Anti-Kim-1 antibody and application thereof

Abstract

The invention discloses an anti-Kim-1 antibody and application thereof, and belongs to the field of immune globulin. Specifically disclosed is an anti-Kim-1 antibody comprising a heavy chain variable region (1st-120th of SEQ ID NO: 1) and a light chain variable region (1st-115th of SEQ ID NO: 2) or an antigen binding fragment thereof, including a murine monoclonal antibody CV18 and a human chimeric antibody hCV18 developed based on the murine monoclonal antibody CV18. The antibodies CV18 and hCV18 provided by the invention have the characteristics of high affinity and specificity, have good sensitivity and specificity when being used for detecting Kim-1 protein, and can be prepared into products for detecting Kim-1 clinically, such as detection reagents or kits. The invention solves the problems of relative shortage of antibody raw materials, poor specificity and low sensitivity of Kim-1 detection items in the current market, and can be used as an auxiliary diagnosis tool for kidney related diseases (such as acute kidney injury and the like).

Description

Technical Field

[0001] The invention belongs to the field of immunoglobulins, and specifically relates to an anti-Kim-1 antibody and an application thereof. Background Art

[0002] Kidney injury molecule 1 (Kim-1), also known as hepatitis A virus cell receptor 1 (HAVCR1), is a transmembrane glycoprotein of renal proximal tubular epithelial cells and belongs to the immunoglobulin gene superfamily. Kim-1 is not expressed in healthy kidneys. When the kidneys are damaged, the expression of KIM-1 will increase significantly. This characteristic makes Kim-1 a sensitive biomarker for diagnosing and monitoring renal injury. Especially in the early stages of renal injury, KIM-1 can show obvious changes, which helps to timely detect and intervene in renal disease, reverse renal function damage, and avoid further deterioration of the disease. KIM-1 can quickly, sensitively and specifically reflect the damage and recovery process of various renal diseases. It is not only a reliable biological marker for detecting early renal injury, but also can be used to evaluate the recovery of renal disease and provide a basis for doctors to formulate personalized treatment plans. Using specific antibodies against Kim-1 to monitor changes in KIM-1 levels can detect renal injury early, evaluate the progression of the disease and the effect of treatment, thereby providing key information for timely treatment and providing a basis for doctors to adjust treatment plans. Although the current Kim-1 detection technology has made certain progress, there is still a relative shortage of antibody raw materials for the Kim-1 detection project, as well as insufficient specificity and sensitivity.

[0003] In view of this, research and development of highly specific and sensitive anti-Kim-1 antibodies and their supporting immunological detection methods and immunoassay kits will help to achieve early and accurate identification of renal injury, improve the accuracy of clinical diagnosis, optimize patient management strategies, and provide strong support for the timely treatment and prognosis evaluation of kidney diseases. Summary of the invention

[0004] One of the purposes of the present invention is to provide an anti-Kim-1 antibody and its application. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] To achieve the above object, the present invention first provides an anti-Kim-1 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 having an amino acid sequence as shown at positions 31-35 of SEQ ID NO:1, a CDR2 having an amino acid sequence as shown at positions 50-66 of SEQ ID NO:1, and a CDR3 having an amino acid sequence as shown at positions 99-109 of SEQ ID NO:1; and the light chain variable region comprises a CDR1 having an amino acid sequence as shown at positions 24-39 of SEQ ID NO:2, a CDR2 having an amino acid sequence as shown at positions 55-61 of SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown at positions 94-105 of SEQ ID NO:2.

[0006] The antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanoantibody), minibody and minimum recognition unit (MRU).

[0007] The sequences of the complementarity determining regions (CDRs) described herein may be defined according to the Kabat numbering system.

[0008] The heavy chain variable region and the light chain variable region both contain a framework region (FR, the region outside the CDR in the variable region). The framework region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, horses, donkeys, chickens, monkeys, camels, etc.).

[0009] Furthermore, the amino acid sequence of the heavy chain variable region may be as shown at positions 1-120 of SEQ ID NO:1, and the amino acid sequence of the light chain variable region may be as shown at positions 1-115 of SEQ ID NO:2.

[0010] Furthermore, the antibody or antigen-binding fragment thereof may also include a heavy chain constant region (CH) and a light chain constant region (CL). The heavy chain constant region may be selected from the heavy chain constant region of IgG, IgA, IgM, IgD or IgE. The heavy chain constant region may also be selected from CH1, Fc and CH3 domains. The light chain constant region may be selected from Kappa (κ) or lambda (λ) type light chain constant regions. The heavy chain constant region and the light chain constant region may be derived from humans or non-human mammals (such as mice, rats, guinea pigs, rabbits, sheep, horses, donkeys, chickens, monkeys, camels, etc.).

[0011] Further, the heavy chain constant region can be selected from the heavy chain constant region of human IgG subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant region can also be selected from the heavy chain constant region of mouse IgG subclasses such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG5 and IgG6.

[0012] Furthermore, the antibody or antigen-binding fragment thereof may further comprise the heavy chain constant region and the light chain constant region shown in A1) or A2) below: A1) the amino acid sequence of the heavy chain constant region is as shown in positions 121-444 of SEQ ID NO: 1, and the amino acid sequence of the light chain constant region is as shown in positions 116-222 of SEQ ID NO: 2; A2) The amino acid sequence of the heavy chain constant region is shown at positions 121-450 of SEQ ID NO:5, and the amino acid sequence of the light chain constant region is shown at positions 116-222 of SEQ ID NO:6.

[0013] Furthermore, the antibody or antigen-binding fragment thereof may comprise the heavy chain and light chain shown in B1) or B2) below: B1) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2; B2) The amino acid sequence of the heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the light chain is shown in SEQ ID NO:6.

[0014] The antibody containing the heavy chain and light chain described in B1) may be a mouse monoclonal antibody and may be named CV18.

[0015] The antibody containing the heavy chain and light chain described in B2) may be a human chimeric antibody and may be named hCV18.

[0016] The present invention also provides a biomaterial, which may be any of the following: C1) a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of any one of the antibodies or antigen-binding fragments thereof described herein; C2) a nucleic acid molecule encoding the heavy chain and light chain of any antibody or antigen-binding fragment thereof described herein; C3) an expression cassette containing the nucleic acid molecule described in C1) or C2); C4) a recombinant vector containing the nucleic acid molecule described in C1) or C2); C5) a recombinant microorganism containing the nucleic acid molecule described in C1) or C2); C6) A recombinant host cell containing the nucleic acid molecule described in C1) or C2).

[0017] Furthermore, the biological materials can express any of the antibodies or antigen-binding fragments thereof described herein.

[0018] In the above biological material, the recombinant vector can be a cloning vector or an expression vector.

[0019] Furthermore, the recombinant vector may be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof described herein into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector).

