participates in myocardial inflammatory changes and process of ventricular remodeling.
  • the expression level of Galectin-3 in heart failure patients continues to increase, which can be used as an index for evaluating heart failure.
  • Myocardial fibrosis is mainly caused by excessive accumulation of collagen fibers in the myocardium, which can cause ventricular remodeling.
  • the expression of Galectin-3 was significantly increased in fibrotic tissues (heart, liver fibrosis models). Galectin-3 can reflect the degree of fibrosis.
  • galectin-3 in body fluids such as blood or serum can reflect the disease status of the human body, such as chronic heart failure, etc., so accurate quantification of galectin-3 can be used to identify or predict diseases, and to evaluate the severity of diseases Clinical or scientific research needs such as severity, staging or prediction of disease consequences, or monitoring of drug effects.
  • Galectin-3 is closely related to 14 other mammalian galectins, especially in the conserved A high degree of sequence similarity is shared in the carbohydrate recognition domain (CRD), and galectin-3 is a substrate for, and cleaved by, MMPs.
  • Another factor hindering the development of specific, reproducible detection assays for Galectin-3 is the propensity of Galectin-3 to bind different proteins, carbohydrates, nucleic acids and lipids.
  • the present application provides an antibody or an antigen-binding portion thereof, which is capable of binding at least a part of the amino acid sequence of human galectin-3, the human galectin-3 At least a portion of the amino acid sequence comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: Amino acid sequence shown in ID NO:10:
  • MLITILGTVKPN positions 1 to 12 of SEQ ID NO:1; SEQ ID NO:3
  • VAFHFNPRFN 26th to 35th of SEQ ID NO:1; SEQ ID NO:6
  • TKLDNNW 45th to 62nd of SEQ ID NO:1; SEQ ID NO:7)
  • GREERQSVFPFESGK (63rd to 78th of SEQ ID NO:1; SEQ ID NO:8)
  • VFPFESGKPFKIQVLVEP 70th to 88th of SEQ ID NO:1; SEQ ID NO:9
  • DHFKVAVNDAHL (89th to 100th of SEQ ID NO: 1; SEQ ID NO: 10).
  • At least a portion of the amino acid sequence of human galectin-3 is located at the C-terminal part of human galectin-3, wherein the C-terminal part comprises the amino acid shown in SEQ ID NO: 1 sequence:
  • the antibody comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO:17 and LCDR3 shown in SEQ ID NO:18.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO: 13 to SEQ ID NO: 18.
  • the antibody comprises HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:21, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO: 19 to SEQ ID NO: 24.
  • the antigen binding portion is a Fab, F(ab)' 2 or single chain Fv.
  • the antibody is a monoclonal antibody.
  • the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.
  • the antibodies, or antigen-binding portions thereof are useful for diagnosing heart failure, but are not limited to such use.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the antibody or antigen-binding portion thereof can be used to assist in the diagnosis of tumors, rheumatoid arthritis, or chronic kidney disease, but is not limited thereto.
  • the present application provides an antibody or an antigen-binding portion thereof, which is capable of binding at least a part of the amino acid sequence of human galectin-3, the human galectin-3 At least a portion of comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12:
  • MADNFSLHDALSGSGNPNPQGWPG positions 1 to 24 of SEQ ID NO:2; SEQ ID NO:11
  • AWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG positions 25 to 56 of SEQ ID NO: 2; SEQ ID NO: 12).
  • At least a portion of the amino acid sequence of human galectin-3 is located at the N-terminal portion of human galectin-3, wherein the N-terminal portion comprises the amino acid shown in SEQ ID NO:2 sequence:
  • the antibody comprises HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29 and LCDR3 shown in SEQ ID NO:30.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO:25 to SEQ ID NO:30.
  • the antibody comprises HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:33, LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35 and LCDR3 shown in SEQ ID NO:36.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO: 31 to SEQ ID NO: 36.
  • the antigen binding portion is a Fab, F(ab)' 2 or single chain Fv.
  • the antibody is a monoclonal antibody.
  • the antibody is a mouse monoclonal antibody or a rabbit monoclonal antibody.
  • the antibodies, or antigen-binding portions thereof are useful for diagnosing heart failure, but are not limited to such use.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the antibody or antigen-binding portion thereof can be used to assist in the diagnosis of tumors, rheumatoid arthritis, or chronic kidney disease, but is not limited thereto.
  • the present application provides an antibody combination product, which comprises the antibody or antigen-binding portion thereof described in the first aspect and the antibody or antigen-binding portion thereof described in the second aspect.
  • the present application provides a kit for detecting the content of galectin-3 in a sample from a human subject, which comprises the antibody or antigen-binding portion thereof described in the first aspect or the antibody or antigen-binding portion thereof described in the second aspect.
  • the detectable label includes an enzyme or a chromogenic substance.
  • the enzyme is alkaline phosphatase or horseradish peroxidase.
  • the chromogenic substance is a chemiluminescent compound or a fluorescent substance.
  • the chemiluminescent compound is ruthenium terpyridine or an acridinium ester.
  • the fluorescent substances are fluorescent pigments, fluorescent microspheres, time-resolved fluorescent microspheres, or quantum dots.
  • the sample is derived from whole blood, serum or plasma.
  • antibodies that are not labeled with a detectable label are pre-attached to the solid surface.
  • the solid surface is selected from nitrocellulose membranes, magnetic particles, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, plastic, and glass.
  • the kit further comprises standards and/or quality controls.
  • the kit further comprises instructions.
  • kits are useful for diagnosing heart failure, but are not limited to such use.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the antibody or its antigen-binding portion can be used to assist in the diagnosis of tumors, rheumatoid arthritis, and chronic kidney disease, but not limited thereto.
  • the present application provides the antibody or its antigen-binding portion described in the first aspect or the antibody or its antigen-binding portion described in the second aspect or the antibody combination product described in the third aspect in the preparation of human samples for detection Use in a Galectin-3 Assay Kit.
  • the sample is derived from whole blood, serum or plasma.
  • the present application provides a method for detecting the content of human galectin-3 in a sample, the method comprising using the antibody or antigen-binding portion thereof described in the first aspect or the second aspect or the third aspect The antibody combination product;
  • the antibody or antigen-binding portion thereof described in the first aspect is used to capture human galectin-3 and the antibody or antigen-binding portion thereof described in the first aspect is labeled with a detectable label, or the antibody or antigen-binding portion thereof described in the second aspect The antibody or antigen-binding portion thereof is used to capture human galectin-3 and the antibody or antigen-binding portion thereof of the first aspect is labeled with a detectable label;
  • the antibody or its antigen-binding part is used to detect the content of human galectin-3 by immunological method.
  • the sample is derived from whole blood, serum or plasma.
  • the immunological method is selected from enzyme-linked immunoassay, immunofluorescence, chemiluminescence, immunochromatography, immunoprecipitation, and combinations thereof.
  • the immunological method is an enzyme-linked immunoassay.
  • the present application provides the use of the antibody or its antigen-binding part described in the first aspect or the second aspect, or the antibody combination product described in the third aspect in detecting the content of human galectin-3 in a sample .
  • the sample is derived from whole blood, serum or plasma.
  • the present application provides a method for diagnosing heart failure in an individual or assisting in the diagnosis of tumors, rheumatoid arthritis, or chronic kidney disease, the method comprising: obtaining a biological sample from an individual; using the reagent described in the fourth aspect
  • the kit detects the content of human galectin-3 in the biological sample; and compares the detected content of human galectin-3 with a reference value.
  • the biological sample is derived from whole blood, serum or plasma.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the present application provides the antibody or its antigen-binding part described in the first aspect or the antibody combination product described in the third aspect for use in the diagnosis of heart failure or auxiliary diagnosis of tumors, rheumatoid arthritis or chronic kidney disease. Use in kits for diseases.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • this application includes the following embodiments:
  • Embodiment 1 An antibody or antigen-binding portion thereof capable of binding at least a portion of the amino acid sequence of human galectin-3, at least a portion of the amino acid sequence of human galectin-3 Comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 amino acid sequence.
  • Embodiment 2 The antibody, or antigen-binding portion thereof, of embodiment 1, wherein at least a portion of the amino acid sequence of human galectin-3 is located at the C-terminal portion of human galectin-3, wherein the The C-terminal portion comprises the amino acid sequence shown in SEQ ID NO:1.
  • Embodiment 3 The antibody or antigen-binding portion thereof according to embodiment 1, wherein the antibody comprises HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, and HCDR2 shown in SEQ ID NO:15 HCDR3, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; or
  • the antibody comprises HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:21, LCDR1 shown in SEQ ID NO:22, and HCDR1 shown in SEQ ID NO:23 LCDR2 shown and LCDR3 shown in SEQ ID NO:24.
  • Embodiment 4 An antibody or antigen-binding portion thereof capable of binding at least a portion of the amino acid sequence of human Galectin-3, said at least a portion of human Galectin-3 comprising SEQ ID The amino acid sequence shown in NO:11 or SEQ ID NO:12.
  • Embodiment 5 The antibody or antigen-binding portion thereof of embodiment 4, wherein at least a portion of the amino acid sequence of human galectin-3 is located at the N-terminal portion of human galectin-3, wherein the The N-terminal portion comprises the amino acid sequence shown in SEQ ID NO:2.
  • Embodiment 6 The antibody or antigen-binding portion thereof according to embodiment 4, wherein the antibody comprises HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, and HCDR2 shown in SEQ ID NO:27 HCDR3, LCDR1 set forth in SEQ ID NO:28, LCDR2 set forth in SEQ ID NO:29, and LCDR3 set forth in SEQ ID NO:30; or
  • the antibody comprises HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:33, LCDR1 shown in SEQ ID NO:34, and HCDR1 shown in SEQ ID NO:35 LCDR2 shown and LCDR3 shown in SEQ ID NO:36.
  • Embodiment 7 The antibody, or antigen binding portion thereof, of any one of embodiments 1 to 6, wherein said antigen binding portion is a Fab, F(ab)'2 or single chain Fv.
  • Embodiment 8 The antibody, or antigen-binding portion thereof, of any one of embodiments 1 to 6, wherein said antibody is a monoclonal antibody.
  • Embodiment 9 The antibody, or antigen-binding portion thereof, of embodiment 8, wherein said antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.
  • Embodiment 10 The antibody or antigen-binding portion thereof according to any one of embodiments 1 to 9, which is useful for diagnosing heart failure, or for aiding in the diagnosis of tumors, rheumatoid arthritis or chronic kidney disease.
  • Embodiment 11 The antibody or antigen-binding portion thereof of embodiment 10, wherein diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • Embodiment 12 An antibody combination comprising the antibody, or antigen-binding portion thereof, of any one of embodiments 1-3 and the antibody, or antigen-binding portion thereof, of any one of embodiments 4-6.
  • Embodiment 13 A kit for detecting the amount of Galectin-3 in a sample from a human subject comprising the antibody or antigen binding portion or embodiment thereof of any one of embodiments 1 to 11 12.
  • Embodiment 14 The kit of embodiment 13, wherein the detectable label comprises an enzyme or a chromogenic substance.
  • Embodiment 15 The kit of embodiment 14, wherein the enzyme is alkaline phosphatase or horseradish peroxidase.
  • Embodiment 16 The kit of embodiment 14, wherein the chromogenic substance is a chemiluminescent compound or a fluorescent substance.
  • Embodiment 17 The kit of Embodiment 16, wherein the chemiluminescent compound is ruthenium terpyridine or an acridinium ester.
  • Embodiment 18 The kit according to embodiment 16, wherein the fluorescent substance is a fluorescent pigment, fluorescent microspheres, time-resolved fluorescent microspheres or quantum dots.
  • Embodiment 19 The kit of embodiment 13, wherein the sample is derived from whole blood, serum or plasma.
  • Embodiment 20 The kit of Embodiment 13, wherein the antibody not labeled with a detectable label is pre-attached to the solid surface.
  • Embodiment 21 The kit of Embodiment 20, wherein the solid surface is selected from the group consisting of nitrocellulose membranes, magnetic particles, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, plastic, and glass.
  • Embodiment 22 The kit of embodiment 13, wherein the kit further comprises standards and/or quality controls.
  • Embodiment 23 The kit of embodiment 13, wherein the kit further comprises instructions.
  • Embodiment 24 The antibody or antigen-binding portion thereof according to any one of embodiments 1-11 or the antibody combination product described in embodiment 12 in the preparation of a kit for detecting the content of human galectin-3 in a sample the use of.
  • Embodiment 25 The use according to embodiment 24, wherein the sample is derived from whole blood, serum or plasma.
  • Embodiment 26 A method for detecting the amount of human galectin-3 in a sample comprising the use of the antibody or antigen-binding portion thereof of any one of embodiments 1-11 or of embodiment 12 antibody combination products;
  • the antibody or antigen-binding portion thereof according to any one of Embodiments 1 to 3 is used to capture human galectin-3 and the antibody or antigen-binding portion thereof according to any one of Embodiments 4 to 6 can be used
  • the label for detection is labeled, or the antibody or antigen-binding portion thereof of any one of embodiments 4 to 6 is used to capture human galectin-3 and the antibody of any one of embodiments 1 to 3 or
  • the antigen-binding portion thereof is labeled with a detectable label;
  • the antibody or its antigen-binding part is used to detect the content of human galectin-3 by immunological method.
  • Embodiment 27 The method of embodiment 26, wherein the sample is derived from whole blood, serum or plasma.
  • Embodiment 28 The method of embodiment 26, wherein the immunological method is selected from the group consisting of enzyme-linked immunoassay, immunofluorescence, chemiluminescence, immunochromatography, immunoprecipitation, and combinations thereof.
  • Embodiment 29 Use of the antibody or antigen-binding portion thereof of any one of embodiments 1-11, or the antibody combination of embodiment 12, for detecting the amount of human galectin-3 in a sample.
  • Embodiment 30 The use of embodiment 29, wherein the sample is derived from whole blood, serum or plasma.
  • Embodiment 31 A method for diagnosing heart failure in an individual or aiding in the diagnosis of tumors, rheumatoid arthritis or chronic kidney disease, said method comprising:
  • the detected human galectin-3 content was compared with the reference value.
  • Embodiment 32 The method of embodiment 31, wherein the biological sample is derived from whole blood, serum or plasma.
  • Embodiment 33 The method of embodiment 31, wherein diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • Embodiment 34 The antibody or antigen-binding portion thereof according to any one of embodiments 1-11 or the antibody combination product described in embodiment 12 is used in the preparation of diagnosis of heart failure or auxiliary diagnosis of tumors, rheumatoid arthritis or Use in a kit for chronic kidney disease.
  • Embodiment 35 The use of embodiment 34, wherein diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • Fig. 1 shows a schematic diagram of obtaining an immunoglobulin antibody that specifically recognizes galectin-3 by using B cell in vitro cloning technology.
  • Figure 2 is a schematic diagram of the test strip/kit, where 1: sample pad; 2: binding pad; 3: detection line (T line); 4: quality control line (C line); 5: NC membrane (nitrocellulose membrane) ; 6: Absorbent pad; 7: PVC bottom plate.
  • SEQ ID NO:3 position 1 to 12 of SEQ ID NO:1: MLITILGTVKPN
  • SEQ ID NO:4 (1st to 14th of SEQ ID NO:1): MLITILGTVKPNAN
  • SEQ ID NO:5 (15th to 25th of SEQ ID NO:1): IALDFQRGND
  • SEQ ID NO:8 (63rd to 78th of SEQ ID NO:1): GREERQSVFPFESGK
  • SEQ ID NO:10 (89th to 100th of SEQ ID NO:1): DHFKVAVNDAHL
  • SEQ ID NO:12 (the 25th to the 56th of SEQ ID NO:2)
  • SEQ ID NO: 13 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 14 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 15 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 16 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 17 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 18 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 19 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 20 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 21 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 22 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 23 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 24 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 25 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO:26 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 27 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 28 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 29 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 30 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 31 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 32 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 33 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 34 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 35 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO: 36 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:
  • SEQ ID NO:37 is primer HC-For:acctattactgtcagcacatta
  • SEQ ID NO:38 is primer HC-Rev: ggatacagttggtgcagcatc
  • SEQ ID NO:39 is primer LC-For: gayattgtgmtsacmcarwct
  • SEQ ID NO: 40 is the primer LC-Rev: acctattactgtcagcacatta
  • HF heart failure
  • Any structural or functional cardiac disorder can cause HF, with the majority of HF patients having impaired left ventricular (LV) myocardial function.
  • LV left ventricular
  • Symptoms of HF include dyspnea (shortness of breath), fatigue, and fluid retention.
  • subject refers to a human or non-human organism. Preferred subjects herein are human subjects.