[0020] The prokaryotic expression vector can be selected from Escherichia coli expression vectors (such as pET series vectors, etc.). The eukaryotic expression vector can be selected from yeast expression vectors (such as pYES2, pPICZaA, etc.), insect cell expression vectors (such as pFastBac1, pMT-Bip-V5-HisA, pAc5.1, etc.) and mammalian cell expression vectors (such as pCMV3, pcDNA3.1, pcDNA3.4, etc.). The viral expression vector can be selected from adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, lentivirus (LV) vectors, poxvirus vectors, retrovirus vectors, rhabdovirus (baculovirus) vectors, papillomavirus vectors, Sendai virus vectors and Simian virus expression vectors.

[0021] In the above biological material, the nucleic acid molecule may be any of the following: D1) a DNA molecule encoding a sequence as shown in positions 1 to 360 of SEQ ID NO: 3 and positions 1 to 345 of SEQ ID NO: 4; D2) a DNA molecule with coding sequences as shown in SEQ ID NO:3 and SEQ ID NO:4; D3) A DNA molecule encoding the sequences shown in SEQ ID NO:7 and SEQ ID NO:8.

[0022] The DNA molecule shown at positions 1-360 of SEQ ID NO:3 may be a gene encoding the heavy chain variable region of the murine monoclonal antibody CV18, which encodes an amino acid sequence as the heavy chain variable region shown at positions 1-120 of SEQ ID NO:1.

[0023] The DNA molecule shown at positions 1-345 of SEQ ID NO:4 may be a gene encoding the light chain variable region of the murine monoclonal antibody CV18, which encodes an amino acid sequence such as the light chain variable region shown at positions 1-115 of SEQ ID NO:2.

[0024] The DNA molecules shown in SEQ ID NO: 3 and SEQ ID NO: 4 can be the coding genes for the heavy chain and light chain of antibody CV18.

[0025] The DNA molecules shown in SEQ ID NO: 7 and SEQ ID NO: 8 can be the genes encoding the heavy chain and light chain of the human chimeric antibody hCV18.

[0026] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by modifying the codon preference based on the nucleotide sequences shown in SEQ ID NO: 3, 1-360, 1-345, SEQ ID NO: 4, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 and / or SEQ ID NO: 8. Considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression of specific species as needed.

[0027] The present invention also provides an antibody conjugate, comprising an antibody portion and a conjugated portion, wherein the antibody portion comprises any one of the antibodies or antigen-binding fragments thereof described herein.

[0028] Furthermore, the antibody portion and the coupling portion can be directly linked, or covalently linked via a linker (eg, a hydrazone bond, a disulfide bond, a thioether bond, a peptide bond).

[0029] Furthermore, the conjugated moiety may be a detectable label.

[0030] Furthermore, the detectable markers include enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, etc.), chemiluminescent agents (such as acridinium ester compounds, acridinium sulfonamide compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium, etc.), fluorescent dyes (such as AMCA, FITC, CFSE, GFP, DAPI, 7-AAD, Hoechst 33342, Pacific Blue, PE, PE-TR, PE-Cy7, PE-Cy5, PI, PerCP-Cy5.5, APC, APC-CY7, APC-H7, V500, Alexa 700, BV605, BV480, BV785, BV510, BV711, BV421, etc.), near-infrared dyes (such as cyanine dyes, BODIPY dyes, rhodamine dyes, squaric acid dyes, porphyrin dyes, etc.), radionuclides (such as 125I, 18F, 11C, 99mTc, 123I, etc.), biotin, nanoparticles for magnetic resonance imaging, quantum dots for magnetic resonance imaging, magnetic substances (such as magnetic beads, nanoparticles containing gadolinium complexes, superparamagnetic iron oxide nanoparticles) and colloidal gold, but not limited to these.

[0031] In one or more embodiments of the invention, the coupling moiety is alkaline phosphatase (ALP).

[0032] The coupling moiety described herein may also be a tag protein.

[0033] In order to facilitate the separation, purification, detection and / or positioning of the antibodies or antigen-binding fragments described herein, a label protein may be connected to the amino or carboxyl terminus of the antibodies or antigen-binding fragments thereof. The label protein includes, but is not limited to, GST (glutathione sulfhydryl transferase) label protein, Trx (thioredoxin) label protein, nitrogen utilization substrate A (NusA) label protein, His label protein (His-tag), MBP (maltose binding protein) label protein, Flag label protein, SUMO label protein, HA (influenza hemagglutinin) label protein, Myc label protein, LacZ label protein, CBD (cellulose binding domain) label protein, bacteriophage T7 protein kinase (T7PK) label protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein. Those skilled in the art know how to select a suitable label protein according to the desired purpose. The use of the label does not change the function of the target protein, and its purpose is to separate, purify, detect or trace, so the label protein suitable for this application is not limited to a specific type. The tag can be separated from the target protein by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site to remove the tag by cleavage with TEV protease).

[0034] The present invention also provides use of any of the antibodies or antigen-binding fragments thereof, or the biomaterial, or the antibody conjugate described herein in any of the following: E1) Use in preparing a product for detecting Kim-1 protein; E2) Use in the preparation of products for diagnosing or screening Kim-1 target related diseases.

[0035] The Kim-1 target-related diseases described in this article may be kidney diseases or kidney damage, including but not limited to acute kidney injury (AKI), diabetic nephropathy (DN), sepsis combined with acute kidney injury, renal fibrosis and inflammation.

[0036] The products described herein may include reagents, kits, chips, test strips, test cards, and immunosensors (such as electrochemical immunosensors).

[0037] The detection of Kim-1 protein includes any in vivo or in vitro detection of Kim-1 protein based on the principle of antigen-antibody specific reaction. The detection of Kim-1 protein can be to detect whether the sample to be tested contains Kim-1 protein and / or to detect the content of Kim-1 protein in the sample to be tested.

[0038] The method for detecting Kim-1 protein can be an immunological detection, such as a magnetic particle chemiluminescence detection method, a precipitation reaction, an agglutination test, a Western Blot, an enzyme immunoassay (such as ELISA), a double antibody sandwich ELISA method, a fluorescence-linked immunosorbent assay (FLISA), an enzyme immunoassay (EIA), a chemiluminescence immunoassay, a fluorescence immunoassay, a radioimmunoassay (RIA), a colloidal gold immunoassay (GIC), a colloidal gold immunochromatography technique (GICA), an immunohistochemistry method (IHC), a complement fixation reaction method, a multiplex immunoassay, and a fluorescence immunochromatography technique.

[0039] Furthermore, the method for detecting Kim-1 protein can be an ELISA method or a magnetic particle chemiluminescence detection method.

[0040] Furthermore, the method for detecting Kim-1 protein comprises the step of coating any of the antibodies or antigen-binding fragments thereof or antibody conjugates described herein on a solid phase carrier to obtain a solid phase carrier coated with antibodies. The coating may refer to being bound to the solid phase carrier by physical adsorption.

[0041] Furthermore, the solid phase carrier may be an ELISA plate, a membrane carrier, a microsphere, a biochip or a magnetic bead, but not limited thereto. The material of the solid phase carrier may be polystyrene, cellulose, cross-linked dextran, polyacrylamide, polyethylene, polypropylene, polyvinyl chloride, cross-linked dextran, glass, silicone rubber or agarose gel, but not limited thereto. The membrane carrier may be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane, but not limited thereto.

[0042] The present invention also provides a reagent or a kit, wherein the reagent or the kit comprises any one of the antibodies or antigen-binding fragments thereof, or the antibody conjugate described herein.

[0043] In one or more embodiments of the present invention, the antibody or antigen-binding fragment thereof is labeled with alkaline phosphatase (ALP) and used as a component of a magnetic particle chemiluminescent detection reagent that specifically recognizes Kim-1.

[0044] The reagent (detection reagent) or kit may have at least one of the following uses: (1) detecting Kim-1 protein; (2) diagnosing or screening Kim-1 target-related diseases.

[0045] The test sample of the reagent or kit can be a blood sample (such as whole blood, plasma, serum), a urine sample, a tissue sample, a cell sample, etc., but is not limited thereto.

[0046] The kit can be a chemiluminescent immunoassay kit (such as a magnetic microparticle chemiluminescent assay kit), an enzyme-linked immunosorbent assay kit, an immunoprecipitation assay kit, an immunoblotting assay kit, an immunochromatography assay kit, a flow cytometry assay kit, an immunohistochemistry assay kit, a colloidal gold immunoassay kit or a fluorescent immunoassay kit, but is not limited thereto.

[0047] Furthermore, the kit may also include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic particles, blocking solution, diluent, washing solution, color developing solution, stop solution, etc., but not limited thereto.

[0048] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0049] The present invention also provides a method for preparing any antibody or antigen-binding fragment thereof described herein, the preparation method comprising expressing any antibody or antigen-binding fragment thereof described herein in a host cell, and recovering or isolating the antibody or antigen-binding fragment thereof.

[0050] Furthermore, the preparation method may include the following steps: cloning a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof described herein into an expression vector (such as a prokaryotic expression vector, a eukaryotic expression vector, and a viral expression vector) to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the antibody or antigen-binding fragment thereof; culturing the recombinant host cell, and recovering or isolating the antibody or antigen-binding fragment thereof from the cultured recombinant host cell culture.

[0051] Furthermore, the recovery or separation can be separated from the culture (including all substances in the culture container) by precipitation method (such as salting out method, organic solvent precipitation method, caprylic acid-saturated ammonium sulfate precipitation method, isoelectric point precipitation method) or chromatography technology (such as ion exchange chromatography, gel filtration chromatography, affinity chromatography).

[0052] The introduction method may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation or metal cation-mediated transformation) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) directly transferring the target gene into plant receptor cells by physical or chemical methods, such as chemical stimulation, electric shock, liposome-mediated method, microinjection, gene gun method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method and vortex method. (4) transferring the target gene into plant receptor cells by vector, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method. (5) Introducing the target gene into in vitro animal cells (transfection) through calcium phosphate co-precipitation, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as adenovirus infection method, lentivirus infection method), etc.

[0053] The host cells include but are not limited to 293F cells, CHO cells, and 293T cells. In one embodiment of the present invention, the host cells are ExpiCHO cells.

[0054] The present invention first screened and identified the high-affinity anti-Kim-1 mouse monoclonal antibody CV18 through hybridoma technology, and the titer of the antibody can reach 1:729000. It was identified that the heavy chain of the antibody CV18 is of IgG1 subtype and the light chain is of kappa subtype. When screening antibodies, the present invention selects a variety of natural cell antigens, compares the antibody specificity detection, and after multiple rounds of screening, obtains CV18 as an antibody with good specificity for Kim-1. Multiple and multi-point subcutaneous injections are used in the animal immunization process to improve the titer of the final animal serum and the positive clone rate. The addition of the HAT screening reagent is postponed from the day of fusion to the second day, so that the cell positive rate is significantly improved. The present invention further humanizes the mouse monoclonal antibody CV18, designs and obtains the human chimeric antibody hCV18, and avoids the HAMA reaction in the natural sample. The titer of the human chimeric antibody hCV18 can reach 1:7290000.

[0055] Experiments show that the antibodies CV18 and hCV18 of the present invention have the characteristics of high affinity and specificity, and have good sensitivity and specificity for detecting Kim-1 protein, and can be used in various immunological detection methods. The anti-Kim-1 antibodies (antibodies CV18 and hCV18) of the present invention can be used to detect Kim-1 on the surface of human peripheral blood lymphocytes, or substances containing Kim-1, and substances that can specifically bind to Kim-1 (such as anti-Kim-1 antibodies). The principle of detection is mainly based on the specific recognition and binding of the Kim-1 antibody of the present invention to Kim-1, and detection is performed using chemical labeling technology (such as labeled antibodies, or labeled specific secondary antibodies) or physical detection technology (such as light scattering technology, plasma resonance technology, etc.). The present invention exemplarily provides the use of antibodies CV18 and hCV18 in ELISA methods and magnetic particle chemiluminescence methods. In the indirect ELISA method, the minimum detection limit of CV18 antibody is 25 ng / mL; hCV18 antibody is 12.5 ng / mL, both significantly higher than the control antibody (rabbit anti-Kim-1 antibody abcam, #228973). In addition, the antibodies CV18 and hCV18 of the present invention can also be used in the magnetic particle chemiluminescence detection method to detect Kim-1 protein in urine samples, and both show good specificity.

[0056] The present invention solves the problems of relative shortage of antibody raw materials, poor specificity and low sensitivity in the Kim-1 detection project in the current market. The antibodies CV18 and hCV18 developed by the present invention can be expressed and produced in prokaryotic cells, eukaryotic cells and any recombinant system, and can be made into products for clinical Kim-1 detection, such as detection reagents or kits, etc., and can be used as auxiliary diagnostic tools for kidney-related diseases, such as acute kidney injury (AKI), diabetic nephropathy (DN), sepsis combined with acute kidney injury, renal fibrosis and inflammation, and kidney treatment prognosis evaluation.

[0057] Definition of terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0058] The term "antibody conjugate" herein includes not only antibody-drug conjugates (ADCs) formed by coupling an antibody to a drug (a substance for preventing, treating, or diagnosing a disease) via a linker, but also conjugates obtained by coupling an antibody to a detectable marker or label.

[0059] The term "detectable label" generally refers to any chemical substance with a tracing effect. The conjugate obtained by coupling an antibody with a detectable label can be called a labeled antibody.