  • antigen-binding portion or "antigen-binding site” or “target-binding site” of an antibody as used herein means one or more of a binding protein (e.g., an antibody or receptor) that retains the ability to specifically bind an antigen or target. Multiple fragments, such as immunoglobulin variable domains (eg, VH or VL).
  • the antigen-binding portion of an antibody can be a fragment of a full-length antibody.
  • binding fragments encompassed by the term "antigen-binding portion” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, comprising a hinge region composed of A bivalent fragment of two Fab fragments connected by a disulfide bridge; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of an antibody single arm, (v) comprising dAb fragments of single variable domains; and (vi) isolated complementarity determining regions (CDRs).
  • Fab fragments monovalent fragments consisting of VL, VH, CL and CH1 domains
  • F(ab')2 fragments comprising a hinge region composed of A bivalent fragment of two Fab fragments connected by a disulfide bridge
  • an Fd fragment consisting of the VH and CH1 domains an Fv fragment consisting of the
  • VL and VH of an Fv encoded by separate genes can be joined by a synthetic linker using recombination to make a single protein chain in which the VH and VL regions pair to form a monovalent molecule (termed a single-chain Fv (scFv)).
  • scFvs are also encompassed within the term "antigen-binding portion", as are other forms of single chain antibodies such as diabodies and "linear antibodies” comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), said one The pair of tandem Fv fragments together with the complementary light chain polypeptides form a pair of antigen binding sites.
  • the variable domains of an antibody comprise complementarity determining regions (CDRs) and framework regions (FRs).
  • CDR as used herein means the complementarity determining regions within the variable domain sequences of an immunoglobulin. There are three CDRs in each of the variable regions of the heavy and light chains, which are designated CDR1, CDR2, and CDR3 for each of the heavy and light chain variable regions.
  • CDR set refers to a set of three CDRs that occur in a single variable domain capable of binding antigen. The exact boundaries of these CDRs have been defined according to different systems. The system described by Kabat (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391; Kabat et al. (1987) Sequences of Proteins of Immunological Interest, Fourth Edition US Govt. Printing Off. No.
  • Antibodies herein, or antigen-binding portions thereof may utilize CDRs defined according to any of these systems.
  • the antibodies herein, or antigen-binding portions thereof may utilize CDRs defined according to Kabat or Chothia.
  • antibody as used herein may also refer to an antigen-binding portion thereof.
  • position a to b of SEQ ID NO: X or "amino acid a to b of SEQ ID NO: X” as used herein refers to the amino acid sequence of SEQ ID NO: X from the N-terminal to the C-terminal amino acid at position a to amino acid at position b.
  • SEQ ID NO: the a to the b of X when the sequence is a nucleic acid sequence, can also refer to the a of the nucleic acid sequence of SEQ ID NO: X from the 5' end to the 3' end base to b base.
  • Galectin-3 (GenBank accession numbers: NC_000014.7 (gene) and NP_002297.2 (protein)) are 15 mammalian beta-galactoside-binding lectins or "galactose agglutinins" that specifically bind galactose. element”. Galectin-3 is known in the literature as LGALS3, MAC-2 antigen, carbohydrate-binding protein (CBP)-35, laminin-binding protein, galactose-specific lectin 3, mL-34, L-29 , hL-31, ⁇ BP and IgE-binding protein.
  • CBP carbohydrate-binding protein
  • Galectin-3 consists of a carboxy-terminal carbohydrate recognition domain (CRD) and an amino-terminal tandem repeat (Liu, F.-T. (2000) Role of Galectin-3 in inflammation.Lectins and Pathology.M.Caron and D. Seve, eds. Harwood Academic Publishers, Amsterdam, The Netherlands, p.51; Liu, F.-T. et al. (1995) Am. J. Pathol. 147:1016). Galectin-3 is normally distributed in the epithelium of many organs and various inflammatory cells, including macrophages as well as dendritic cells and Kupffer cells (Flotte, TJ. et al. (1983) Am.J.Pathol.111:112 )middle.
  • Galectin-3 has been shown to function in many cellular processes, including cell-cell adhesion, cell-matrix interaction, phagocytosis, cell cycle, apoptosis, angiogenesis, and mRNA splicing. Galectin-3 has been shown to act through both intracellular and extracellular actions (Sano, H. et al. (2000) The Journal of Immunology, 165:2156-2164). It is a component of nuclear heterogeneous ribonucleoprotein (hnRNP) (Laing, J.G. et al. (1998) Biochemistry 27:5329), a factor in pre-mRNA splicing (Dagher, S.F. et al. (1995) Proc.
  • hnRNP nuclear heterogeneous ribonucleoprotein
  • Galectin-3 secreted galectin-3 in the activation of different types of cells such as monocytes/macrophages (Liu, F.-T. (1993) Immunol Today 14:486), mast cells, neutrophils In granulocytes and lymphocytes (Hsu, D.K., S.R. et al. (1996) Am. J. Pathol. 148:1661) act as extracellular molecules.
  • Galectin-3 has been shown to act as a novel chemoattractant for monocytes and macrophages (Sano, H. et al. (2000) The Journal of Immunology, 2000, 165:2156-2164).
  • Galectin-3 has been implicated in diseases such as cancer, inflammation and heart failure (HF). Quantification of Galectin-3, as disclosed in International Patent Publication No. WO2005/040817, is particularly suitable for use in assays for diagnosing and detecting the severity and predicting consequences of HF.
  • Galectin-3 can be reliably distinguished from other mammalian galectins by targeting the Galectin-3 C-terminal and/or N-terminal domains.
  • the antibodies disclosed herein in combination with immunoassays known in the art, such as ELISA, the galectin-3 content in a sample can be reliably and reproducibly detected, and compared with the subject Among them are the presence, severity and stage of HF.
  • the present application provides an antibody or an antigen-binding portion thereof, which is capable of binding at least a part of the amino acid sequence of human galectin-3, the human galectin-3 At least a portion of the amino acid sequence comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: Amino acid sequence shown in ID NO:10 or any combination thereof:
  • MLITILGTVKPN positions 1 to 12 of SEQ ID NO:1; SEQ ID NO:3
  • VAFHFNPRFN 26th to 35th of SEQ ID NO:1; SEQ ID NO:6
  • TKLDNNW 45th to 62nd of SEQ ID NO:1; SEQ ID NO:7)
  • GREERQSVFPFESGK (63rd to 78th of SEQ ID NO:1; SEQ ID NO:8)
  • VFPFESGKPFKIQVLVEP 70th to 88th of SEQ ID NO:1; SEQ ID NO:9
  • DHFKVAVNDAHL (89th to 100th of SEQ ID NO: 1; SEQ ID NO: 10).
  • the antibody, or antigen-binding portion thereof binds to human galectin-3 at a site within SEQ ID NO: 3; in some embodiments, the antibody, or antigen-binding portion thereof, binds to human galectin-3 The site at which galectin-3 binds is within SEQ ID NO: 4; in some embodiments, the site at which the antibody or antigen-binding portion thereof binds to human galectin-3 is within SEQ ID NO: 5 In some embodiments, the antibody or antigen-binding portion thereof binds to human galectin-3 at a site within SEQ ID NO: 6; in some embodiments, the antibody or antigen-binding portion thereof The site that binds to human galectin-3 is within SEQ ID NO: 7; in some embodiments, the site that the antibody or antigen-binding portion thereof binds to human galectin-3 is within SEQ ID NO: within SEQ ID NO: 8; in some embodiments, the antibody or antigen-binding portion thereof binds to human ga
  • At least a portion of the amino acid sequence of human galectin-3 is located at the C-terminal part of human galectin-3, wherein the C-terminal part comprises the amino acid shown in SEQ ID NO: 1 sequence:
  • the antibody, or antigen-binding portion thereof binds human galectin-3 at a site within SEQ ID NO: 1.
  • the two C-terminal and/or N-terminal epitopes for binding to any two antibodies disclosed herein should be separated by at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, At least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, or a distance that does not interfere with the binding of the two antibodies to the epitope any distance.
  • the distance between two epitopes can be routinely determined by a person skilled in the art.
  • the antibody comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO:17 and LCDR3 shown in SEQ ID NO:18.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO: 13 to SEQ ID NO: 18.
  • the antibody comprising the amino acid sequence set forth in SEQ ID NO: 13 to SEQ ID NO: 18 is the primary antibody.
  • the antibody comprises HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:21, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO: 19 to SEQ ID NO: 24.
  • the antibody comprising the amino acid sequence set forth in SEQ ID NO: 19 to SEQ ID NO: 24 is a secondary antibody.
  • the antigen binding portion is a Fab, F(ab)' 2 or single chain Fv.
  • the antibody is a monoclonal antibody.
  • the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.
  • Monoclonal Antibody Techniques eg, hybridoma, recombinant and phage display techniques
  • monoclonal antibodies are known in the art.
  • Hybridoma monoclonal antibody is a highly uniform antibody produced by a single B cell clone that only targets a specific epitope.
  • B cells with the ability to secrete specific antibodies and myeloma cells with the ability to reproduce indefinitely are fused into B cell hybridomas.
  • a specific antibody against an antigenic epitope that is, a monoclonal antibody, can be prepared.
  • galectin-3 containing recombinant full-length sequence or C-terminal and N-terminal domains or polypeptide fragments was used as an immunogen to select and immunize BALB/C female mice.
  • mice Initially use complete adjuvant mixed with antigen 1:1 for subcutaneous multi-point injection, then use incomplete adjuvant and antigen 1:1 mixed for injection into mice. Stimulate B lymphocytes to produce corresponding antibodies. The lymphocytes are then fused with a mouse or rat myeloma cell line using PEG fusion agent to form a hybridoma cell line. HAT selective medium is generally used. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyltransferase and cannot use the salvage pathway to synthesize DNA and die. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive long-term in vitro and die gradually.
  • the antibodies, or antigen-binding portions thereof are useful in the diagnosis of heart failure.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the antibody or its antigen-binding portion can be used to assist in the diagnosis of tumors, rheumatoid arthritis, and chronic kidney disease, but not limited thereto.
  • the present application provides an antibody or an antigen-binding portion thereof, which is capable of binding at least a part of the amino acid sequence of human galectin-3, the human galectin-3 At least a portion of comprising the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 or both of SEQ ID NO: 11 and SEQ ID NO: 12:
  • MADNFSLHDALSGSGNPNPQGWPG positions 1 to 24 of SEQ ID NO:2; SEQ ID NO:11
  • AWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG positions 25 to 56 of SEQ ID NO: 2; SEQ ID NO: 12).
  • At least a portion of the amino acid sequence of human galectin-3 is located at the N-terminal portion of human galectin-3, wherein the N-terminal portion comprises the amino acid shown in SEQ ID NO:2 sequence:
  • the antibody comprises HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29 and LCDR3 shown in SEQ ID NO:30.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO:25 to SEQ ID NO:30.
  • the antibody comprising the amino acid sequence set forth in SEQ ID NO: 25 to SEQ ID NO: 30 is a third antibody.
  • the antibody comprises HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:33, LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35 and LCDR3 shown in SEQ ID NO:36.
  • the antibody comprises the amino acid sequence set forth in SEQ ID NO: 31 to SEQ ID NO: 36.
  • the antibody comprising the amino acid sequence set forth in SEQ ID NO: 31 to SEQ ID NO: 36 is a fourth antibody.
  • the antigen binding portion is a Fab, F(ab)' 2 or single chain Fv.
  • the antibody is a monoclonal antibody.
  • the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.
  • the antibodies, or antigen-binding portions thereof are useful for diagnosing heart failure, but are not limited to such use.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the antibody or its antigen-binding portion can be used to assist in the diagnosis of tumors, rheumatoid arthritis, and chronic kidney disease, but not limited thereto.
  • the present application provides an antibody combination product, which comprises the antibody or antigen-binding portion thereof described in the first aspect and the antibody or antigen-binding portion thereof described in the second aspect.
  • the antibody combination includes an antibody comprising the amino acid sequence set forth in SEQ ID NO: 13 to SEQ ID NO: 18 and an antibody comprising the amino acid sequence set forth in SEQ ID NO: 25 to SEQ ID NO: 30.
  • the antibody combination includes an antibody comprising the amino acid sequence set forth in SEQ ID NO: 13 to SEQ ID NO: 18 and an antibody comprising the amino acid sequence set forth in SEQ ID NO: 31 to SEQ ID NO: 36.
  • the antibody combination includes an antibody comprising the amino acid sequence set forth in SEQ ID NO: 19 to SEQ ID NO: 24 and an antibody comprising the amino acid sequence set forth in SEQ ID NO: 25 to SEQ ID NO: 30.
  • the antibody combination includes an antibody comprising the amino acid sequence set forth in SEQ ID NO: 19 to SEQ ID NO: 24 and an antibody comprising the amino acid sequence set forth in SEQ ID NO: 31 to SEQ ID NO: 36.
  • the antibody combination product includes any combination of the first antibody to the fourth antibody, for example, the antibody combination product includes the first antibody and the third antibody; or the first antibody and the fourth antibody; or the second antibody and the second antibody A tetrabody; or a second antibody and a third antibody; or a first antibody, a second antibody, and a fourth antibody; or a first antibody, a second antibody, and a third antibody; or a first antibody, a third antibody, and a fourth antibody or a second antibody, a third antibody, and a fourth antibody; or a first antibody, a second antibody, a third antibody, and a fourth antibody.
  • antibody combinations comprise a combination of a first or second antibody and a third or fourth antibody.
  • antibody combination products comprise a combination of a solution comprising a first antibody or a second antibody and a solution comprising a third antibody or a fourth antibody.
  • the solution containing the first antibody or the second antibody further includes a buffer, such as phosphate buffer; in some embodiments, the solution containing the third antibody or the fourth antibody further includes a buffer, such as phosphate buffer liquid.
  • an antibody combination product comprises a powder comprising a first antibody or a second antibody in combination with a powder comprising a third antibody or a fourth antibody.
  • a detectable label is bound to the first antibody or the second antibody; in some embodiments, a detectable label is bound to the third antibody or the fourth antibody.
  • the present application also provides an antibody combination product, which comprises multiple/multiple antibodies or antigen-binding parts thereof according to the first aspect.
  • the present application also provides an antibody combination product, which comprises multiple/multiple antibodies or antigen-binding parts thereof according to the second aspect.
  • the present application also provides an antibody combination product, which comprises the antibody or antigen-binding portion thereof described in the first aspect and the antibody or antigen-binding portion thereof described in the second aspect.
  • the present application also provides an antibody combination product, which comprises multiple/multiple antibodies or antigen-binding portions thereof described in the first aspect and antibodies or antigen-binding portions thereof described in the second aspect.
  • the present application also provides an antibody combination product, which comprises the antibody or antigen-binding portion thereof described in the first aspect and multiple/multiple antibodies or antigen-binding portions thereof described in the second aspect.
  • the present application also provides an antibody combination product, which comprises multiple/multiple antibodies or antigen-binding portions thereof described in the first aspect and multiple/multiple antibodies or antigens thereof described in the second aspect Combined part.
  • One of the antibodies in the kit, or an antigen-binding portion thereof, is used as a capture antibody and can be immobilized on a solid surface for capturing Galectin-3.
  • Yet another antibody, or antigen-binding portion thereof, is used as the detection antibody and is linked to a detectable label.
  • the present application provides a kit for detecting the content of galectin-3 in a sample from a human subject, which comprises the antibody or antigen-binding portion thereof described in the first aspect or the antibody or antigen-binding portion thereof described in the second aspect.
  • the kit comprises the antibody or antigen-binding portion thereof of the first aspect.
  • the kit comprises the antibody or antigen-binding portion thereof of the second aspect.
  • the kit comprises the antibody combination product of the third aspect.
  • the detectable label includes an enzyme or a chromogenic substance.
  • the enzyme is alkaline phosphatase or horseradish peroxidase.
  • the chromogenic substance is a chemiluminescent compound or a fluorescent substance.
  • the chemiluminescent compound is ruthenium terpyridine or an acridinium ester.
  • the fluorescent substances are fluorescent pigments, fluorescent microspheres, time-resolved fluorescent microspheres or quantum dots.
  • the sample is derived from whole blood, serum or plasma.
  • the sample is derived from blood.
  • the sample is derived from a bodily fluid.
  • Bodily fluids can be processed or untreated. Methods of obtaining bodily fluids from a subject are known to those skilled in the art.
  • the sample is derived from urine, gastric juice, bile, saliva, sweat, and spinal fluid, feces, or muscle biopsy.
  • antibodies that are not labeled with a detectable label are pre-attached to the solid surface.
  • the solid surface is selected from nitrocellulose membranes, magnetic particles, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, plastic, and glass.
  • the kit further comprises standards and/or quality controls.
  • the kit further comprises instructions.