[0060] The term "antigen binding fragment" generally refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Typically, when the activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parental binding activity. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target. It is well known to those skilled in the art that the antigen binding fragment can be an antigen binding fragment of an antibody produced by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody.

[0061] The term "chimeric antibody" generally refers to replacing the constant region (C region) in the antibody molecule with a human one, while retaining the mouse variable region (V region) of the light and heavy chains. Chimeric antibodies may also include antibodies in which the heavy chain variable region and light chain variable region of the antibody are from a first antibody, while the heavy chain constant region and light chain constant region of the antibody are from a second antibody.

[0062] The term "complementarity determining region" or "CDR" generally refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, a person skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art.

[0063] The term "comprising" is not intended to be limiting, but is intended to be inclusive and means that there may be other elements besides the listed elements, which can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "comprising" and "including" are used interchangeably herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Construction map for pCDNA3.1-Kim-1 plasmid.

[0065] Figure 2 To detect the purity of Kim-1 target protein by SDS-PAGE experiment.

[0066] Figure 3 This is the subtype identification of the antibody expressed by the hybridoma cell line CV18 and the cloning gel image of the antibody variable region.

[0067] Figure 4This is the plasmid map of the humanized chimeric antibody expression plasmid pGmab-K-hCV18 constructed using antibody CV18.

[0068] Figure 5 This is the plasmid map of the humanized chimeric antibody expression plasmid pGmab-H-hCV18 constructed using antibody CV18.

[0069] Figure 6 This is the standard curve of Kim-1 protein concentration-luminescence value. DETAILED DESCRIPTION

[0070] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0071] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0072] Example 1. Preparation of Kim-1 recombinant protein 1. Construction of Kim-1 recombinant protein expression plasmid The nucleic acid sequence of Kim-1 is based on the sequence shown in NCBI Gene ID: NM_001173393.3 (Kim-1). When synthesizing the DNA sequence, the extracellular region sequence of Kim-1 (1Met-295Gly) was connected to a protein purification tag sequence (6×His) at the 3´ end, and EcoRI and XbaI sites were designed upstream and downstream of the DNA sequence, respectively. The designed DNA sequence was synthesized by a third-party gene synthesis company and constructed into the pUC57 plasmid. The constructed plasmid was named pUC57-Kim-1. Using the NEBCutsmart endonuclease system, 2 μg of pUC57-Kim-1 and pCDNA3.1 plasmids were added with 2 U EcoRI and 2 UXbaI endonucleases respectively. After digestion at 37°C for 2 hours, the positive bands were separated and recovered by 1% agarose gel, and the concentration of the recovered DNA fragments was detected by A260. 2 nmol of the double-digested fragments of pUC57-Kim-1 and pCDNA3.1 plasmids were taken respectively, 1 U T4 ligase was added, and the volume was made up to 20 μL with T4 ligase buffer and ultrapure water. After overnight connection at 16°C, the cells were transformed into DH5α competent cells and spread on LB plates. Single colonies were picked and sequenced with FCMV and SV40 universal primers. The gene sequences were correct after sequencing. The constructed plasmid was named pCDNA3.1-Kim-1, and its structure is as follows Figure 1 shown.

[0073] 2. Expression and identification of Kim-1 recombinant protein First, the expression plasmid pCDNA3.1-Kim-1 was transfected into 293F cells. The transfection reagent used for transfection of suspension cultured 293F cells was Free Style MAX (Invitrogen, 16447100), and the transfection procedure was carried out according to the manufacturer's product instructions.

[0074] Subsequently, the transfected 293F cells were transferred to a 125 mL Erlenmeyer shake flask and cultured at a cell density of 1 × 10 6 cells / mL. The cells were cultured in a CO2 incubator (37°C, 5% CO2) with a shaker speed of 130 rpm. The culture supernatant was collected after 7 days of cell culture, and the concentration of Kim-1 recombinant protein (expressed and secreted by 293F cells) in the supernatant was detected by sandwich ELISA.

[0075] Specifically, purified antibodies (abcam, #ab228973, #ab195534) were used as coating antibodies and detection antibodies in sandwich ELISA, respectively. To determine the active concentration of Kim-1, a Kim-1 control antigen (origene, #TP317289) with a known concentration (detected by A280 method) was used to make a concentration standard curve. Rabbit anti-Kim-1 antibody (abcam, # 228973) 1 μg / mL diluted with pH 9.5 carbonate buffer, 100 μL / well coated 96-well ELISA plate, overnight at 4℃; washed twice with PBST; added 200 μL of 1% BSA in PBS to each well, blocked for 2 hours at room temperature, and then placed on trifold paper to pat dry; added samples: PTB (1% BSA added to 10 mM PBS buffer) was used to dilute Kim-1 transfection supernatant and control antigen in different proportions or concentrations, and 0.1 mL of the diluted sample was added to the reaction well, incubated at 37℃ for 1 hour, and then washed. At the same time, blank wells (no sample added), negative control wells and positive control wells were made. Add 0.1 mL of the newly diluted antibody to each reaction well, incubated at 37℃ for 1 hour, and then washed 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of the newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37℃ for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H2SO4 to each reaction well. Measure the OD value to determine the result. Use a microplate reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 1.

[0076] Finally, ELISA CALC was used to calculate the 4-parameter logistic curve of the control protein as the standard curve, where R 2 =0.990. The expression concentration of Kim-1 protein in the supernatant of the cells to be tested was calculated to be about 20.8 ng / mL. The results showed that the target protein was obviously expressed in the supernatant of 293F cells transfected with pCDNA3.1-Kim-1.

[0077]

[0078] 3. Screening and identification of 293F stable cell lines of Kim-1 To screen for cell lines that stably express Kim-1, ExpiCHO cells transfected with pCDNA3.1-Kim-1 were diluted to a density of 100 cells / well and added to the microwells of a 96-well plate, with 200 µL CD-CHO medium / well containing 12.5 µM MSX reagent (glutamine synthetase GS inhibitor, used to screen for GS resistance genes). The microwell culture plate was incubated in a CO2 incubator (37°C, 5% CO2) for about 20 days to allow resistant cells to grow. The cells in the microwells with resistant cell growth were transferred to a 24-well cell culture plate for expansion culture (and quantitative determination of the expression level of Kim-1 protein in different cell lines), with about 1000 cells / well at the time of transfer. After 7 days of culture, the cell supernatant in the plate was taken out for Kim-1 protein concentration detection.