  • kits are useful for diagnosing heart failure, but are not limited to such use.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the antibody or its antigen-binding portion can be used to assist in the diagnosis of tumors, rheumatoid arthritis, and chronic kidney disease, but not limited thereto.
  • the antibody of the first aspect is labeled with a detectable label.
  • the antibody of the second aspect is labeled with a detectable label.
  • the antibody of the first aspect is labeled with a detectable label and the antibody of the second aspect is not labeled with a detectable label.
  • the antibody of the second aspect is labeled with a detectable label and the antibody of the first aspect is not labeled with a detectable label.
  • the kit includes coupling magnetic microspheres with an antibody to galectin-3 to form a solid phase conjugate.
  • An enzyme involved in catalyzing a chemiluminescent reaction such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP)
  • HRP horseradish peroxidase
  • ALP alkaline phosphatase
  • Coating antibody-antigen to be tested-enzyme-labeled antibody complex after washing, add a luminescent agent, the enzyme catalyzes and decomposes the substrate to emit light, and is read by a chemiluminescence instrument.
  • the kit includes an immunochromatographic test strip with a fluorescent material as a marker.
  • a fluorescent material is used as a label, and the fluorescent material is covalently coupled with another antibody to form a fluorescent probe, which can form a fluorescent probe after reacting with the antigen.
  • the fluorescent signal on the test strip is then read using an immunofluorescence analyzer.
  • the present application provides the antibody or its antigen-binding portion described in the first aspect or the antibody or its antigen-binding portion described in the second aspect or the antibody combination product described in the third aspect in the preparation of human samples for detection Use in a Galectin-3 Assay Kit.
  • the present application also provides the use of the antibody or antigen-binding portion thereof according to the first aspect in preparing a kit for detecting the content of human galectin-3 in a sample.
  • the present application also provides the use of the antibody or antigen-binding portion thereof according to the second aspect in preparing a kit for detecting the content of human galectin-3 in a sample.
  • the sample is an ex vivo sample.
  • the present application also provides the use of the antibody combination product described in the third aspect in preparing a kit for detecting the content of human galectin-3 in a sample.
  • the antibody combination product comprises any combination of the antibody or antigen-binding portion thereof of the first aspect and the antibody or antigen-binding portion thereof of the second aspect, or any number or type of the first An antibody or antigen-binding portion thereof of one aspect in combination with any number or species of antibodies or antigen-binding portions thereof of the second aspect.
  • the sample is derived from whole blood, serum or plasma.
  • the sample is derived from blood.
  • the sample is derived from a bodily fluid.
  • Bodily fluids can be processed or untreated. Methods of obtaining bodily fluids from a subject are known to those skilled in the art. A sample can be an ex vivo sample.
  • the sample is derived from urine, gastric juice, bile, saliva, sweat, and spinal fluid, feces, or muscle biopsy.
  • antibodies that bind to the C-terminal and/or N-terminal portion of human galectin-3 can be used to quantify the amount of galectin-3 in a sample.
  • the present application provides a method for detecting the content of human galectin-3 in a sample, the method using the antibody or antigen-binding portion thereof described in the first aspect or the second aspect or the antibody or antigen-binding portion thereof described in the third aspect.
  • the antibody or antigen-binding portion thereof described in the first aspect is used to capture human galectin-3 and the antibody or antigen-binding portion thereof described in the first aspect is labeled with a detectable label, or the antibody or antigen-binding portion thereof described in the second aspect The antibody or antigen-binding portion thereof is used to capture human galectin-3 and the antibody or antigen-binding portion thereof of the first aspect is labeled with a detectable label;
  • the antibody or its antigen-binding part is used to detect the content of human galectin-3 by immunological method.
  • the sample is derived from whole blood, serum or plasma.
  • the sample is derived from a bodily fluid.
  • the sample can be an ex vivo sample; the assay can be an in vitro assay.
  • Bodily fluids can be processed or untreated. Methods of obtaining bodily fluids from a subject are known to those skilled in the art.
  • the sample is derived from urine, gastric juice, bile, saliva, sweat, and spinal fluid, feces, or muscle biopsy.
  • the sample is derived from blood.
  • the immunological method is selected from enzyme-linked immunoassay, immunofluorescence, chemiluminescence, immunochromatography, immunoprecipitation, and combinations thereof.
  • the immunological method is an enzyme-linked immunoassay.
  • Galectin-3 can be detected and quantified using a "sandwich” assay.
  • an antibody is immobilized on a solid surface to bind and capture Galectin-3.
  • capture antibodies Such antibodies are thus referred to herein as capture antibodies.
  • the second antibody is detectably labeled with a chromogenic substance (e.g., a fluorophore or chemiluminescent compound) or an enzyme so that binding of the second antibody to the Galectin-3-complex indicates Galectin-3 has been captured.
  • the intensity of the signal is proportional to the concentration of Galectin-3 in the sample.
  • the second antibody is therefore also referred to herein as a detection antibody or a marker antibody.
  • Such assay methods may be referred to as two-site immunoassays, "sandwich” methods, or “sandwich immunoassays.”
  • Capture and detection antibodies can be contacted with the test sample simultaneously or sequentially, as is known in the art. Sequential methods, sometimes called “forward” methods, can be accomplished by incubating a capture antibody with the sample followed by the addition of a labeled detection antibody at predetermined times. Alternatively, a labeled detection antibody can first be incubated with the sample, and then the sample can be contacted with the capture antibody (sometimes referred to as the "reverse” method).
  • Such assays can be performed in a number of specific formats known to those skilled in the art, including through the use of various high throughput clinical laboratory analyzers or with point of care or home testing equipment.
  • ELISA enzyme immunoassay
  • ELISA is a technique that uses labeled (eg, enzyme-linked) forms of antibodies to detect and measure the concentration of antigens.
  • labeled eg, enzyme-linked
  • an antibody eg, anti-galectin-3
  • a solid phase ie, microtiter plate
  • a biological sample containing the antigen eg, galectin-3
  • the solid phase is then washed to remove unbound antigen.
  • a labeled antibody eg, enzyme-linked
  • enzymes that can be linked to antibodies are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and beta-galactosidase.
  • the enzyme-linked antibody reacts with the substrate to produce a chromogenic reaction product that can be measured. This measurement can be used to deduce the concentration of Galectin-3 present in the sample, for example by comparing the measured value to a Galectin-3 standard curve.
  • Immunofluorescence is based on the principle of antigen-antibody reaction. Firstly, known antigens or antibodies are labeled with fluorescein to make fluorescent markers, and then this fluorescent antibody (or antigen) is used as a molecular probe to check the corresponding cells or tissues. Antigen (or Antibody). The antigen-antibody complexes formed in cells or tissues contain fluorescein. Use a fluorescence microscope to observe the specimen. The fluorescein emits bright fluorescence when irradiated by excitation light, and the cells or tissues where the fluorescence is located can be seen to determine the identity of the antigen or antibody. properties, location, and determination of content using quantitative techniques.
  • Chemiluminescence immunoassay is a combination of highly sensitive chemiluminescent assay technology and highly specific immune response for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs and other detection and analysis techniques. It is a newest immunoassay technique developed after radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and time-resolved fluorescent immunoassay. Chemiluminescent immunoassays use chemiluminescent agents to directly label antigens or antibodies for immunoassays.
  • Chemiluminescent substances commonly used for labeling include acridinium ester compounds, which are effective luminescent markers, which emit light by initiating the action of a luminescent reagent, and the intense direct luminescence is completed within one second, which is a fast flashing luminescence.
  • Acridine ester is used as a marker for immunoassay. Its chemical reaction is simple, fast, and does not require a catalyst.
  • the competition method is used to detect small molecule antigens, and the sandwich method is used to detect macromolecular antigens. It has less non-specific binding and low background; Binding does not reduce the amount of light produced, increasing sensitivity.
  • Immunoprecipitation is a method of purifying and enriching the target protein by using antibody-specific reaction. After the antibody binds to the corresponding protein in the sample, it is incubated with protein A/G (ProteinA/G) or secondary antibody-coupled agarose or Sepharose beads, and the beads-protein A/G or secondary antibody-antibody-purpose are obtained by centrifugation For protein complexes, the precipitate is washed, resuspended in electrophoresis loading buffer, boiled, and under the action of high temperature and reducing agent, the antigen and antibody are dissociated, and the supernatant is collected by centrifugation, which includes antibody, target protein and a small amount miscellaneous protein.
  • protein A/G ProteinA/G
  • secondary antibody-coupled agarose or Sepharose beads secondary antibody-coupled agarose or Sepharose beads
  • the beads-protein A/G or secondary antibody-antibody-purpose are obtained by centrifugation
  • the precipitate is washed,
  • the markers used in the present application can be selected from any markers generally known in the art. Preferred markers are those that allow more precise quantification.
  • labels include, but are not limited to, fluorescent moieties, enzymes, electrochemically active species, radioisotopes, chemiluminescent molecules, latex or gold particles, detectable ligands, and the like.
  • the label is an enzyme or a fluorescent molecule.
  • Methods for attaching labels to antibodies are well known in the art and include covalent and non-covalent attachments.
  • antibodies can be labeled with fluorescent compounds.
  • fluorescent compounds When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by the emitted fluorescence.
  • fluorescent labeling compounds are Cy3 and Cy5 (water-soluble fluorescent dyes of the cyanine dye family - "Cy” dyes), fluorescein isothiocyanate, rhodamine, phycocyanin, allophycocyanin, phthalate Formaldehyde and Fluorescamine.
  • the detection antibody is detectably labeled by linking the antibody to an enzyme.
  • the enzyme will thus react with the substrate upon contact with it, which reaction can be detected, for example, by spectrophotometric measurements, fluorometric methods or visual inspection.
  • Enzymes that can be used to detectably label antibodies of the invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, Triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, ⁇ -galactosidase, ribonuclease, urease, catalase, glucose- 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Detection can also be achieved using radiolabeled antibodies. Antibodies can then be detected by using radioimmunoassays. Radioisotopes can be detected by such methods as the use of gamma counters or scintillation counters or by autoradiography. Particularly useful isotopes for the purposes of this application are3H , 131I , 35S , 14C , and preferably125I.
  • Antibodies can also be detectably labeled by coupling the antibodies to chemiluminescent moieties. The presence of chemiluminescent antibodies is then determined by detecting the presence of luminescence produced during the chemical reaction.
  • chemiluminescent labeling compounds are luminol, luciferin, isophthalamide, imidazoles, acridinium salts and oxalates.
  • the present application provides the use of the antibody or its antigen-binding part described in the first aspect or the second aspect, or the antibody combination product described in the third aspect in detecting the content of human galectin-3 in a sample .
  • the sample is derived from whole blood, serum or plasma.
  • the sample is derived from blood.
  • a sample can be an ex vivo sample.
  • the present application provides a method for diagnosing heart failure in an individual or assisting in the diagnosis of tumors, rheumatoid arthritis, or chronic kidney disease, the method comprising: obtaining a biological sample from an individual; using the reagent described in the fourth aspect
  • the kit detects the content of human galectin-3 in the biological sample; and compares the detected content of human galectin-3 with a reference value.
  • using the kit described in the fourth aspect to detect the content of human galectin-3 in the biological sample includes detecting the amount of a detectable marker, and comparing the detection result with a standard curve.
  • the reference value is the content of human galectin-3 in a biological sample from healthy humans.
  • the reference value is the conventional or known human galectin-3 content in the art.
  • the biological sample is derived from whole blood, serum or plasma.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosticating heart failure.
  • the present application provides the antibody or its antigen-binding part described in the first aspect or the antibody combination product described in the third aspect for use in the diagnosis of heart failure or auxiliary diagnosis of tumors, rheumatoid arthritis or chronic kidney disease. Use in kits for diseases.
  • the diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing the prediction of heart failure.
  • galectin-3 polypeptides Using recombinant galectin-3 polypeptides to prepare and screen antibodies that can be used for diagnosis, with high yield and high sensitivity; and/or
  • the galectin-3 protein content in serum of patients with heart failure is determined by immunological technology. This indicator can be used as a key basis for the recovery of patients' prognosis or early detection of diseases, and has high detection sensitivity and specificity.
  • Balb/c mice were immunized with human galectin-3 to obtain antibody-expressing hybridoma cells, which were detected and screened to obtain antibodies that specifically recognize galectin-3.
  • the specific method is shown in Figure 1.
  • the recombinant galectin-3 full-length antigen was used to immunize Balb/c mice, and 6-8 weeks old female Balb/c mice were selected to immunize six mice in total.
  • the dose of antigen used was 100 ⁇ g/monkey.
  • Antigen and complete adjuvant were emulsified at a ratio of 1:1, and injected subcutaneously at multiple points; the immunization cycle was six times. One week between each needle.
  • the titer When the titer reaches 1:10000 or more, it is fused with mouse myeloma cells, and the absorbance OD value of the cell supernatant is detected by ELISA indirect method.
  • One cell per well is seeded in a culture dish, and the cells proliferate to form a monoclonal cell line.
  • mice 46 mouse monoclonal antibody hybridoma cells were screened. Antibodies were produced using in vivo induction. Male Balb/c mice over 6 weeks old were selected and sensitized with Freund's incomplete adjuvant (500 ⁇ L/mouse). Inoculate the cell lines about 7 days to make the density reach 2X10 6 . After about 7 days, abdominal swelling can be clearly seen; the ascites was extracted and sterilized by filtration with a filter membrane with a pore size of 0.45 ⁇ m, and then packed and frozen at -20°C.
  • Freund's incomplete adjuvant 500 ⁇ L/mouse
  • Expansion culture and cryopreservation The screened cell lines with strong specificity were expanded and cultured in time, using 10% DMSO cryopreservation medium; 1ml/tube was cryopreserved until liquid nitrogen storage.
  • Example 1 The 46 mouse monoclonal antibodies obtained in Example 1 were further identified. Capture antibody and detection antibody pairs were identified using the checkerboard method. In short, all 46 antibodies were plated on a 384-well microtiter plate at a concentration of 2 ng/mL (antibody concentration adjusted in PBS), 100 ⁇ L per well as capture antibodies, and each capture antibody was plated on 47 wells.
  • the obtained 46 antibodies were detected by ELISA, and the antibodies with weak positive signals were eliminated. Two pairs (4) of mouse monoclonal antibodies specific to human Galectin-3 were obtained.
  • the screened cell lines with strong specificity were expanded and cultured in time, and 10% DMSO freezing medium was used; 1ml/tube was frozen and stored in liquid nitrogen.
  • a protein A column purchased from LIFE TECH
  • Four kinds of antibodies were obtained, namely G3-5-5, G3-12-1, G3-2-1 and G3-2-3, namely the first antibody to the fourth antibody of the present application.
  • RNA from positive hybridoma cells was isolated with RNA miniprep kit (purchased from Qiagen), and reverse-transcribed into cDNA with reverse transcriptase (purchased from Biosystems). PCR was then performed using the mouse antibody-specific primers described below. The amplification products of mouse antibody Ig heavy and light chains were obtained and then sequenced.
  • G3-5-5 (primary antibody) comprises HCDR1 shown in SEQ ID NO:13, the heavy chain variable region of HCDR2 shown in SEQ ID NO:14 and HCDR3 shown in SEQ ID NO:15, And the light chain variable region comprising LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17 and LCDR3 shown in SEQ ID NO:18.
  • G3-12-1 (secondary antibody) comprises HCDR1 shown in SEQ ID NO:19, the heavy chain variable region of HCDR2 shown in SEQ ID NO:20 and HCDR3 shown in SEQ ID NO:21, and contains SEQ ID NO:21 LCDR1 shown in ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 light chain variable region shown in SEQ ID NO:24.
  • G3-2-1 (third antibody) comprises HCDR1 shown in SEQ ID NO:25, the heavy chain variable region of HCDR2 shown in SEQ ID NO:26 and HCDR3 shown in SEQ ID NO:27, and contains SEQ ID NO:27 LCDR1 shown in ID NO:28, LCDR2 shown in SEQ ID NO:29 and LCDR3 light chain variable region shown in SEQ ID NO:30.
  • G3-2-3 (the fourth antibody) comprises HCDR1 shown in SEQ ID NO:31, the heavy chain variable region of HCDR2 shown in SEQ ID NO:32 and HCDR3 shown in SEQ ID NO:33, and contains SEQ ID NO:33 LCDR1 shown in ID NO:34, LCDR2 shown in SEQ ID NO:35 and LCDR3 light chain variable region shown in SEQ ID NO:36.
  • Bioinformatics analysis was performed on Galectin-3, the binding site was predicted, and a total of 88 corresponding short overlapping peptides were synthesized. After the short peptides were coupled to the carrier protein, they were detected by enzyme-linked immunosorbent assay.
  • Example 2 Label the primary antibody, secondary antibody, third antibody, and fourth antibody obtained in Example 2 at 4 ng/ml, and add 50 ⁇ L per well to 96 wells; wash with PBST 5 times.
  • An antibody other than itself labeled with biotin was used as a detection antibody.
  • the corresponding wells were assayed at a volume of 50 ⁇ L per well.
  • the specific antigen-binding epitope of the antibody is obtained according to the interaction between the fusion short peptide and the antibody, and the degree of binding firmness.
  • the primary and secondary antibodies were detected to be able to bind non-contiguous epitopes on the C-terminal portion of human galectin-3.
  • the third antibody and the fourth antibody are capable of binding non-contiguous epitopes on the N-terminal portion of human galectin-3.
  • the fluorescent immunochromatography kit includes a sample pad, a binding pad, a nitrocellulose membrane, an absorbent pad and a polyvinyl chloride (PVC) bottom plate.
  • a sample pad As shown in Figure 2, the fluorescent immunochromatography kit includes a sample pad, a binding pad, a nitrocellulose membrane, an absorbent pad and a polyvinyl chloride (PVC) bottom plate.
  • PVC polyvinyl chloride
  • the primary antibody G3-5-5 and the secondary antibody of human galectin-3 were pre-immobilized on the nitrocellulose membrane to form a test line or a control line, and after adding the sample containing the analyte , the analyte is combined with the labeled antibody on the binding pad, and is further captured by the monoclonal antibody immobilized on the T line with the action of chromatography.
  • the excess labeled antibody is captured by the secondary antibody on the C line, and the signal intensity of the marker on the T/C line is read by a specific detection instrument, and substituted into the pre-drawn standard curve.
  • Coat the fluorescein particle-antibody complex on the binding pad connect the primary antibody G3-5-5 and the secondary antibody to the antibody-carrying membrane in a linear form to form T-lines and C-lines, and attach them with an adhesive Bond the sample pad, binding pad, antibody-carrying membrane, and absorbent paper to each other on the liner.
  • the human galectin-3 protein was configured with a standard buffer solution (50mM Tris-HCl, 1% BSA, 0.9% NaCl, pH7.4) to a concentration of 0, 5, 20, 40, 60, 100ng/mL, Pack 1mL per bottle and store at 4°C for future use.
  • a standard buffer solution 50mM Tris-HCl, 1% BSA, 0.9% NaCl, pH7.4
  • the operating procedure of the kit described in this application is as follows: return the reagent and the sample to be tested to room temperature, and turn on the immunofluorescence analyzer according to the instructions; use a dropper to vertically add the sample to be tested to the sample hole of the test card corresponding to the sample pad Incubate for 15 minutes. After the incubation is completed, use an immunofluorescence analyzer to detect the signal of the detection card, and the content of human galectin-3 in the sample is proportional to its relative fluorescence signal.
  • the antibody immobilized on the nitrocellulose membrane may be the primary antibody or the secondary antibody, or the tertiary antibody or the quaternary antibody.
  • the antibody coupled to the fluorescent particle can be the third antibody or the fourth antibody, or the first antibody or the second antibody.
  • the third antibody G3-2-1 and the secondary antibody of human galectin-3 were also pre-immobilized on the nitrocellulose membrane to form a test line or a control line, and a human galactose agglutination Fluorescent particles of G3-12-1, the second antibody of prime-3, to prepare a fluorescent immunochromatography kit and detect it.
  • Example 4 Using the method in Example 4 and the prepared test paper, the human galectin-3 calibrator was detected, and a standard curve was drawn. Similarly, the sample to be tested is measured, and the concentration of human galectin-3 in the sample is calculated according to the luminous intensity of the sample.
  • Detection of sensitivity Refer to the experimental scheme recommended by CLSI EP17-A document, calculate the sensitivity of human galectin-3 fluorescent immunochromatography reagent, and the sensitivity is 1.5ng/mL.
  • Precision measurement Take two human galectin-3 samples with a concentration of 15ng/mL and 60ng/mL, make three parallels for each concentration of each sample, use three batches of kits for detection, and calculate the intra-batch accuracy of the kit. And the difference between batches, the results show that the difference between batches and batches of the kit is less than 10%.
  • Magnetic particle chemiluminescence kit includes magnetic particle coating, enzyme label, chemiluminescent substrate solution, analysis buffer, washing solution, standard and quality control.
  • the primary antibody G3-5-5 of human galectin-3 was coated on the magnetic particles, and the fourth antibody G3-2-3 of human galectin-3 was labeled on the base
  • the analyte will combine with the magnetic particle coating and the enzyme label to form an immune complex
  • the chemiluminescent substrate solution after washing with the washing solution, add the chemiluminescent substrate solution, the luminous intensity of the instrument was detected by chemiluminescence, and substituted into the pre-drawn standard curve.
  • Disperse the carboxylated magnetic particles on the surface in phosphate buffer add the primary antibody G3-5-5 to human galectin-3, mix for 30 minutes, add carbodiamine and place at 4 degrees Celsius for 12-15 Hours, separated with a magnetic frame, washed to obtain magnetic particle-antibody complexes, stored in phosphate buffer containing 0.5% BSA and 0.1% Tween 20.
  • chemiluminescence substrate solution contains 10% 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1, 2-Dioxyethane (AMPPD) in Tris buffer.
  • Assay buffer is phosphate buffer containing 1% BSA and 0.1% Tween20.
  • washing solution is phosphate buffer solution containing 1% Tween20.
  • the standard product is to prepare the human galectin-3 protein with a concentration of 0, 5, 20, 40, 60, 100ng/mL, each bottle is divided into 1mL, and stored at 4°C for later use.
  • the quality control product is to prepare the human galectin-3 protein with the standard buffer solution to a concentration of 10 and 50 ng/mL respectively, aliquot 1 mL in each bottle, and store at 4°C for later use.
  • the magnetic particle coating, enzyme markers, chemiluminescent substrate solution, analysis buffer, washing solution, standard and quality control are assembled into an adult galectin-3 magnetic particle chemiluminescent kit.
  • the operating procedure of the magnetic particle chemiluminescence kit described in this application is as follows: Take 50 ⁇ L of the sample to be tested or standard/quality control, 50 ⁇ L of analysis buffer, 50 ⁇ L of magnetic particle coating, and 50 ⁇ L of enzyme marker in a reaction cup Carry out the reaction and incubate at 37°C for 10 minutes. After the reaction is completed, perform magnetic separation and washing on the above-mentioned reactant for 3 times on the magnetic plate.
  • the antibody coated on the magnetic particles may be the first antibody or the second antibody, or the third antibody or the fourth antibody.
  • the antibody conjugated to alkaline phosphatase may be a third antibody or a fourth antibody, or may be a first antibody or a second antibody.
  • Example 6 Using the method and prepared kit in Example 6, the human galectin-3 calibrator was detected, and a standard curve was drawn. Similarly, the sample to be tested is measured, and the concentration of human galectin-3 in the sample is calculated according to the luminescence intensity of the sample.
  • Detection of sensitivity Refer to the experimental scheme recommended by CLSI EP17-A document, calculate the sensitivity of human galectin-3 fluorescent immunochromatography reagent, and the sensitivity is 1.0ng/mL.
  • Precision measurement Take two human galectin-3 samples with a concentration of 15ng/mL and 60ng/mL, make three parallels for each concentration of each sample, use three batches of kits for detection, and calculate the intra-batch accuracy of the kit. And the difference between batches, the results show that the difference between batches and batches of the kit is less than 5%.

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Abstract

本申请提供了用于结合人半乳糖凝集素-3的抗体或其抗原结合部分以及抗体组合产品。本申请还提供了用于检测来自人受试者的样品中的半乳糖凝集素-3含量的试剂盒及其用途。

Description

半乳糖凝集素-3的免疫测定
相关申请的交叉引用
本申请要求于2021年10月15日向中国国家知识产权局提交的第202111203392.8号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请大体上涉及生物检测领域，具体而言，本申请提供了用于结合人半乳糖凝集素-3的抗体或其抗原结合部分以及抗体组合产品。本申请还提供了用于检测来自人受试者的样品中的半乳糖凝集素-3含量的试剂盒及其用途。
背景技术