[0079] The specific operation of Kim-1 protein concentration detection is as follows: 1 μg / mL rabbit anti-Kim-1 antibody (abcam, # 228973) diluted with pH 9.6 carbonate buffer solution, 100 μL / well coated 96-well ELISA plate, 4℃ overnight; PBST washed twice; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper to pat dry; sample addition: PTB diluted the culture supernatant of different Kim-1 transfected ExpiCHO stable cell lines and control antigen (origene, #TP317289) in different proportions or concentrations, and 0.1 mL of the diluted sample was placed in the reaction well, incubated at 37℃ for 1 hour, and then washed, and blank wells (no sample added), negative control wells and positive control wells were made at the same time. 0.1 mL of the newly diluted antibody was added to each reaction well, incubated at 37℃ for 1 hour, and then washed 3 times. Add enzyme-labeled secondary antibody: 0.1 mL of the newly diluted enzyme-labeled antibody was added to each reaction well. Incubate at 37℃ for 1 hour, wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1 M H2SO4 to each reaction well. Measure the OD value to determine the result, and use a microplate reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 2.

[0080] The 4-parameter logistic curve of the control protein was calculated using ELISA CALC as the standard curve, where R 2 =0.990. The expression concentration of Kim-1 protein in the supernatant of the cells to be tested was calculated, and the results showed that the supernatant of 293F 3F5 cell line transfected with pCDNA3.1-Kim-1 had obvious expression of the target protein of 228.1 ng / mL. Combined with the expression level of Kim-1 and the cell growth characteristics, the 3F5 cell line was finally selected for the later production of Kim-1 protein.

[0081]

[0082] 4. Production and purification of Kim-1 recombinant protein Cell culture stage: Kim-1 stable cell line 3F5 was cultured at 2×10 5 The cells were cultured at a concentration of 10 cells / ml in a 1 L shake flask containing 400 mL CD-CHO medium containing 12.5 µM MSX reagent. The shake flask was placed in a shaker (130 rpm) in a CO2 incubator (37°C, 5% CO2) and cultured for 10 days. The cell culture medium after 10 days of culture was removed, centrifuged at 200 g for 5 minutes (to separate cells and coarse cell debris), and the supernatant was transferred to a new bottle and centrifuged at 3000 g for 15 minutes (to separate fine cell debris). The separated supernatant was sterilized by filtration using a 0.22 µM filter cup and stored at 4°C.

[0083] Protein purification stage: The sterilized cell supernatant was balanced to room temperature and then purified using a Ni column. Ni column filler (medium), company GE Healthcare, catalog number: #17-3712-02. Take 30 mL of the chromatography column and pack it, and the final volume of the column medium is 10 mL. The cell supernatant was loaded at a volume of 400 mL, the flow rate was 1.0 mL / min, and then eluted with different concentrations of imidazole eluent (20 mM Tris-HCl, pH 8.5, 50-1000 mM imidazole concentration gradient). Kim-1 recombinant protein was eluted in 100 mM imidazole eluent. Samples from each elution step of the purification experiment were taken, and the purity of the Kim-1 target protein was detected by SDS-PAGE experiment. The results are shown in Figure 2 , where lanes 1-9 were loaded with: supernatant, flow-through, washing solution, marker, 10 mM imidazole eluent, 50 mM imidazole eluent, 100 mM imidazole eluent, 200 mM imidazole eluent, 500 mM imidazole eluent; the purified Kim-1 protein was mainly present in Lane 7 (i.e., in the 100 mM imidazole eluent). Figure 2 The results showed that the purified Kim-1 recombinant protein was mainly present in Lane 7, with a molecular weight of 32.4 KDa, which was consistent with the theoretical value; the visual purity was 90%.

[0084] The eluate (Lane 7) containing Kim-1 recombinant protein was concentrated using a 10KDa ultrafiltration tube (Millipore, UFC900396) and dialyzed overnight using a 10 KDa dialysis bag. The dialyzate was 10 mM pH7.4 PBS buffer. The protein concentration of the dialyzed recombinant protein was detected by the A280 method. The total protein concentration was 1.12 mg / mL. The purified Kim-1 recombinant protein was stored at 4°C.

[0085] Example 2. Preparation and identification of anti-Kim-1 monoclonal antibodies 1. Immunization of mice with Kim-1 recombinant protein BALB / c mice aged 6 to 8 weeks were selected for the following immunization procedure: for the first immunization, 25 µg of purified Kim-1 recombinant protein was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously at multiple points after emulsification; 14 days after the first immunization, 12.5 µg of purified Kim-1 recombinant protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the second immunization, 12.5 µg of purified Kim-1 recombinant protein was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the third immunization, blood was collected and serum was separated. ELISA plates were coated with Kim-1 recombinant protein, and indirect ELISA tests were performed to determine the serum titer. The results showed that the titer of the prepared mouse antiserum was 1:72900. The test results of the immunized mouse serum are shown in Table 3.

[0086]

[0087] 2. Cell fusion After the third immunization, the mice were boosted with the following steps: 12.5 µg of purified Kim-1 recombinant protein was mixed with PBS to a volume of 200 μL for intraperitoneal injection. Cell fusion was performed 3 days after the boosted immunization of mice. After removing the eyeballs of the mice to collect blood, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied to find the spleen, which was removed with tweezers and gently crushed in a 200-mesh stainless steel filter. The cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. The supernatant was discarded to obtain spleen cells for use.

[0088] When preparing feeder layer cells, mice were killed by dislocation and placed in a 70% alcohol bottle for 2 minutes. The mice were then fixed on a foam board in a biosafety cabinet, the abdominal skin was untied, PBS was drawn up with a syringe and gently injected into the peritoneum, and the liquid containing the feeder layer cells was washed out from the other side. The cells were then centrifuged at 200 g for 10 minutes at room temperature, and the supernatant was discarded to obtain the feeder layer cells for use. 2.0×10 7 FO myeloma cells (ATCC CRL-1646) and 2.0×10 8Mix the spleen cells, centrifuge at 200 g for 10 minutes, discard the supernatant, gently vortex to mix, add 1 mL of 50% PEG-1450 (Merk, P1458) aqueous solution within 90 seconds in a 37°C water bath, then add 20 mL of DMEM culture medium, centrifuge at 200 g for 10 minutes, discard the supernatant, repeat the wash, centrifuge at 200 g for 10 minutes, discard the supernatant, obtain hybridoma cells, and plate the hybridoma cells into 10 96-well culture plates with 150 μL in each well. Add 10,000 cells / well of the feeder layer cells to the above 10 96-well cell culture plates, 100 μL per well, mark the culture plates, and place them in a cell culture incubator at 37°C containing 5% CO2. Add HAT selection medium on the second day, and then use HAT selection culture for 1 to 2 days. A large number of tumor cells will die. After 3 to 4 days, the tumor cells disappear and hybrid cells form small colonies. After 7 to 10 days of HAT selection culture medium, switch to HT culture medium (Merk, H0137), and then maintain for 2 weeks. Continue to culture with 20% FBS (ExCell, FSP500) DMEM culture medium. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom of the well, you can start to detect specific antibodies and screen out the required hybridoma cell lines. During the selection culture period, change half of the culture medium every 2 to 3 days.