半乳糖凝集素-3(Galectin-3、Gal-3)是一种具有特殊的氨基酸序列的半乳糖结合蛋白，广泛存在于从低级到高级的动物中。半乳糖凝集素(Galectins)是一大类蛋白家族，已有的研究证实了该蛋白家族的15种成员，根据其结构上的差异和在其多肽链内保守的糖识别域的数量，把它们分为三组：(1)原型半乳凝集素(Galectin-1、2、5、7、10、11、13、14和15)，其包含一个CRD；(2)串联重复序列半乳凝集素(Galectin-4、6、8、9和12)，其由一个连接区域连接两个CRDs；(3)嵌合型半乳凝集素(Galectin-3)，其包含一个CRD，并连接到一个扩展的非凝集素的N末端结构域。Galectin-3是半乳凝集素家族中是研究最多的成员之一。人类Galectin-3基因定位于染色体14q21-22，总长约17kb，含有251个氨基酸残基，分子量约为29.31KD，含有6个外显子和5个内含子。人类Galectin-3有3个结构域：(1)1-12氨基酸NH2-(氨基)末端，此部位决定细胞定位，也是高度保守的ser6残基参与Galectin-3抗凋亡活动所必须的；(2)富含甘氨酸、酪氨酸、脯氨酸的胶原蛋白重复序列，功能是基质金属蛋白酶类(Matrix Metalloproteinases,MMPs)的底物；(3)是一个呈球形结构碳水化合物结合部位，于COOH-(羧基)末端，可与细胞外基质及糖蛋白相互作用。
Galectin-3广泛分布于肿瘤细胞、上皮细胞、成纤维细胞和巨噬细胞及其他炎性细胞，在心脏、肾脏、肝脏、大脑、胰腺中也均有少量表达，Galectin-3在细胞质、细胞核和细胞表面均有表达，以细胞质为主。Galectin-3的生物学功能与细胞内定位有关。当细胞处于静息状态时它主要位于细胞浆，处于增殖状态时则主要分布于细胞核内。Galectin-3的分泌不依赖内质网和高尔基器，通过一种类似细胞胞吐的非经典机制转移至细胞外，可溶性的Galectin-3可进入全身循环。
Galectin-3是MMPs的底物，MMP-2或MMP-9将Galectin-3进行蛋白分解后，生成一个22kU的糖识别域和一个9kU的氨基酸残基片段。Galectin-3更容易在肿瘤的转移、侵袭过程中发挥作用。有文献报道，在乳腺癌的研究中，被分解后的Galectin-3通过激活PKC或诱导GTP酶，上调pFAK途径，增强血管内皮细胞的趋化性，促进血管内皮细胞的迁移、趋化，从而促进新生血管的形成。
Galectin-3参与体内许多生理和病理过程，包括细胞生长、细胞黏附、炎症反应、免疫调节、细胞凋亡以及肿瘤转化转移。在许多癌症如黑色素瘤、肺癌、乳腺癌、结肠直肠癌、头颈部癌和非霍奇金淋巴瘤中表达显著增加。Galectin-3可参与肾脏、肺、肝脏等器官纤维化的形成。Galectin-3与风湿免疫性疾病，支气管哮喘联系较为密切。Galectin-3是一种促炎症信号因子，在多种炎症因子中表达，参与巨噬细胞吞噬、细胞黏附等。目前与Galectin-3相关的疾病研究最多的是心力衰竭，心力衰竭(HF)是各种心脏疾病的严重表现或晚期阶段，发病率有不断增高的趋势，且死亡率和再住院率居高不下，是目前主要的心血管疾病之一。心力衰竭是一种 累及全身各个系统的复杂临床综合征，其病理生理机制主要涉及血流动力学的障碍和神经内分泌系统的异常激活。
目前已有研究证实心力衰竭发生、发展与正常心肌细胞被肌原纤维替代密切相关，同期研究表明半乳糖凝集素-3(Galectin-3、Gal-3)促进心肌纤维化，参与心肌炎性改变和心室重构等过程。相对于非心力衰竭患者，心力衰竭患者的Galectin-3表达水平持续升高，可作为评价心力衰竭的指标。心肌纤维化主要是心肌内胶原纤维过量堆积导致的，可引起心室重构。Galectin-3在纤维化组织(心脏、肝脏纤维化模型)中表达量明显增高。Galectin-3可以反应纤维化的程度。
半乳糖凝集素-3在血液或血清等体液中的含量能够反映人体的疾病状况，譬如慢性心脏衰竭等，所以准确定量半乳糖凝集素-3可以用来鉴定或预测疾病，用来评估疾病的严重程度，分期或预测疾病后果，或监视用药效果等临床或科研需要。
迄今为止的测定方法难以明确将半乳糖凝集素-3与其它半乳糖凝集素或其蛋白片段相区别，因为半乳糖凝集素-3与其它14种哺乳动物半乳糖凝集素，特别是在保守的糖类识别结构域(CRD)中共有高度的序列相似性，且半乳糖凝集素-3是MMPs的底物，可被其裂解。阻碍开发特异性、可重现的检测分析的半乳糖凝集素-3的另一因素是半乳糖凝集素-3结合不同的蛋白质、糖类、核酸和脂质的倾向性。
发明概述
第一方面，本申请提供了一种抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的氨基酸序列的至少一部分包含SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列：
MLITILGTVKPN(SEQ ID NO:1的第1位至第12位；SEQ ID NO:3)，
MLITILGTVKPNAN(SEQ ID NO:1的第1位至第14位；SEQ ID NO:4)，
IALDFQRGND(SEQ ID NO:1的第15位至第25位；SEQ ID NO:5)，
VAFHFNPRFN(SEQ ID NO:1的第26位至第35位；SEQ ID NO:6)，
TKLDNNW(SEQ ID NO:1的第45位至第62位；SEQ ID NO:7)，
GREERQSVFPFESGK(SEQ ID NO:1的第63位至第78位；SEQ ID NO:8)，
VFPFESGKPFKIQVLVEP(SEQ ID NO:1的第70位至第88位；SEQ ID NO:9)，
DHFKVAVNDAHL(SEQ ID NO:1的第89位至第100位；SEQ ID NO:10)。
在一些实施方案中，所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的C末端部分，其中所述C末端部分包含SEQ ID NO:1所示的氨基酸序列：
MLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI(SEQ IDNO:1)。
在一些实施方案中，所述抗体包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2和SEQ ID NO:18所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:13至SEQ ID NO:18所示的氨基酸序列。
在一些实施方案中，所述抗体包含SEQ ID NO:19所示的HCDR1、SEQ ID NO:20所示的HCDR2、SEQ ID NO:21所示的HCDR3、SEQ ID NO:22所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:24所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:19至SEQ ID NO:24所示的氨基酸序列。
在一些实施方案中，所述抗原结合部分是Fab、F(ab)’ 2或单链Fv。
在一些实施方案中，所述抗体为单克隆抗体。
在一些实施方案中，所述抗体为鼠单克隆抗体或兔单克隆抗体。
在一些实施方案中，所述抗体或其抗原结合部分可用于诊断心力衰竭，但不仅限于此用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
在一些实施方案中，所述抗体或其抗原结合部分可用于辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病，但不仅限于此用途。
第二方面，本申请提供了一种抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的至少一部分包含SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列：
MADNFSLHDALSGSGNPNPQGWPG(SEQ ID NO:2的第1位至第24位；SEQ ID NO:11)，
AWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG(SEQ ID NO:2的第25位至第56位；SEQ ID NO:12)。
在一些实施方案中，所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的N末端部分，其中所述N末端部分包含SEQ ID NO:2所示的氨基酸序列：
MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG
(SEQ ID NO:2)。
在一些实施方案中，所述抗体包含SEQ ID NO:25所示的HCDR1、SEQ ID NO:26所示的HCDR2、SEQ ID NO:27所示的HCDR3、SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2和SEQ ID NO:30所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:25至SEQ ID NO:30所示的氨基酸序列。
在一些实施方案中，所述抗体包含SEQ ID NO:31所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:33所示的HCDR3、SEQ ID NO:34所示的LCDR1、SEQ ID NO:35所示的LCDR2和SEQ ID NO:36所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:31至SEQ ID NO:36所示的氨基酸序列。
在一些实施方案中，所述抗原结合部分是Fab、F(ab)’ 2或单链Fv。
在一些实施方案中，所述抗体为单克隆抗体。
在一些实施方案中，所述抗体为鼠单克隆抗体或兔单克隆抗体，。
在一些实施方案中，所述抗体或其抗原结合部分可用于诊断心力衰竭，但不仅限于此用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
在一些实施方案中，所述抗体或其抗原结合部分可用于辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病，但不仅限于此用途。
第三方面，本申请提供了一种抗体组合产品，其包含第一方面所述的抗体或其抗原结合部分以及第二方面所述的抗体或其抗原结合部分。
第四方面，本申请提供了用于检测来自人受试者的样品中的半乳糖凝集素-3含量的试剂盒，其包含第一方面所述的抗体或其抗原结合部分或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品，其中所述抗体或其抗原结合部分用可检测的标记物标记。
在一些实施方案中，所述可检测的标记物包括酶或显色物质。
在一些实施方案中，所述酶为碱性磷酸酶或辣根过氧化物酶。
在一些实施方案中，所述显色物质为化学发光化合物或荧光物质。
在一些实施方案中，所述化学发光化合物为三联吡啶钌或吖啶酯。
在一些实施方案中，所述荧光物质为荧光色素、荧光微球、时间分辨荧光微球或量子点等。
在一些实施方案中，所述样品来源于全血、血清或血浆。
在一些实施方案中，未用可检测的标记物标记的抗体被预先连接在固体表面上。
在一些实施方案中，所述固体表面选自硝酸纤维膜、磁微粒、荧光微球、时间分辨荧光微球、量子点、塑料和玻璃。
在一些实施方案中，所述试剂盒还包含标准品和/或质控品。
在一些实施方案中，所述试剂盒还包含说明书。
在一些实施方案中，所述试剂盒可用于诊断心力衰竭，但不仅限于此用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
在一些实施方案中，所述抗体或其抗原结合部分可用于辅助诊断肿瘤，类风湿性关节炎，慢性肾脏病，但不仅限于此用途。
第五方面，本申请提供了第一方面所述的抗体或其抗原结合部分或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
在一些实施方案中，所述样品来源于全血、血清或血浆。
第六方面，本申请提供了用于检测样品中的人半乳糖凝集素-3含量的方法，所述方法包括利用第一方面或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品；
其中第一方面所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且第一方面所述的抗体或其抗原结合部分用可检测的标记物标记，或者第二方面所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且第一方面所述的抗体或其抗原结合部分用可检测的标记物标记；和
其中所述抗体或其抗原结合部分通过免疫学方法来检测人半乳糖凝集素-3含量。
在一些实施方案中，所述样品来源于全血、血清或血浆。
在一些实施方案中，所述免疫学方法选自酶联免疫法、免疫荧光法、化学发光法、免疫层析法、免疫沉淀法以及以上的组合。
在一些实施方案中，所述免疫学方法为酶联免疫法。
第七方面，本申请提供了第一方面或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品在检测样品中的人半乳糖凝集素-3含量中的用途。
在一些实施方案中，所述样品来源于全血、血清或血浆。
第八方面，本申请提供了用于诊断个体心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的方法，所述方法包括：从个体获得生物样品；使用第四方面所述的试剂盒检测所述生物样品中人半乳糖凝集素-3的含量；和将所检测到的人半乳糖凝集素-3的含量与参考值比较。
在一些实施方案中，所述生物样品来源于全血、血清或血浆。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
第九方面，本申请提供了第一方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品在制备用于诊断心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的试剂盒中的用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
作为非限制性实施方案，本申请包括以下实施方案：
实施方案1.抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的氨基酸序列的至少一部分包含SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9 或SEQ ID NO:10所示的氨基酸序列。
实施方案2.如实施方案1所述的抗体或其抗原结合部分，其中所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的C末端部分，其中所述C末端部分包含SEQ ID NO:1所示的氨基酸序列。
实施方案3.如实施方案1所述的抗体或其抗原结合部分，其中所述抗体包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2和SEQ ID NO:18所示的LCDR3；或者
所述抗体包含SEQ ID NO:19所示的HCDR1、SEQ ID NO:20所示的HCDR2、SEQ ID NO:21所示的HCDR3、SEQ ID NO:22所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:24所示的LCDR3。
实施方案4.抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的至少一部分包含SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列。
实施方案5.如实施方案4所述的抗体或其抗原结合部分，其中所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的N末端部分，其中所述N末端部分包含SEQ ID NO:2所示的氨基酸序列。
实施方案6.如实施方案4所述的抗体或其抗原结合部分，其中所述抗体包含SEQ ID NO:25所示的HCDR1、SEQ ID NO:26所示的HCDR2、SEQ ID NO:27所示的HCDR3、SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2和SEQ ID NO:30所示的LCDR3；或者
所述抗体包含SEQ ID NO:31所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:33所示的HCDR3、SEQ ID NO:34所示的LCDR1、SEQ ID NO:35所示的LCDR2和SEQ ID NO:36所示的LCDR3。
实施方案7.如实施方案1至6中任一项所述的抗体或其抗原结合部分，其中所述抗原结合部分是Fab、F(ab)’2或单链Fv。
实施方案8.如实施方案1至6中任一项所述的抗体或其抗原结合部分，其中所述抗体为单克隆抗体。
实施方案9.如实施方案8所述的抗体或其抗原结合部分，其中所述抗体为鼠单克隆抗体或兔单克隆抗体。
实施方案10.如实施方案1至9中任一项所述的抗体或其抗原结合部分，其可用于诊断心力衰竭，或者用于辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病。
实施方案11.如实施方案10所述的抗体或其抗原结合部分，其中所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
实施方案12.抗体组合产品，其包含实施方案1至3中任一项所述的抗体或其抗原结合部分以及实施方案4至6中任一项所述的抗体或其抗原结合部分。
实施方案13.用于检测来自人受试者的样品中的半乳糖凝集素-3含量的试剂盒，其包含实施方案1至11中任一项所述的抗体或其抗原结合部分或实施方案12所述的抗体组合产品，其中所述抗体或其抗原结合部分用可检测的标记物标记。
实施方案14.如实施方案13所述的试剂盒，其中所述可检测的标记物包括酶或显色物质。
实施方案15.如实施方案14所述的试剂盒，其中所述酶为碱性磷酸酶或辣根过氧化物酶。
实施方案16.如实施方案14所述的试剂盒，其中所述显色物质为化学发光化合物或荧光物质。
实施方案17.如实施方案16所述的试剂盒，其中所述化学发光化合物为三联吡啶钌或吖啶酯。
实施方案18.如实施方案16所述的试剂盒，其中所述荧光物质为荧光色素、荧光微球、时间分辨荧光微球或量子点。
实施方案19如实施方案13所述的试剂盒，其中所述样品来源于全血、血清或血浆。
实施方案20.如实施方案13所述的试剂盒，其中未用可检测的标记物标记的抗体被预先连接在固体表面上。
实施方案21.如实施方案20所述的试剂盒，其中所述固体表面选自硝酸纤维膜、磁微粒、荧光微球、时间分辨荧光微球、量子点、塑料和玻璃。
实施方案22.如实施方案13所述的试剂盒，其中所述试剂盒还包含标准品和/或质控品。
实施方案23.如实施方案13所述的试剂盒，其中所述试剂盒还包含说明书。
实施方案24.实施方案1-11中任一项所述的抗体或其抗原结合部分或实施方案12所述的抗体组合产品在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
实施方案25.如实施方案24所述的用途，其中所述样品来源于全血、血清或血浆。
实施方案26.用于检测样品中的人半乳糖凝集素-3含量的方法，所述方法包括利用实施方案1-11中任一项所述的抗体或其抗原结合部分或实施方案12所述的抗体组合产品；
其中实施方案1至3中任一项所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且实施方案4至6中任一项所述的抗体或其抗原结合部分用可检测的标记物标记，或者实施方案4至6中任一项所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且实施方案1至3中任一项所述的抗体或其抗原结合部分用可检测的标记物标记；和
其中所述抗体或其抗原结合部分通过免疫学方法来检测人半乳糖凝集素-3含量。
实施方案27.如实施方案26所述的方法，其中所述样品来源于全血、血清或血浆。
实施方案28.如实施方案26所述的方法，其中所述免疫学方法选自酶联免疫法、免疫荧光法、化学发光法、免疫层析法、免疫沉淀法以及以上的组合。
实施方案29.实施方案1-11中任一项所述的抗体或其抗原结合部分或实施方案12所述的抗体组合产品在检测样品中的人半乳糖凝集素-3含量中的用途。
实施方案30.如实施方案29所述的用途，其中所述样品来源于全血、血清或血浆。
实施方案31.用于个体诊断心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的方法，所述方法包括：
从所述个体获得生物样品；
使用实施方案13至23中任一项所述的试剂盒检测所述生物样品中人半乳糖凝集素-3的含量；和
将所检测到的人半乳糖凝集素-3的含量与参考值比较。
实施方案32.如实施方案31所述的方法，其中所述生物样品来源于全血、血清或血浆。
实施方案33.如实施方案31所述的方法，其中所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
实施方案34.实施方案1-11中任一项所述的抗体或其抗原结合部分或实施方案12所述的抗体组合产品在制备用于诊断心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的试剂盒中的用途。
实施方案35.如实施方案34所述的用途，其中所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
附图说明
图1示出了采用B细胞体外克隆技术获得特异性识别半乳糖凝集素-3的免疫球蛋白抗体的示意图。
图2为试纸条/试剂盒示意图，其中1：样品垫；2：结合垫；3：检测线(T线)；4：质控线(C线)；5：NC膜(硝酸纤维膜)；6：吸水垫；7：PVC底板。
序列说明
SEQ ID NO:1
Figure PCTCN2022124770-appb-000001
SEQ ID NO:2
Figure PCTCN2022124770-appb-000002
SEQ ID NO:3(SEQ ID NO:1的第1位至第12位)：MLITILGTVKPN
SEQ ID NO:4(SEQ ID NO:1的第1位至第14位)：MLITILGTVKPNAN
SEQ ID NO:5(SEQ ID NO:1的第15位至第25位)：IALDFQRGND
SEQ ID NO:6(SEQ ID NO:1的第26位至第35位)：VAFHFNPRFN
SEQ ID NO:7(SEQ ID NO:1的第45位至第62位)：TKLDNNW
SEQ ID NO:8(SEQ ID NO:1的第63位至第78位)：GREERQSVFPFESGK
SEQ ID NO:9(SEQ ID NO:1的第70位至第88位)：VFPFESGKPFKIQVLVEP
SEQ ID NO:10(SEQ ID NO:1的第89位至第100位)：DHFKVAVNDAHL
SEQ ID NO:11(SEQ ID NO:2的第1位至第24位)：
Figure PCTCN2022124770-appb-000003
SEQ ID NO:12(SEQ ID NO:2的第25位至第56位)
Figure PCTCN2022124770-appb-000004
SEQ ID NO:13为能够结合人半乳糖凝集素-3的抗体的HCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000005
SEQ ID NO:14为能够结合人半乳糖凝集素-3的抗体的HCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000006
SEQ ID NO:15为能够结合人半乳糖凝集素-3的抗体的HCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000007
SEQ ID NO:16为能够结合人半乳糖凝集素-3的抗体的LCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000008
SEQ ID NO:17为能够结合人半乳糖凝集素-3的抗体的LCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000009
SEQ ID NO:18为能够结合人半乳糖凝集素-3的抗体的LCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000010
SEQ ID NO:19为能够结合人半乳糖凝集素-3的抗体的HCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000011
SEQ ID NO:20为能够结合人半乳糖凝集素-3的抗体的HCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000012
SEQ ID NO:21为能够结合人半乳糖凝集素-3的抗体的HCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000013
SEQ ID NO:22为能够结合人半乳糖凝集素-3的抗体的LCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000014
SEQ ID NO:23为能够结合人半乳糖凝集素-3的抗体的LCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000015
SEQ ID NO:24为能够结合人半乳糖凝集素-3的抗体的LCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000016
SEQ ID NO:25为能够结合人半乳糖凝集素-3的抗体的HCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000017
SEQ ID NO:26为能够结合人半乳糖凝集素-3的抗体的HCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000018
SEQ ID NO:27为能够结合人半乳糖凝集素-3的抗体的HCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000019
SEQ ID NO:28为能够结合人半乳糖凝集素-3的抗体的LCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000020
SEQ ID NO:29为能够结合人半乳糖凝集素-3的抗体的LCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000021
SEQ ID NO:30为能够结合人半乳糖凝集素-3的抗体的LCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000022
SEQ ID NO:31为能够结合人半乳糖凝集素-3的抗体的HCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000023
SEQ ID NO:32为能够结合人半乳糖凝集素-3的抗体的HCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000024
SEQ ID NO:33为能够结合人半乳糖凝集素-3的抗体的HCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000025
SEQ ID NO:34为能够结合人半乳糖凝集素-3的抗体的LCDR1的氨基酸序列：
Figure PCTCN2022124770-appb-000026
SEQ ID NO:35为能够结合人半乳糖凝集素-3的抗体的LCDR2的氨基酸序列：
Figure PCTCN2022124770-appb-000027
SEQ ID NO:36为能够结合人半乳糖凝集素-3的抗体的LCDR3的氨基酸序列：
Figure PCTCN2022124770-appb-000028
SEQ ID NO:37为引物HC-For：acctattactgtcagcacatta
SEQ ID NO:38为引物HC-Rev：ggatacagttggtgcagcatc
SEQ ID NO:39为引物LC-For：gayattgtgmtsacmcarwct
SEQ ID NO:40为引物LC-Rev：acctattactgtcagcacatta
发明详细描述
除非另外指明，否则本发明的实施将采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，否则本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
本文中所用的术语“心力衰竭”或“HF”是指损害心室充满或射出血液的能力的复杂临床综合征。任何结构或功能性心脏障碍可引起HF，其中大部分HF患者具有受损的左心室(LV)心肌功能。HF的综合征包括呼吸困难(呼吸短促)、疲劳和液体潴留。
本文所用的术语“受试者”指人类或非人类生物体。本文优选的受试者是人受试者。
本文所用的术语抗体的“抗原结合部分”或“抗原结合位点”或“靶结合位点”意指保留与抗原或靶特异性结合的能力的结合蛋白(例如抗体或受体)的一个或多个片段，例如免疫球蛋白可变结构域(例如VH或VL)。抗体的抗原结合部分可为全长抗体的片段。术语“抗原结合部分”包含的结合片段的例子包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab’)2片段，包含在铰链区由二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段，(v)包含单个可变结构域的dAb片段；和(vi)分离的互补决定区(CDR)。此外，由分开基因编码的Fv的VL和VH可使用重组法通过合成接头进行连接，使其成为单条蛋白质链，其中VH和VL区配对以形成单价分子(称为单链Fv(scFv))。此类scFv也包含在术语“抗原结合部分”内，与其他形式的单链抗体例如包含一对串联Fv片段(VH-CH1-VH-CH1)的双抗体和“线性抗体”一样，所述一对串联Fv片段连同互补轻链多肽一起形成一对抗原结合位点。并非抗原结合部分的每一个氨基酸均可与抗原结合。例如，抗体的可变结构域包含互补决定区(CDR)和构架区(FR)。
本文所用的术语“CDR”意指在免疫球蛋白可变区序列内的互补决定区。在重链和轻链的可变区中各自存在三个CDR，其对于重链和轻链可变区各自指定为CDR1、CDR2和CDR3。术语“CDR组”指在能够结合抗原的单个可变区中出现的一组三个CDR。这些CDR的确切边界已根据不同的系统进行限定。由Kabat描述的系统(Kabat等人(1971)Ann.NY Acad.Sci.190:382-391；Kabat等人(1987)Sequences of Proteins of Immunological Interest,第四版US Govt.Printing Off.No.165-492；Kabat等人(1991)Sequences of Proteins of Immunological Interest,第五版.NIH公开号91-3242)不仅提供了可应用于任何抗体可变区的明确的残基编号系统，还提供了限定三个CDR的精确残基边界。这些CDR可被称为Kabat CDR。CDR区的氨基酸残基比抗体的重链和轻链可变区中的其他氨基酸残基更可变(例如高变)。Chothia及同事(Chothia和Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:877-883)发现Kabat CDR内的某些亚部分采取几乎相同的肽主链构象，尽管在氨基酸序列水平上具有大的多样性。这些亚部分被指定为L1(LCDR1)、L2(LCDR2)和L3(LCDR3)或H1(HCDR1)、H2(HCDR2)和H3(HCDR3)，其中“L”和 “H”分别指定轻链和重链区。这些区可被称为Chothia CDR，其具有与Kabat CDR重叠的边界。限定与Kabat CDR重叠的CDR的其他边界已由Padlan(1995)FASEB J.9:133-139和MacCallum(1996)J.Mol.Biol.262(5):732-45描述。另外其他CDR边界定义可能不严格遵循本文系统之一，但仍与Kabat CDR重叠。本文的抗体或其抗原结合部分可利用根据这些系统中的任一个限定的CDR。例如，本文的抗体或其抗原结合部分可利用根据Kabat或Chothia限定的CDR。
本文中所用的术语“抗体”也可以是指其抗原结合部分。
本文中所用的术语“SEQ ID NO:X的第a位至第b位”或“SEQ ID NO:X的氨基酸a至b”是指SEQ ID NO:X的氨基酸序列从N端至C端的第a位氨基酸至第b位氨基酸。
本文中所用的术语“SEQ ID NO:X的第a位至第b位”，当序列为核酸序列时，还可以是指SEQ ID NO:X的核酸序列从5’端至3’端的第a位碱基至第b位碱基。
半乳糖凝集素-3及其抗体
半乳糖凝集素-3(GenBank登录号：NC_000014.7(基因)和NP_002297.2(蛋白))是能特异性结合半乳糖的15种哺乳动物β半乳糖苷-结合凝集素或“半乳糖凝集素”之一。半乳糖凝集素-3在文献中被称为LGALS3、MAC-2抗原、糖结合蛋白(CBP)-35、层粘连蛋白结合蛋白、半乳糖-特异性凝集素3、mL-34，L-29，hL-31，εBP和IgE-结合蛋白。半乳糖凝集素-3由羧基末端糖类识别结构域(CRD)和氨基末端串联重复组成(Liu，F.-T.(2000)Role of Galectin-3 in inflammation.Lectins and Patho1ogy.M.Caron和D.Seve，eds.Harwood Academic Publishers，Amsterdam，The Netherlands，p.51；Liu，F.-T.等人(1995)Am.J.Pathol.147：1016)。半乳糖凝集素-3通常分布在许多器官的上皮和各种炎症细胞，包括巨噬细胞以及树突细胞和枯否细胞(Flotte，TJ.等人(1983)Am.J.Pathol.111：112)中。
半乳糖凝集素-3已显示在许多细胞过程中起作用，包括细胞间粘着、细胞-基质相互作用、吞噬、细胞周期、细胞凋亡、血管生成和mRNA剪接。半乳糖凝集素-3已显示通过细胞内和细胞外作用起作用(Sano，H.等人(2000)The Journal of Immunology，165：2156-2164)。其是核不均一核糖核蛋白(hnRNP)的组分(Laing，J.G.等人(1998)Biochemistry 27：5329)，一种pre-mRNA剪接中的因子(Dagher，S.F.等人(1995)Proc.Natl.Acad.ScL USA 92：1213)，并且已发现其控制细胞周期(Kim，H.-R.C.等人(1999)Cancer Res.59：4148)和通过与Bc1-2家族成员相互作用而阻止T细胞凋亡(Yang，R.-Y.等人(1996)Proc.Natl.Acad.Sci.USA 93：6737)。另一方面，已显示从单核/巨噬细胞(Sato，S.等人(1994)J.Biol.Chem.269：4424)和上皮细胞(Lindstedt，R.G.等人(1993)J.Biol.Chem.268：11750)分泌的半乳糖凝集素-3在活化不同类型的细胞例如单核细胞/巨噬细胞(Liu，F.-T.(1993)Immunol Today 14：486)、肥大细胞、中性粒细胞和淋巴细胞(Hsu，D.K.，S.R.等(1996)Am.J.Pathol.148：1661)中充当细胞外分子。已显示半乳糖凝集素-3充当单核细胞和巨噬细胞的新型化学引诱物(Sano，H.等人(2000)The Journal of Immunology，2000，165：2156-2164)。半乳糖凝集素-3牵涉诸如癌症、炎症和心力衰竭(HF)的疾病。如国际专利公开案No.WO2005/040817中所公开的，半乳糖凝集素-3的定量特别适合用于诊断和检测HF的严重程度和预测后果的测定法。
本发明人发现，通过靶向半乳糖凝集素-3C末端和/或N末端结构域可将半乳糖凝集素-3与其它哺乳动物半乳糖凝集素可靠地区分开来。通过使用本文中所公开的抗体，并结合本领域内公知的免疫测定法，例如ELISA，能够可靠地且可重复地检测出样品中的半乳糖凝集素-3含量，并将其与受试者中例如HF的存在，严重程度和分期等相关联。
第一方面，本申请提供了一种抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的氨基酸序列的至少一部分包含SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10或者它们的任意组合所示的氨基酸序列：
MLITILGTVKPN(SEQ ID NO:1的第1位至第12位；SEQ ID NO:3)，
MLITILGTVKPNAN(SEQ ID NO:1的第1位至第14位；SEQ ID NO:4)，
IALDFQRGND(SEQ ID NO:1的第15位至第25位；SEQ ID NO:5)，
VAFHFNPRFN(SEQ ID NO:1的第26位至第35位；SEQ ID NO:6)，
TKLDNNW(SEQ ID NO:1的第45位至第62位；SEQ ID NO:7)，
GREERQSVFPFESGK(SEQ ID NO:1的第63位至第78位；SEQ ID NO:8)，
VFPFESGKPFKIQVLVEP(SEQ ID NO:1的第70位至第88位；SEQ ID NO:9)，
DHFKVAVNDAHL(SEQ ID NO:1的第89位至第100位；SEQ ID NO:10)。
在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:3内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:4内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:5内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:6内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:7内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:8内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:9内；在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:10内。
在一些实施方案中，所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的C末端部分，其中所述C末端部分包含SEQ ID NO:1所示的氨基酸序列：
Figure PCTCN2022124770-appb-000029
在一些实施方案中，所述抗体或其抗原结合部分与人半乳糖凝集素-3结合的位点在SEQ ID NO:1内。
字母表示标准氨基酸代码。虽然这些氨基酸序列描述了线性表位，但也可使用由N端的二级和三级结构产生的并且包含来自序列非连续的各段的氨基酸的表位。而且，本领域技术人员应理解，用于与本文所公开的任意两个抗体结合的两个C末端和/或N末端表位应当相距至少5个氨基酸、至少6个氨基酸、至少7个氨基酸、至少8个氨基酸、至少9个氨基酸、至少10个氨基酸、至少11个氨基酸，至少12个氨基酸，至少13个氨基酸、至少14个氨基酸、至少15个氨基酸或者相距不影响两个抗体与表位相结合的任何距离。两个表位的距离是本领域技术人员能够常规地确定的。
在一些实施方案中，所述抗体包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2和SEQ ID NO:18所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:13至SEQ ID NO:18所示的氨基酸序列。
在一些实施方案中，包含SEQ ID NO:13至SEQ ID NO:18所示的氨基酸序列的抗体为第一抗体。
在一些实施方案中，所述抗体包含SEQ ID NO:19所示的HCDR1、SEQ ID NO:20所示的HCDR2、SEQ ID NO:21所示的HCDR3、SEQ ID NO:22所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:24所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:19至SEQ ID NO:24所示的氨基酸序列。
在一些实施方案中，包含SEQ ID NO:19至SEQ ID NO:24所示的氨基酸序列的抗体为第二抗体。
在一些实施方案中，所述抗原结合部分是Fab、F(ab)’ 2或单链Fv。
在一些实施方案中，所述抗体为单克隆抗体。
在一些实施方案中，所述抗体为鼠单克隆抗体或兔单克隆抗体。
单克隆抗体技术(例如，杂交瘤、重组和噬菌体展示技术)制备单克隆抗体是本领域已知的。