[0089] 3. Screening and subcloning of positive hybridoma cell lines First, the optimal coating amount of Kim-1 recombinant protein (also referred to as Kim-1 protein in this article) as an antigen was determined by the square array titration method. 0.5, 1.0, 2.0, and 4.0 μg of Kim-1 protein were coated on a 96-well plate, and 6 wells of each concentration were set as 3 positive and 3 negative. Array titration was performed with positive serum from mice immunized with Kim-1 protein at different dilutions, and negative serum from unimmunized mice was used as a negative control. 96-well ELISA plates were coated with 0.5 μg of purified Kim-1 protein per well and incubated at 4°C overnight; washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: 0.1 mL of the sample to be tested was added to the reaction well, incubated at 37°C for 1 hour, and then washed, and blank wells (no sample added), negative control wells, and positive control wells were made at the same time. Add 0.1 mL of freshly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody (Solebol, #SE131) to each reaction well. Incubate at 37°C for 1 hour, and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1 M H2SO4 to each reaction well. Measure the OD value to determine the result, and use an enzyme reader to measure the absorbance value (A450) at 450 nm. A value that is 2.1 times or more of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select the hybridoma cell monoclonal clone against Kim-1.

[0090] According to the above method, the positive hybridoma cells obtained by screening were subcloned, and the original wells were diluted with HAT selection medium by limiting dilution method and then redistributed into 96-well culture plates, and then the morphology and number of cells were observed. The cells were adjusted to 3-10 cells / mL. Take the cell culture plate of feeder layer cells prepared the day before, and add 100µL of diluted cells to each well. Culture statically in a 37°C, 5% CO2 incubator. Change the medium on the 7th day, and change the medium once every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity is detected in time. The cells in the positive wells were transferred to a 24-well plate for expansion culture. The binding activity of antibodies and Kim-1 protein in the culture supernatants of different cell lines was compared by indirect ELISA detection, and the one with the highest OD value (OD 450 =1.856) was used for later experiments. The cells should be frozen as soon as possible, and the hybridoma cell line with clone number CV18 was finally selected for antibody production. The monoclonal antibody secreted by the hybridoma cell line CV18 was named monoclonal antibody CV18 (also referred to as anti-Kim-1 antibody CV18 or antibody CV18 in this article).

[0091] 4. Mass preparation of monoclonal antibodies and antibody titer determination (1) Large-scale preparation of monoclonal antibodies 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1×10 6 Hybridoma cell CV18 can produce ascites 7 to 10 days after inoculation of cells. Closely observe the health status of the animals and the signs of ascites. When the ascites is as much as possible and the mice are about to die, kill the mice and use a dropper to suck the ascites into a test tube. 5 to 10 mL of ascites can be obtained from one mouse. Ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained was centrifuged at 3000 g for 10 minutes, the upper layer of fat and the bottom sediment were discarded, the supernatant was collected and aliquoted at -20℃, and after the ascites supernatant was thawed and equilibrated to room temperature, 1 / 10 volume of 1 M Tris-HCl pH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was equilibrated with 20 column volumes of 100 mM Tris-HCl at pH 8.0, and the ascites supernatant after adjusting the pH to 8.0 was applied to the column, followed by washing with 20 column volumes of 100 mM Tris-HCl at pH 8.0, and finally the antibody was eluted with 100 mM Glycine-HCL pH 2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged in a centrifuge (Xiangyi, L550) at room temperature for 20 minutes at 3000×g. Batch centrifugation was performed until the solution volume reached 1 mL / concentrator tube (2 tubes), 4 mL of 10 mM PBS pH 7.4 buffer was added, and centrifugation at room temperature for 20 minutes at 3000×g was continued. The centrifugation was repeated 3 times to make the antibody buffer 10 mM PBS pH 7.4, and 10 mM PBS pH 7.4 was added to a total volume of 10 mL. Finally, the concentrated antibody solution was dispensed into centrifuge tubes at 2 mL / tube and stored at -80°C. The antibody concentration was determined using a BCA kit (Solabo, PC0020). The concentration of the purified monoclonal antibody CV18 was 0.89 mg / mL.

[0092] (2) Antibody titer determination The titer of anti-Kim-1 antibody CV18 was detected by indirect ELISA. Kim-1 protein was diluted with PBS and coated in a 96-well ELISA plate at 0.2 µg / mL and 100 µL / well. After overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300 µL / well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA in PBS solution, 200 µL / well, at room temperature for 2 hours, and patted dry after blocking; anti-Kim-1 antibody CV18 diluted to 20 ng / mL in PTB was added, reacted in a 37°C incubator for 1 hour, and the ELISA plate was washed twice with PBST solution, 300 µL / well each time. Patted dry after washing. HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB was added, reacted in a 37°C incubator for 1 hour, and the ELISA plate was washed twice with PBST solution, 300 µL / well each time. Patted dry after washing. TMB substrate solution was added, 100 µL / well was reacted at room temperature for 10 minutes, and 100 µL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 4, and the titer of the purified anti-Kim-1 antibody was 1:729000.

[0093]

[0094] 5. Sequence and subtype identification of monoclonal antibodies (1) Monoclonal antibody subtype identification The hybridoma cell line CV18 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO2) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15 mL centrifuge tube and 4 × 10 6 After centrifugation at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. After total RNA was extracted from the cells in the tube, cDNA was synthesized using the reverse transcription kit of QIAGEN (Qiagen, 74134). The antibody subtype was determined by PCR using antibody subtype-specific primers. The above-synthesized cDNA was used as a PCR reaction template. The primer sequence information used for PCR is shown in Table 5.

[0095]

[0096] Note: In the table, S = C or G, M = A or C, R = A or G, W = A or T.

[0097] PCR reaction solution system: TAKARA Ex Taq (5 U / µL, TAKARA, RR001B), 0.25 µL; 10×ExTaq Buffer, 5 µL; dNTP mixture (2.5 mM each), 4 µL; template cDNA, 1 µL; upstream primer (100 µM), 1 µL; downstream primer (100 µM), 1 µL; add double distilled water to a total volume of 50 µL.

[0098] PCR reaction temperature program: 94°C for 5 minutes of pre-denaturation, 30 temperature cycles (94°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minute), and extension at 72°C for 10 minutes.