杂交瘤单克隆抗体是由单一的B细胞克隆产生的高度均一、仅针对某一特定抗原表位的抗体。将具有分泌特异性抗体能力的B细胞和具有无限繁殖能力的骨髓瘤细胞融合为B细胞杂交瘤。用具备这种特性的单个杂交瘤细胞培养成细胞群，可制备针对一种抗原表位的特异性抗体即单克隆抗体。
本申请中使用含重组全长序列或C末端和N末端结构域或多肽片段的半乳糖凝集素-3作为免疫原，选择免疫BALB/C雌性小鼠。
初次使用完全佐剂与抗原1:1混合皮下多点注射，之后使用不完全佐剂与抗原1:1混合注射小鼠体内。刺激B淋巴细胞产生相应抗体。然后使用PEG聚乙二醇融合剂将淋巴细胞与小鼠或大鼠的骨髓瘤细胞系相融合，形成杂交瘤细胞系。一般采用HAT选择性培养基。在HAT培养基中，未融合的骨髓瘤细胞因缺乏次黄嘌呤-鸟嘌呤-磷酸核糖转移酶，不能利用补救途径合成DNA而死亡。未融合的淋巴细胞虽具有次黄嘌呤-鸟嘌呤-磷酸核糖转移酶，但其本身不能在体外长期存活也逐渐死亡。只有融合的杂交瘤细胞由于获得了次黄嘌呤-鸟嘌呤-磷酸核糖转移酶且具有骨髓瘤细胞能无限增殖的特性，因此能在HAT培养基中存活和增殖。使用全长或者截短的包含N端和C端的乳糖凝集素-3抗原进行筛选，通过酶联免疫来检测杂交瘤细胞上清液的特异性。
在一些实施方案中，所述抗体或其抗原结合部分可用于诊断心力衰竭。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
在一些实施方案中，所述抗体或其抗原结合部分可用于辅助诊断肿瘤，类风湿性关节炎，慢性肾脏病，但不仅限于此用途。
第二方面，本申请提供了一种抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的至少一部分包含SEQ ID NO:11或SEQ ID NO:12或者SEQ ID NO:11和SEQ ID NO:12二者所示的氨基酸序列：
MADNFSLHDALSGSGNPNPQGWPG(SEQ ID NO:2的第1位至第24位；SEQ ID NO:11)，
AWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG(SEQ ID NO:2的第25位至第56位；SEQ ID NO:12)。
在一些实施方案中，所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的N末端部分，其中所述N末端部分包含SEQ ID NO:2所示的氨基酸序列：
Figure PCTCN2022124770-appb-000030
在一些实施方案中，所述抗体包含SEQ ID NO:25所示的HCDR1、SEQ ID NO:26所示的HCDR2、SEQ ID NO:27所示的HCDR3、SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2和SEQ ID NO:30所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:25至SEQ ID NO:30所示的氨基酸序列。
在一些实施方案中，包含SEQ ID NO:25至SEQ ID NO:30所示的氨基酸序列的抗体为第三抗体。
在一些实施方案中，所述抗体包含SEQ ID NO:31所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:33所示的HCDR3、SEQ ID NO:34所示的LCDR1、SEQ ID NO:35所示的LCDR2和SEQ ID NO:36所示的LCDR3。
在一些实施方案中，所述抗体包含SEQ ID NO:31至SEQ ID NO:36所示的氨基酸序列。
在一些实施方案中，包含SEQ ID NO:31至SEQ ID NO:36所示的氨基酸序列的抗体为第四 抗体。
在一些实施方案中，所述抗原结合部分是Fab、F(ab)’ 2或单链Fv。
在一些实施方案中，所述抗体为单克隆抗体。
在一些实施方案中，所述抗体为鼠单克隆抗体或兔单克隆抗体。
在一些实施方案中，所述抗体或其抗原结合部分可用于诊断心力衰竭，但不仅限于此用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
在一些实施方案中，所述抗体或其抗原结合部分可用于辅助诊断肿瘤，类风湿性关节炎，慢性肾脏病，但不仅限于此用途。
第三方面，本申请提供了一种抗体组合产品，其包含第一方面所述的抗体或其抗原结合部分以及第二方面所述的抗体或其抗原结合部分。
在一些实施方案中，抗体组合产品包括包含SEQ ID NO:13至SEQ ID NO:18所示的氨基酸序列的抗体以及包含SEQ ID NO:25至SEQ ID NO:30所示的氨基酸序列的抗体。
在一些实施方案中，抗体组合产品包括包含SEQ ID NO:13至SEQ ID NO:18所示的氨基酸序列的抗体以及包含SEQ ID NO:31至SEQ ID NO:36所示的氨基酸序列的抗体。
在一些实施方案中，抗体组合产品包括包含SEQ ID NO:19至SEQ ID NO:24所示的氨基酸序列的抗体以及包含SEQ ID NO:25至SEQ ID NO:30所示的氨基酸序列的抗体。
在一些实施方案中，抗体组合产品包括包含SEQ ID NO:19至SEQ ID NO:24所示的氨基酸序列的抗体以及包含SEQ ID NO:31至SEQ ID NO:36所示的氨基酸序列的抗体。
在一些实施方案中，抗体组合产品包括第一抗体至第四抗体的任意组合，例如，抗体组合产品包括第一抗体和第三抗体；或第一抗体和第四抗体；或第二抗体和第四抗体；或第二抗体和第三抗体；或第一抗体、第二抗体和第四抗体；或第一抗体、第二抗体和第三抗体；或第一抗体、第三抗体和第四抗体；或第二抗体、第三抗体和第四抗体；或第一抗体、第二抗体、第三抗体和第四抗体。
在一些实施方案中，抗体组合产品包括第一抗体或第二抗体与第三抗体或第四抗体的组合。
在一些实施方案中，抗体组合产品包括含有第一抗体或第二抗体的溶液与含有第三抗体或第四抗体的溶液的组合。
在一些实施方案中，含有第一抗体或第二抗体的溶液还包括缓冲液，例如磷酸缓冲液；在一些实施方案中，含有第三抗体或第四抗体的溶液还包括缓冲液，例如磷酸缓冲液。
在一些实施方案中，抗体组合产品包括含有第一抗体或第二抗体的粉末与含有第三抗体或第四抗体的粉末的组合。
在一些实施方案中，第一抗体或第二抗体上结合有可检测的标记物；在一些实施方案中，第三抗体或第四抗体上结合有可检测的标记物。
本申请还提供了一种抗体组合产品，所述抗体组合产品包含多个/多种第一方面所述的抗体或其抗原结合部分。
本申请还提供了一种抗体组合产品，所述抗体组合产品包含多个/多种第二方面所述的抗体或其抗原结合部分。
本申请还提供了一种抗体组合产品，所述抗体组合产品包含第一方面所述的抗体或其抗原结合部分以及第二方面所述的抗体或其抗原结合部分。
本申请还提供了一种抗体组合产品，所述抗体组合产品包含多个/多种第一方面所述的抗体或其抗原结合部分以及第二方面所述的抗体或其抗原结合部分。
本申请还提供了一种抗体组合产品，所述抗体组合产品包含第一方面所述的抗体或其抗原结合部分以及多个/多种第二方面所述的抗体或其抗原结合部分。
本申请还提供了一种抗体组合产品，所述抗体组合产品包含多个/多种第一方面所述的抗体或其抗原结合部分以及多个/多种第二方面所述的抗体或其抗原结合部分。
试剂盒
试剂盒中的一种抗体或其抗原结合部分用作捕获抗体，并可被固定在固体表面上以用于捕获半乳糖凝集素-3。而另一种抗体或其抗原结合部分用作检测抗体，并与可检测的标记物连接。
第四方面，本申请提供了用于检测来自人受试者的样品中的半乳糖凝集素-3含量的试剂盒，其包含第一方面所述的抗体或其抗原结合部分或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品，其中所述抗体或其抗原结合部分用可检测的标记物标记。
在一些实施方案中，试剂盒包含第一方面所述的抗体或其抗原结合部分。
在一些实施方案中，试剂盒包含第二方面所述的抗体或其抗原结合部分。
在一些实施方案中，试剂盒包含第三方面所述的抗体组合产品。
在一些实施方案中，所述可检测的标记物包括酶或显色物质。
在一些实施方案中，所述酶为碱性磷酸酶或辣根过氧化物酶。
在一些实施方案中，所述显色物质为化学发光化合物或荧光物质。
在一些实施方案中，所述化学发光化合物为三联吡啶钌或吖啶酯。
在一些实施方案中，所述荧光物质为荧光色素、荧光微球、时间分辨荧光微球或量子点。
在一些实施方案中，所述样品来源于全血、血清或血浆。
在一些实施方案中，所述样品来源于血液。
在一些实施方案中，所述样品来源于体液。
体液可以是经过处理的或未经处理的。从受试者获得体液的方法对于本领域技术人员来说是已知的。
在一些实施方案中，所述样品来源于尿、胃汁、胆汁、唾液、汗液和脊髓液、粪便或肌肉活检组织。
在一些实施方案中，未用可检测的标记物标记的抗体被预先连接在固体表面上。
在一些实施方案中，所述固体表面选自硝酸纤维膜、磁微粒、荧光微球、时间分辨荧光微球、量子点、塑料和玻璃。
在一些实施方案中，所述试剂盒还包含标准品和/或质控品。
在一些实施方案中，所述试剂盒还包含说明书。
在一些实施方案中，所述试剂盒可用于诊断心力衰竭，但不仅限于此用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
在一些实施方案中，所述抗体或其抗原结合部分可用于辅助诊断肿瘤，类风湿性关节炎，慢性肾脏病，但不仅限于此用途。
在一些实施方案中，第一方面所述的抗体用可检测的标记物标记。
在一些实施方案中，第二方面所述的抗体用可检测的标记物标记。
在一些实施方案中，第一方面所述的抗体用可检测的标记物标记且第二方面所述的抗体未用可检测的标记物标记。
在一些实施方案中，第二方面所述的抗体用可检测的标记物标记且第一方面所述的抗体未用可检测的标记物标记。
在一些实施方案中，试剂盒包括用磁性微球和半乳糖凝集素-3的一个抗体偶联形成固相偶联物。参与催化化学发光反应的酶(如辣根过氧化物酶(HRP)或碱性磷酸酶(ALP))来标记另一个抗体，在与待测样品中相应的抗原发生免疫反应后，形成固相包被抗体-待测抗原- 酶标记抗体复合物，经洗涤后，加入发光剂,酶催化和分解底物发光，由化学发光仪仪器读数。
在一些实施方案中，试剂盒包括以荧光材料为标记物建立的免疫层析试纸条。半乳糖凝集素-3的一个抗体包被于硝酸纤维膜上，使用荧光材料作为标记物，将荧光材料与另一个抗体通过共价键偶联形成荧光探针，与抗原反应后，可形成含有标记荧光信号的抗原抗体复合物。然后使用免疫荧光分析仪读取试纸条上的荧光信号。
第五方面，本申请提供了第一方面所述的抗体或其抗原结合部分或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
本申请还提供了第一方面所述的抗体或其抗原结合部分在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
本申请还提供了第二方面所述的抗体或其抗原结合部分在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
在一些实施方案中，所述样品是离体样品。
本申请还提供了第三方面所述的抗体组合产品在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
在一些实施方案中，所述抗体组合产品包含第一方面所述的抗体或其抗原结合部分以及第二方面所述的抗体或其抗原结合部分的任意组合，或包含任意数量或种类的第一方面所述的抗体或其抗原结合部分以及任意数量或种类的第二方面所述的抗体或其抗原结合部分的组合。
在一些实施方案中，所述样品来源于全血、血清或血浆。
在一些实施方案中，所述样品来源于血液。
在一些实施方案中，所述样品来源于体液。
体液可以是经过处理的或未经处理的。从受试者获得体液的方法对于本领域技术人员来说是已知的。样品可以是离体样品。
在一些实施方案中，所述样品来源于尿、胃汁、胆汁、唾液、汗液和脊髓液、粪便或肌肉活检组织。
用于检测体外样品中人半乳糖凝集素-3含量的方法
根据上文的描述，可使用能结合人半乳糖凝集素-3的C末端和/或N末端部分的抗体来定量样品中半乳糖凝集素-3含量。
第六方面，本申请提供了用于检测样品中的人半乳糖凝集素-3含量的方法，所述方法利用第一方面或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品；
其中第一方面所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且第一方面所述的抗体或其抗原结合部分用可检测的标记物标记，或者第二方面所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且第一方面所述的抗体或其抗原结合部分用可检测的标记物标记；和
其中所述抗体或其抗原结合部分通过免疫学方法来检测人半乳糖凝集素-3含量。
在一些实施方案中，所述样品来源于全血、血清或血浆。
在一些实施方案中，所述样品来源于体液。
在一些实施方案中，样品可以是离体样品；检测可以是体外检测。
体液可以是经过处理的或未经处理的。从受试者获得体液的方法对于本领域技术人员来说是已知的。
在一些实施方案中，所述样品来源于尿、胃汁、胆汁、唾液、汗液和脊髓液、粪便或肌肉活检组织。
在一些实施方案中，所述样品来源于血液。
在一些实施方案中，所述免疫学方法选自酶联免疫法、免疫荧光法、化学发光法、免疫层析法、免疫沉淀法以及以上的组合。
在一些实施方案中，所述免疫学方法为酶联免疫法。
在一些实施方案中，可以使用“夹心”测定法检测和定量半乳糖凝集素-3。在该方法中，通常，一个抗体被固定在固体表面上，以便结合并且捕获半乳糖凝集素-3。该抗体因而在本文中称为捕获抗体。第二个抗体被显色物质(例如，荧光基团或化学发光化合物)或酶可检测地标记，以便第二个抗体与半乳糖凝集素-3-复合物的结合表示半乳糖凝集素-3已被捕获。信号的强度与样品中半乳糖凝集素-3的浓度成比例。因此第二个抗体在本文中也称为检测抗体或标记抗体。
此类测定方法可称为双位点免疫测定法、“夹心”法或“夹心免疫测定”。如本领域内已知的，可将捕获和检测抗体同时或相继地与测试样品接触。序贯法(Sequential method)，有时称为“正向(forward)”法，可通过将捕获抗体与样品一起温育和此后在预定的时间上加入标记的检测抗体来完成。可选地，首先可将标记的检测抗体与样品一起温育，然后可将样品与捕获抗体接触(有时称为“反向”法)。此类测定可以以本领域技术人员已知的许多特定形式来执行，包括通过不同的高通量临床实验室分析仪的使用或利用床旁检测(point of care)或家用测试设备来执行。
最常用的酶免疫测定是ELISA。ELISA是使用抗体的标记(例如，酶连接的)形式检测和测量抗原的浓度的技术。存在本领域技术人员公知的不同ELISA形式。本领域内已知的用于ELISA的标准技术描述于“Methods in Immunodiagnosis”，第2版，Rose and Bigazzi，eds.John Wiley&Sons，1980；Campbell等人，“Methods and Immunology”，W.A.Benjamin，Inc.，1964；和Oelleric，M.(1984，J.Clin.Chem.Clin.Biochem.22：895-904中。
在“夹心ELISA”中，将抗体(例如，抗半乳糖凝集素-3)连接至固相(即，微量滴定板)并且与含有抗原(例如，半乳糖凝集素-3)的生物样品接触。然后洗涤固相以除去未结合的抗原。然后将标记的抗体(例如，酶连接的)与结合抗原结合，从而形成抗体-抗原-抗体夹心。可被连接至抗体的酶的实例是碱性磷酸酶、辣根过氧化物酶、荧光素酶、尿素酶和β-半乳糖苷酶。酶联抗体与底物反应产生可被测量的显色反应产物。该测量可用于例如通过将测量值与半乳糖凝集素-3标准曲线相比较来推导存在于样品中的半乳糖凝集素-3的浓度。
免疫荧光法是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光素制成荧光标记物，再用这种荧光抗体(或抗原)作为分子探针检查细胞或组织内的相应抗原(或抗体)。在细胞或组织中形成的抗原抗体复合物上含有荧光素，利用荧光显微镜观察标本，荧光素受激发光的照射而发出明亮的荧光，可以看见荧光所在的细胞或组织，从而确定抗原或抗体的性质、定位，以及利用定量技术测定含量。
化学发光免疫分析(chemiluminescence immunoassay,CLIA)是将具有高灵敏度的化学发光测定技术与高特异性的免疫反应相结合，用于各种抗原、半抗原、抗体、激素、酶、脂肪酸、维生素和药物等的检测分析技术。是继放免分析、酶免分析、荧光免疫分析和时间分辨荧光免疫分析之后发展起来的一项最新免疫测定技术。化学发光免疫分析用化学发光剂直接标记抗原或抗体进行免疫分析。常用于标记的化学发光物质有吖啶酯类化合物，其是有效的发光标记物，其通过起动发光试剂作用而发光，强烈的直接发光在一秒钟内完成，为快速的闪烁发光。吖啶酯作为标记物用于免疫分析，其化学反应简单、快速、无须催化剂，检测小分子抗原采用竞争法，大分子抗原则采用夹心法，非特异性结合少，本底低；与大分子的结合不会减小所产生的光量，从而增加灵敏度。
免疫沉淀是利用抗体特异性反应纯化富集目的蛋白的一种方法。抗体与样品中相应的蛋白结合后，再与蛋白A/G(ProteinA/G)或二抗偶联的琼脂糖或Sepharose珠子孵育，通过离心得到珠子-蛋白A/G或二抗-抗体-目的蛋白复合物，沉淀经过洗涤后，重悬于电泳上样缓冲液，煮沸，在高 温及还原剂的作用下，抗原与抗体解离，离心收集上清，上清中包括抗体、目的蛋白和少量的杂蛋白。
本申请中使用的标记物可选自本领域内通常已知的任意标记物。优选标记物是允许更精确定量的标记物。标记物的实例包括但不限于荧光部分、酶、电化学活性种类、放射性同位素、化学发光分子、胶乳粒子或金颗粒、可检测的配体等。在一个优选实施方案中，标记物是酶或荧光分子。用于将标记物附着至抗体的方法在本领域内是公知的，并且包括共价和非共价连接。
在一些实施方案中，可用荧光化合物标记抗体。当将荧光标记的抗体暴露于适当波长的光时，则可通过发射的荧光检测其存在。其中最常用的荧光标记化合物是Cy3和Cy5(花青染料家族的水溶性荧光染料-“Cy”染料)、异硫氰酸荧光素、罗丹明、藻蓝蛋白、别藻蓝蛋白、邻苯二甲醛和荧光胺。
在一些实施方案中，通过将抗体连接至酶来可检测地标记检测抗体。从而所述酶在与其底物接触时将与底物反应，这种反应可以例如通过分光光度计测量法、荧光测定法或目测法检测。可用于可检测地标记本发明的抗体的酶包括但不限于苹果酸脱氢酶、葡萄球菌核酸酶、δ-V-类固醇异构酶、酵母乙醇脱氢酶、α-甘油磷酸脱氢酶、磷酸丙糖异构酶、辣根过氧化物酶、碱性磷酸酶、天冬酰胺酶、葡糖氧化酶、β-半乳糖苷酶、核糖核酸酶、尿素酶、过氧化氢酶、葡萄糖-6-磷酸脱氢酶、葡糖淀粉酶和乙酰胆碱酯酶。
还可使用放射性标记的抗体来实现检测。然后可通过使用放射免疫测定法检测抗体。可利用此类方法如γ计数器或闪烁计数器的使用或利用放射自显影术来检测放射性同位素。对于本申请的目的特别有用的同位素是 3H、 131I、 35S、 14C，和优选的 125I。
还可通过将抗体偶联至化学发光部分来可检测地标记抗体。然后通过检测化学反应过程中产生的发光的存在来测定化学发光抗体的存在。特别有用的化学发光标记化合物的实例是鲁米诺，萤光素、异氨基苯二酰肼、咪唑、吖啶盐和草酸酯。
第七方面，本申请提供了第一方面或第二方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品在检测样品中的人半乳糖凝集素-3含量中的用途。
在一些实施方案中，所述样品来源于全血、血清或血浆。
在一些实施方案中，所述样品来源于血液。
在一些实施方案中，样品可以是离体样品。
用于诊断心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的方法
第八方面，本申请提供了用于诊断个体心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的方法，所述方法包括：从个体获得生物样品；使用第四方面所述的试剂盒检测所述生物样品中人半乳糖凝集素-3的含量；和将所检测到的人半乳糖凝集素-3的含量与参考值比较。
在一些实施方案中，使用第四方面所述的试剂盒检测所述生物样品中人半乳糖凝集素-3的含量包括检测可检测的标记物的量，将检测结果与标准曲线比较。
在一些实施方案中，所述参考值是来自健康人类的生物样品中人半乳糖凝集素-3的含量。
在一些实施方案中，所述参考值是本领域常规的或已知的人半乳糖凝集素-3含量。
在一些实施方案中，所述生物样品来源于全血、血清或血浆。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
第九方面，本申请提供了第一方面所述的抗体或其抗原结合部分或第三方面所述的抗体组合产品在制备用于诊断心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的试剂盒中的用途。