[0099] After the reaction, 10 μL of each PCR product was loaded into 1% agarose gel for electrophoresis. The electrophoresis diagram is shown in Figure 3 As shown, through electrophoresis analysis, Lane 2 and Lane 6 amplified positive bands of 650 bp for IgG1 heavy chain and kappa light chain, respectively, thereby confirming that the heavy chain of the monoclonal antibody CV18 obtained in the present invention is of IgG1 subtype, and the light chain is of kappa subtype. According to the PCR product results, the subtype of the antibody can be inferred, and the monoclonal antibody CV18 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0100] (2) Sequencing of the variable region (V region) of the hybridoma cell line CV18 antibody The fragment obtained after PCR amplification of the V region of the antibody of the cell line CV18 in the above step (1) was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB medium and DNA sequencing was performed after overnight culture. The sequencing results showed that the sequence of the anti-Kim-1 antibody CV18 was as follows: The amino acid sequence of the heavy chain of antibody CV18 is shown in SEQ ID NO: 1, and the nucleotide sequence of the heavy chain encoding gene is shown in SEQ ID NO: 3; The amino acid sequence of the light chain of antibody CV18 is shown in SEQ ID NO:2, and the nucleotide sequence of the light chain encoding gene is shown in SEQ ID NO:4; The amino acid sequence of the heavy chain variable region of antibody CV18 is shown at positions 1-120 of SEQ ID NO: 1, and the nucleotide sequence of the heavy chain variable region encoding gene is shown at positions 1-360 of SEQ ID NO: 3; The amino acid sequence of the light chain variable region of antibody CV18 is shown at positions 1-115 of SEQ ID NO: 2, and the nucleotide sequence of the gene encoding the light chain variable region is shown at positions 1-345 of SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region CDR1 of antibody CV18 is shown at positions 31-35 of SEQ ID NO: 1; The amino acid sequence of the heavy chain variable region CDR2 of antibody CV18 is shown at positions 50-66 of SEQ ID NO: 1; The amino acid sequence of the heavy chain variable region CDR3 of antibody CV18 is shown at positions 99-109 of SEQ ID NO: 1; The amino acid sequence of the light chain variable region CDR1 of antibody CV18 is shown at positions 24-39 of SEQ ID NO: 2; The amino acid sequence of the light chain variable region CDR2 of antibody CV18 is shown at positions 55-61 of SEQ ID NO: 2; The amino acid sequence of the light chain variable region CDR3 of antibody CV18 is shown at positions 94-105 of SEQ ID NO:2.

[0101] The above CDR sequences were obtained according to the IMGT database; the sequences of the CDRs were defined according to the Kabat numbering system.

[0102] Example 3: Transformation, expression and purification of human chimeric antibody CV18 1. Design and sequence of human chimeric antibodies The murine monoclonal antibody CV18 was humanized by replacing the constant region (C region) of the antibody CV18 with the human region, while retaining the murine variable region (V region) of the light and heavy chains. The designed human chimeric antibody was named hCV18, and its sequence is as follows: The amino acid sequence of the heavy chain of antibody hCV18 is shown in SEQ ID NO:5, and the nucleotide sequence of the heavy chain encoding gene is shown in SEQ ID NO:7; wherein positions 1-120 of SEQ ID NO:5 are the heavy chain variable region, and positions 121-450 of SEQ ID NO:5 are the heavy chain constant region; The amino acid sequence of the light chain of antibody hCV18 is shown in SEQ ID NO:6, and the nucleotide sequence of the light chain encoding gene is shown in SEQ ID NO:8; wherein positions 1-115 of SEQ ID NO:6 are the light chain variable region, and positions 116-222 of SEQ ID NO:6 are the light chain constant region.

[0103] 2. Construction of pGmab-K-hCV18 / pGmab-H-hCV18 plasmid The gene encoding the light chain variable region (VL) of antibody CV18 (the nucleotide sequence is shown in positions 1-345 of SEQ ID NO:4) and the gene encoding the heavy chain variable region (VH) (the nucleotide sequence is shown in positions 1-360 of SEQ ID NO:3) were constructed into pUC57 plasmid, and the constructed plasmids were named pUC57-CV18-VL and pUC57-CV18-VH. 2 μg of pUC57-CV18-VL or pUC57-CV18-VH and pGmab-K (containing human antibody kappa chain constant region) or pGmab-H (containing human antibody IgG1 chain constant region) plasmids were added with 2 U XbaI and 2 U PCR products respectively using NEB Cutsmart endonuclease system. BamHI endonuclease, digested at 37℃ for 2 hours, separated and recovered positive bands by 1% agarose gel, and the concentration of recovered DNA fragments was detected by A260. 2nmol of the insert fragment (CV18-VL or CV18-VH) and the expression plasmid double digestion fragment (pGmab-K or pGmab-H) were taken, 1 U T4 ligase was added, and the volume was adjusted to 20 μL with T4 ligase buffer and ultrapure water. After overnight connection at 16℃, it was transformed into DH5α competent cells and spread on LB plates. Single colonies were picked and sequenced with FCMV and SV40 universal primers to confirm that the gene sequence was correct. The constructed expression plasmids were named pGmab-K-hCV18 (antibody hCV18 light chain expression plasmid) and pGmab-H-hCV18 (antibody hCV18 heavy chain expression plasmid), respectively. See the plasmid map for details. Figure 4 and Figure 5 .

[0104] 3. Expression and purification of human chimeric antibody hCV18 3-1. Expression and titer detection of antibody hCV18: The two plasmids pGmab-K-hCV18 and pGmab-H-hCV18 constructed above were mixed at a ratio of 1:1 and transfected into ExpiCHO cells. The transfection reagent used for transfection of suspension cultured ExpiCHO cells was Free Style MAX (Invitrogen, 16447100), and the transfection procedure was performed as per the manufacturer’s product instructions. Subsequently, the transfected ExpiCHO cells were transferred to a 125 mL triangular shake flask and cultured in 30 mL FreeStyleCHO medium at a cell density of 1×10 6cells / mL. The cells were cultured in a CO2 incubator (37°C, 5% CO2) with a shaking speed of 130 rpm. Finally, the culture supernatant was collected after 7 days of cell culture to detect the activity concentration of human chimeric antibody hCV18 (expressed and secreted by ExpiCHO cells) in the supernatant. The detection method was indirect ELISA, and the steps were as follows: Kim-1 protein was diluted with PBS as an antigen and coated in a 96-well ELISA plate at a coating concentration of 1 µg / mL and a volume of 100 µL / well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 µL / well each time. After washing, pat dry, and the ELISA plate was blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. Pat dry, add the supernatant of cells expressing hCV18 diluted with PTB at different ratios, add it to the above blocked ELISA plate, 100 µL / well, and react in a 37°C incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry, add TMB substrate solution to the ELISA plate, 100 µL / well, at room temperature for 10 minutes, and then add 100 µL / well of 1 M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450 nm. The results are shown in Table 6, the activity titer of the anti-Kim-1 humanized antibody hCV18 in the cell supernatant was detected, and the titer of the antibody hCV18 in the cell supernatant was 1:72900.