在一些实施方案中，所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测 定心力衰竭的严重程度或预后心力衰竭的预测。
本申请存在众多优势和有益效果，例如：
利用重组的半乳糖凝集素-3多肽来制备并筛选可用于诊断的抗体，产量高且灵敏度高；和/或
通过免疫技术测定心力衰竭患者血清中的半乳糖凝集素-3蛋白含量，该指标可作为患者预后恢复或者疾病早查中的关键依据，具有较高的检测灵敏度与特异性。
实施例
提供以下实施例仅是对本申请的一些实施方案进行举例说明，没有任何限制的目的或性质。此外，如没有特别说明，实施例中的方法将按照本领域的常规方案进行。
实施例1：鼠单克隆抗体的获得
将人半乳糖凝集素-3免疫Balb/c小鼠获取表达抗体的杂交瘤细胞，检测并筛选以获得特异性识别半乳糖凝集素-3的抗体。具体方法如图1所示。
采用重组半乳糖凝集素-3全长抗原免疫Balb/c小鼠，选用6-8周雌性Balb/c小鼠，共免疫六只。抗原使用剂量为100μg/只。抗原与完全佐剂1:1混合比例进行乳化，皮下多点注射；免疫周期为六次。每针间隔一周。
待效价达到1:10000以上与小鼠骨髓瘤细胞融合，使用ELISA间接法检测细胞上清液吸光度OD值，根据OD值结果选定具有阳性孔的细胞，采用有限稀释法克隆细胞，按每孔1个细胞接种在培养皿中，细胞增殖后成为单克隆细胞系。
筛选获得46个小鼠单克隆抗体杂交瘤细胞。使用体内诱生法产生抗体。选择雄性6周龄以上的Balb/c小鼠，使用弗氏不完全佐剂致敏(500μL/只)。7天左右接种细胞株，使密度达到2X10 6。7天左右可明显看见腹部膨大；抽取腹水并进行过滤除菌，滤膜孔径为0.45μm，分装并冻存于-20℃。
扩大培养及冻存：将筛选出的特异性强的细胞株及时扩大培养，使用10％DMSO冻存液；按1ml/管冻存至液氮保存。
实施例2：抗体的鉴定
对实施例1中所获得的46个小鼠单克隆抗体进行进一步鉴定。利用棋盘法鉴定捕捉抗体和检测抗体对。简言之，将所有46个抗体以2ng/mL(PBS调整抗体浓度)的浓度，每孔100μL的量铺到384孔微量滴定板作为捕捉抗体，每个捕捉抗体铺47孔，18小时后，每孔添加100μL的含4％BSA的PBS进行封闭，然后将标准品Galectin-3(购自BBI Solution)以每孔100μL的量添加到384孔微量滴定板内，PBST清洗5遍后用生物素标记的除其本身之外的抗体，作为检测抗体(浓度为2ng/mL，每孔加100μL)来检测对应的孔。
分别用ELISA检测所获得的46个抗体，剔除阳性信号弱的抗体。获得两对(4个)对人Galectin-3具有专一性的小鼠单克隆抗体。
将筛选出的特异性强的细胞株及时扩大培养，使用10％DMSO冻存液；按1ml/管冻存至液氮保存。采用蛋白A柱子(购自LIFE TECH)分离纯化特异性小鼠抗体。获得四种抗体，分别为G3-5-5、G3-12-1、G3-2-1和G3-2-3，即本申请的第一抗体至第四抗体。
用RNA miniprep试剂盒(购自Qiagen)分离阳性杂交瘤细胞的RNA，并用逆转录酶(购自Biosystems)逆转录成cDNA。然后采用下述小鼠抗体特异性引物进行PCR。获得小鼠抗体Ig重链和轻链的扩增产物后进行测序。
HC-For：acctattactgtcagcacatta(SEQ ID NO:37)
HC-Rev：ggatacagttggtgcagcatc(SEQ ID NO:38)
LC-For：gayattgtgmtsacmcarwct(SEQ ID NO:39)
LC-Rev：acctattactgtcagcacatta(SEQ ID NO:40)
经测序，G3-5-5(第一抗体)包含SEQ ID NO:13所示的HCDR1，SEQ ID NO:14所示的HCDR2和SEQ ID NO:15所示的HCDR3的重链可变区，以及含SEQ ID NO:16所示的LCDR1，SEQ ID NO:17所示的LCDR2和SEQ ID NO:18所示的LCDR3的轻链可变区。
G3-12-1(第二抗体)包含SEQ ID NO:19所示的HCDR1，SEQ ID NO:20所示的HCDR2和SEQ ID NO:21所示的HCDR3的重链可变区，以及含SEQ ID NO:22所示的LCDR1，SEQ ID NO:23所示的LCDR2和SEQ ID NO:24所示的LCDR3轻链可变区。
G3-2-1(第三抗体)包含SEQ ID NO:25所示的HCDR1，SEQ ID NO:26所示的HCDR2和SEQ ID NO:27所示的HCDR3的重链可变区，以及含SEQ ID NO:28所示的LCDR1，SEQ ID NO:29所示的LCDR2和SEQ ID NO:30所示的LCDR3轻链可变区。
G3-2-3(第四抗体)包含SEQ ID NO:31所示的HCDR1，SEQ ID NO:32所示的HCDR2和SEQ ID NO:33所示的HCDR3的重链可变区，以及含SEQ ID NO:34所示的LCDR1，SEQ ID NO:35所示的LCDR2和SEQ ID NO:36所示的LCDR3轻链可变区。
实施例3：抗原表位的确定
对Galectin-3进行生物信息学分析，预测结合位点，并合成相应的短重叠肽共88条，短肽偶联至载体蛋白后，利用酶联免疫法进行检测。
以5μg/ml(PBS调整抗体浓度)的浓度包被短肽融合蛋白，每孔100μL的量铺到96孔酶标板中，4℃过夜反应，每孔添加130μL的含有1％的BSA封闭2小时。
将4ng/ml的实施例2中获得的第一抗体、第二抗体、第三抗体、第四抗体进行标记，每孔50μL的量添加到96孔中；PBST清洗5遍。用生物素标记的除其本身之外的抗体，作为检测抗体。以每孔50μL的量来检测对应的孔。根据融合短肽和抗体的相互作用，和结合牢固程度来获得抗体的具体抗原结合表位。
经检测，第一抗体和第二抗体能够结合人半乳糖凝集素-3的C末端部分上的非连续的表位。第三抗体和第四抗体能够结合人半乳糖凝集素-3的N末端部分上的非连续的表位。
实施例4.用于人半乳糖凝集素-3的定量检测的荧光免疫层析测定
如图2所示，荧光免疫层析试剂盒包括样品垫、结合垫、硝酸纤维膜、吸水垫和聚氯乙烯(PVC)底板。
在本实施例中，将人半乳糖凝集素-3的第一抗体G3-5-5和二抗被预先固定在硝酸纤维膜上以形成测试线或对照线，加入含待测物的样品后，待测物与结合垫上的标记抗体结合，随层析作用进一步被固定在T线上的单抗捕获。过量的标记抗体被C线上的二抗捕获，通过特定检测仪器读取T/C线上的标记物信号强度，并代入预先绘制的标准曲线。
制备偶联有人半乳糖凝集素-3的第四抗体G3-2-3的荧光微粒：
将表面氨基化的荧光素颗粒分散在磷酸缓冲液中，加入戊二醛反应4-8小时，离心除去戊二醛，将荧光素颗粒重新分散在磷酸缓冲液中,加入人半乳糖凝集素-3的第四抗体G3-2-3，反应4-12小时，洗涤后获得荧光素颗粒-抗体复合物,保存在含0.1％BSA和0.1％Tween 20的磷酸缓冲液中。
荧光免疫层析试纸的组装：
将荧光素颗粒-抗体复合物涂覆在结合垫上，将第一抗体G3-5-5和二抗分别以线状连接于抗体承载膜上形成T线和C线，并在带有粘合剂的衬板上相互搭接地粘合样品垫、结合垫、抗体承载膜、吸水纸。
人半乳糖凝集素-3定标品的制备：
用标准品缓冲液(50mM Tris-HCl、1％BSA、0.9％NaCl，pH7.4)将人半乳糖凝集素-3蛋白配置成浓度为0、5、20、40、60、100ng/mL，每瓶1mL分装，4℃保存备用。
离体样品中的人半乳糖凝集素-3检测：
本申请所述的试剂盒的使用操作程序如下：将试剂和待检样品恢复至室温，按照说明书开启免疫荧光分析仪；使用滴管垂直加入待测样品至样品垫对应的检测卡加样孔内孵育15分钟。孵育完成后，使用免疫荧光分析仪对检测卡进行信号检测，样品中的人半乳糖凝集素-3含量与其相对荧光信号成正比例关系。
此外，固定在硝酸纤维膜上的抗体可以是第一抗体或第二抗体，也可以是第三抗体或第四抗体。同样地，与荧光微粒偶联的抗体可以是第三抗体或第四抗体，也可以是第一抗体或第二抗体。
使用与上述相同的方法，还制备了以不同抗体进行组合的荧光免疫层析试剂盒，并检测所制备的试剂盒。
在本实施例中，还将人半乳糖凝集素-3的第三抗体G3-2-1和二抗预先固定在硝酸纤维膜上以形成测试线或对照线，并且制备偶联有人半乳糖凝集素-3的第二抗体G3-12-1的荧光微粒，以制备荧光免疫层析试剂盒并对其进行检测。
实施例5：人半乳糖凝集素-3荧光免疫层析性能评价
采用实施例4中的方法及制备的试纸，对人半乳糖凝集素-3定标品进行检测，绘制标准曲线。同样地，对待测样品进行测量，根据样品发光强度计算样品中的人半乳糖凝集素-3浓度。
灵敏度的检测：参照CLSI EP17-A文件推荐的实验方案，计算人半乳糖凝集素-3荧光免疫层析试剂的灵敏度，灵敏度为1.5ng/mL。
线性的检测：定标品做线性分析，对第一抗体和第四抗体的组合计算线性相关系数，r＝0.9996，并且试剂盒对人半乳糖凝集素-3样品检测的线性范围为1.5ng/mL至80ng/mL。对第三抗体和第二抗体的组合计算线性相关系数，r＝0.9987，且试剂盒对人半乳糖凝集素-3样品检测的线性范围为2ng/mL至100ng/mL。
精密度测定：取浓度为15ng/mL和60ng/mL的两个人半乳糖凝集素-3样品，每个样品每个浓度各做3个平行，用三批试剂盒进行检测，计算试剂盒批内及批间差，结果表明该试剂盒批内及批间差均小于10％。
实施例6.用于人半乳糖凝集素-3的定量检测的磁微粒化学发光测定
磁微粒化学发光试剂盒包括磁微粒包被物、酶标记物、化学发光底物液、分析缓冲液、洗涤液及标准品和质控品。
在本实施例中，将人半乳糖凝集素-3的第一抗体G3-5-5包被在磁微粒上，将人半乳糖凝集素-3的第四抗体G3-2-3标记在碱性磷酸酶上，加入经分析缓冲液处理的含待测物的样品后，待测物与磁微粒包被物及酶标记物结合，形成免疫复合物，经过洗涤液清洗后加入化学发光底物液，通过化学发光检测仪器发光强度，并代入预先绘制的标准曲线。
制备包被有人半乳糖凝集素-3的第一抗体G3-5-5的磁微粒包被物：
将表面羧基化的磁微粒分散在磷酸缓冲液中，加入人半乳糖凝集素-3的第一抗体G3-5-5，混匀30分钟，加入碳亚二胺置于4摄氏度反应12-15小时，用磁力架分离，洗涤后获得磁微粒-抗体复合物,保存在含0.5％BSA和0.1％Tween 20的磷酸缓冲液中。
制备人半乳糖凝集素-3的第四抗体G3-2-3的酶标记物：
在碱性磷酸酶溶液中加入戊二醛反应4-8小时，透析除去戊二醛，加入人半乳糖凝集素-3的第四抗体G3-2-3，反应4-12小时，保存在含50％甘油、0.1％BSA和0.1％Tween 20的磷酸缓冲 液中。
制备化学发光底物液：化学发光底物液为含有10％的3-(2’-螺旋金刚烷)-4-甲氧基-4-(3’-磷酰氧基)苯基-1,2-二氧乙烷(AMPPD)的Tris缓冲液。
制备分析缓冲液：分析缓冲液为含有1％BSA和0.1％Tween20的磷酸缓冲液。
制备洗涤液：洗涤液为含1％的Tween20的磷酸缓冲液。
制备标准品和质控品：标准品为用标准品缓冲液(50mM Tris-HCl、1％BSA、0.9％NaCl，pH7.4)将人半乳糖凝集素-3蛋白分别配置成浓度为0、5、20、40、60、100ng/mL，每瓶1mL分装，4℃保存备用。
质控品为用标准品缓冲液将人半乳糖凝集素-3蛋白分别配置成浓度为10、50ng/mL，每瓶1mL分装，4℃保存备用。
磁微粒化学发光试剂盒的组装：
将磁微粒包被物、酶标记物、化学发光底物液、分析缓冲液、洗涤液及标准品和质控品组装成人半乳糖凝集素-3磁微粒化学发光试剂盒。
离体样品中的人半乳糖凝集素-3检测：
本申请所述的磁微粒化学发光试剂盒的使用操作程序如下：取50μL待检测样本或标准品/质控品、50μL分析缓冲液、50μL磁微粒包被物、50μL酶标记物于反应杯一起进行反应，37℃孵育10分钟，反应完成后，在磁板上对上述反应物进行磁分离清洗3次，清洗完成后加入100μL化学发光底物液，混匀后即可进行检测。
此外，包被在磁微粒上的抗体可以是第一抗体或第二抗体，也可以是第三抗体或第四抗体。同样地，与碱性磷酸酶偶联的抗体可以是第三抗体或第四抗体，也可以是第一抗体或第二抗体。
使用与上述相同的方法，还制备了以不同抗体进行组合的磁微粒化学发光试剂盒，并检测所制备的试剂盒。
实施例7：人半乳糖凝集素-3磁微粒化学发光试剂盒性能评价
采用实施例6中的方法及制备的试剂盒，对人半乳糖凝集素-3定标品进行检测，绘制标准曲线。同样地，对待测样品进行测量，根据样品发光强度计算样品中的人半乳糖凝集素-3浓度。
灵敏度的检测：参照CLSI EP17-A文件推荐的实验方案，计算人半乳糖凝集素-3荧光免疫层析试剂的灵敏度，灵敏度为1.0ng/mL。
线性的检测：定标品做线性分析，对第一抗体和第四抗体的组合计算线性相关系数，r＝0.9996，并且试剂盒对人半乳糖凝集素-3样品检测的线性范围为1.0ng/mL至100ng/mL。对第三抗体和第二抗体的组合计算线性相关系数，r＝0.9997，且试剂盒对人半乳糖凝集素-3样品检测的线性范围为1.0ng/mL至100ng/mL。
精密度测定：取浓度为15ng/mL和60ng/mL的两个人半乳糖凝集素-3样品，每个样品每个浓度各做3个平行，用三批试剂盒进行检测，计算试剂盒批内及批间差，结果表明该试剂盒批内及批间差均小于5％。

Claims (35)

  1. 抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的氨基酸序列的至少一部分包含SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10所示的氨基酸序列。
  2. 如权利要求1所述的抗体或其抗原结合部分，其中所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的C末端部分，其中所述C末端部分包含SEQ ID NO:1所示的氨基酸序列。
  3. 如权利要求1所述的抗体或其抗原结合部分，其中所述抗体包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2和SEQ ID NO:18所示的LCDR3；或者
    所述抗体包含SEQ ID NO:19所示的HCDR1、SEQ ID NO:20所示的HCDR2、SEQ ID NO:21所示的HCDR3、SEQ ID NO:22所示的LCDR1、SEQ ID NO:23所示的LCDR2和SEQ ID NO:24所示的LCDR3。
  4. 抗体或其抗原结合部分，所述抗体或其抗原结合部分能够结合人半乳糖凝集素-3的氨基酸序列的至少一部分，所述人半乳糖凝集素-3的至少一部分包含SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列。
  5. 如权利要求4所述的抗体或其抗原结合部分，其中所述人半乳糖凝集素-3的氨基酸序列的至少一部分位于人半乳糖凝集素-3的N末端部分，其中所述N末端部分包含SEQ ID NO:2所示的氨基酸序列。
  6. 如权利要求4所述的抗体或其抗原结合部分，其中所述抗体包含SEQ ID NO:25所示的HCDR1、SEQ ID NO:26所示的HCDR2、SEQ ID NO:27所示的HCDR3、SEQ ID NO:28所示的LCDR1、SEQ ID NO:29所示的LCDR2和SEQ ID NO:30所示的LCDR3；或者
    所述抗体包含SEQ ID NO:31所示的HCDR1、SEQ ID NO:32所示的HCDR2、SEQ ID NO:33所示的HCDR3、SEQ ID NO:34所示的LCDR1、SEQ ID NO:35所示的LCDR2和SEQ ID NO:36所示的LCDR3。
  7. 如权利要求1至6中任一项所述的抗体或其抗原结合部分，其中所述抗原结合部分是Fab、F(ab)’ 2或单链Fv。
  8. 如权利要求1至6中任一项所述的抗体或其抗原结合部分，其中所述抗体为单克隆抗体。
  9. 如权利要求8所述的抗体或其抗原结合部分，其中所述抗体为鼠单克隆抗体或兔单克隆抗体。
  10. 如权利要求1至9中任一项所述的抗体或其抗原结合部分，其用于诊断心力衰竭，或者用于辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病。
  11. 如权利要求10所述的抗体或其抗原结合部分，其中所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
  12. 抗体组合产品，其包含权利要求1至3中任一项所述的抗体或其抗原结合部分以及权利要求4至6中任一项所述的抗体或其抗原结合部分。
  13. 用于检测来自人受试者的样品中的半乳糖凝集素-3含量的试剂盒，其包含权利要求1至11中任一项所述的抗体或其抗原结合部分或权利要求12所述的抗体组合产品，其中所述抗体或其抗原结合部分用可检测的标记物标记。
  14. 如权利要求13所述的试剂盒，其中所述可检测的标记物包括酶或显色物质。
  15. 如权利要求14所述的试剂盒，其中所述酶为碱性磷酸酶或辣根过氧化物酶。
  16. 如权利要求14所述的试剂盒，其中所述显色物质为化学发光化合物或荧光物质。
  17. 如权利要求16所述的试剂盒，其中所述化学发光化合物为三联吡啶钌或吖啶酯。
  18. 如权利要求16所述的试剂盒，其中所述荧光物质为荧光色素、荧光微球、时间分辨荧光微球或量子点。
  19. 如权利要求13所述的试剂盒，其中所述样品来源于全血、血清或血浆。
  20. 如权利要求13所述的试剂盒，其中未用可检测的标记物标记的抗体被预先连接在固体表面上。
  21. 如权利要求20所述的试剂盒，其中所述固体表面选自硝酸纤维膜、磁微粒、荧光微球、时间分辨荧光微球、量子点、塑料和玻璃。
  22. 如权利要求13所述的试剂盒，其中所述试剂盒还包含标准品和/或质控品。
  23. 如权利要求13所述的试剂盒，其中所述试剂盒还包含说明书。
  24. 权利要求1-11中任一项所述的抗体或其抗原结合部分或权利要求12所述的抗体组合产品在制备检测样品中的人半乳糖凝集素-3含量的试剂盒中的用途。
  25. 如权利要求24所述的用途，其中所述样品来源于全血、血清或血浆。
  26. 用于检测样品中的人半乳糖凝集素-3含量的方法，所述方法包括利用权利要求1-11中任一项所述的抗体或其抗原结合部分或权利要求12所述的抗体组合产品；
    其中权利要求1至3中任一项所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且权利要求4至6中任一项所述的抗体或其抗原结合部分用可检测的标记物标记，或者权利要求4至6中任一项所述的抗体或其抗原结合部分用于捕获人半乳糖凝集素-3且权利要求1至3中任一项所述的抗体或其抗原结合部分用可检测的标记物标记；和
    其中所述抗体或其抗原结合部分通过免疫学方法来检测人半乳糖凝集素-3含量。
  27. 如权利要求26所述的方法，其中所述样品来源于全血、血清或血浆。
  28. 如权利要求26所述的方法，其中所述免疫学方法选自酶联免疫法、免疫荧光法、化学发光法、免疫层析法、免疫沉淀法以及以上的组合。
  29. 权利要求1-11中任一项所述的抗体或其抗原结合部分或权利要求12所述的抗体组合产品在检测离体样品中的人半乳糖凝集素-3含量中的用途。
  30. 如权利要求29所述的用途，其中所述离体样品来源于全血、血清或血浆。
  31. 用于诊断个体心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的方法，所述方法包括：
    从所述个体获得生物样品；
    使用权利要求13至23中任一项所述的试剂盒检测生物样品中人半乳糖凝集素-3的含量；和
    将所检测到的人半乳糖凝集素-3的含量与参考值比较。
  32. 如权利要求31所述的方法，其中所述生物样品来源于全血、血清或血浆。
  33. 如权利要求31所述的方法，其中所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
  34. 权利要求1-11中任一项所述的抗体或其抗原结合部分或权利要求12所述的抗体组合产品在制备用于诊断心力衰竭或辅助诊断肿瘤、类风湿性关节炎或慢性肾脏病的试剂盒中的用途。
  35. 如权利要求34所述的用途，其中所述诊断心力衰竭包括评估心力衰竭的风险、诊断心力衰竭的存在、测定心力衰竭的严重程度或预后心力衰竭的预测。
PCT/CN2022/124770 2021-10-15 2022-10-12 半乳糖凝集素-3的免疫测定 WO2023061388A1 (zh)

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