[0105]

[0106] 3-2. Purification of hCV18 antibody and titer detection of purified antibody: The cell supernatant was centrifuged at 3000×g for 10 minutes, and 1 / 10 volume of 1 M Tris–HCl pH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was equilibrated with 20 column volumes of 100 mM Tris–HCl pH8.0, and the cell supernatant after adjusting the pH to 8.0 was loaded onto the column, followed by washing with 20 column volumes of 100 mM Tris–HCl pH 8.0, and finally the antibody was eluted with 100 mM Glycine–HCL pH2.5. The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at 3000×g for 20 minutes at room temperature in a centrifuge (Xiangyi, L550). Centrifuge in batches until the solution volume reaches 1 mL / concentrator tube (2 tubes), add 4 mL of 10 mM PBS pH 7.4 buffer, and continue centrifugation at room temperature for 20 minutes at 3000×g. Repeat centrifugation 3 times to make the antibody buffer 10 mM PBS pH7.4, and add 10 mM PBS pH 7.4 to a total volume of 10 mL. Finally, the concentrated antibody solution 2 mL / tube was divided into centrifuge tubes and stored at -80°C. The antibody concentration was determined using the BCA kit (Solebo, PC0020), and the concentration of the purified human chimeric antibody hCV18 was 4.1 mg / mL. The activity titer of the purified anti-Kim-1 humanized antibody hCV18 was detected by indirect ELISA, the steps are as follows: Kim-1 protein was diluted with PBS as an antigen and coated in a 96-well ELISA plate with a coating concentration of 1 µg / mL and a volume of 100 µL / well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 µL / well each time. After washing, the plate was patted dry and blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. Patted dry, different concentrations of antibody hCV18 diluted with PTB were added to the blocked ELISA plate, 100 µL / well, and reacted in a 37°C incubator for 1 hour. Patted dry, the plate was washed 3 times with PBST, 300 µL / well each time. Patted dry, HRP-labeled goat anti-human antibody diluted 5000 times with PTB was added to the ELISA plate, and reacted in a 37°C incubator for 1 hour. Washed the plate 3 times with PBST, 300 µL / well. Patted dry, TMB substrate solution was added to the ELISA plate, 100 µL / well, at room temperature for 10 minutes, and then 100 µL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 7. The titer of hCV18 in the cell supernatant was 1:7290000.

[0107]

[0108] Example 4: Identification of the sensitivity and specificity of antibodies hCV18 and CV18 for detecting Kim-1 (1) Identification of the sensitivity of antibodies hCV18 and CV18 for detecting Kim-1 The sensitivity of hCV18 and CV18 in detecting Kim-1 was evaluated by indirect ELISA. The sensitivity was compared with that of the control antibody (rabbit anti-Kim-1 antibody abcam, #228973). The specific steps are as follows: Kim-1 protein was diluted with PBS as an antigen and coated in a 96-well ELISA plate at a coating concentration of 1 µg / mL and a volume of 100 µL / well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 µL / well each time. After washing, pat dry, and the ELISA plate was blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. Pat dry, add different concentrations of CV18, hCV18, and control antibody diluted with PTB, add to the above blocked ELISA plate, 100 µL / well, and react in a 37°C incubator for 1 hour. Pat dry, and wash the plate 3 times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled goat anti-human antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry, add TMB substrate solution to the ELISA plate, 100 µL / well, incubate at room temperature for 10 minutes, then add 100 µL / well of 1 M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance (A450) at 450 nm.

[0109] The experimental results are shown in Table 8. The sensitivity of antibody CV18 and antibody hCV18 in detecting Kim-1 protein is better than that of the control antibody. In addition, the background value of CV18 antibody is lower than that of hCV18 and Kim-1 protein (0.052). The standard curve of Kim-1 protein R 2 =0.981, IC50=25 ng / mL. The standard curve range of this method is 0-1000 ng / mL, and the minimum detection limit of the control antibody is 100 ng / mL; CV18 antibody is 25 ng / mL; hCV18 antibody is 12.5 ng / mL. The results are shown in Table 8.

[0110]

[0111] (2) Identification of the specificity of antibodies hCV18 and CV18 for detecting Kim-1 Preparation of Kim-1 overexpressing 293T cells: The gene encoding Kim-1 protein (nucleotide sequence as shown in GenBank accession number: AF043724.1) was cloned into the vector pCDNA3.1 (purchased from Thermo Company, catalog number V79520) to obtain a recombinant vector, and the recombinant vector was transfected into 293T cells (ATCC CRL-11268) to obtain Kim-1 overexpressing 293T cells.

[0112] The specificity of antibodies hCV18 and CV18 for detecting Kim-1 was evaluated by indirect ELISA. The specific steps were as follows: culture cells (293T, as negative control in this experiment), cultured in DMEM medium with 10% serum for 3 generations, and then lysed with cell lysis buffer (Biyuntian, # P0013); Kim-1 overexpressing 293T cells (as positive cells in this experiment) were lysed with lysis buffer (abcam, # ab94224). The lysates obtained after lysing the cells with the above two cell lysis buffers were diluted to 1 μg / mL with carbonate buffer solution at pH 9.5 and transferred to 96-well cell culture plates with a volume of 100 µL / well for overnight coating. Wash twice with PBST solution, 300 µL / well each time. After washing, pat dry, and block the 96-well plate with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours, and then pat dry. Add different concentrations of hCV18 and CV18 antibodies diluted with PTB, and control antibody (rabbit anti-Kim-1 antibody abcam, # 228973) 100 µL / well, and react in a 37℃ incubator for 1 hour. Pat dry, wash the plate 3 times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled secondary antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37℃ incubator for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry, add TMB substrate solution to the ELISA plate, 100 µL / well, and incubate at room temperature for 10 minutes, then add 100 µL / well of 1M H2SO4 to stop the reaction. Measure the absorbance (A450) at 450 nm using an ELISA reader. The experimental results are shown in Table 9. Compared with the control antibody, both antibody CV18 and antibody hCV18 have good specificity, and antibody hCV18 has better specificity than antibody CV18 in detecting Kim-1 protein.

[0113]

[0114] Example 5: Application of antibodies hCV18 and CV18 in detecting Kim-1 in human urine by magnetic microparticle chemiluminescence detection method (1) Alkaline phosphatase (ALP) labeling of antibodies hCV18 and CV18 Antibodies hCV18 and CV18 (20 nmol, 3 mg) were taken to a volume of 0.5 mL and added to a dialysis bag (10 KDa, 1 cm width), and dialyzed in 2 L of 10 mM PBS solution (pH 7.4) at 4°C overnight. The next day, the dialysis bag containing the antibody solution was placed in 1 L of 10 mM carbonate buffer (pH 9.5) and dialyzed at room temperature for 2 hours to prepare for coupling with activated ALP.

[0115] At the same time, use an analytical balance to accurately weigh 0.1 mg of ALP, add it to the dialyzed antibody solution, add ALP while stirring to prevent the powder from adhering to the wall, and place it in a 4°C refrigerator or icehouse and continue stirring for 12 to 18 hours. After the binding is completed, first centrifuge the conjugate at 3000×g for 20 minutes, remove a small amount of precipitate, put it into a dialysis bag, and then dialyze it overnight in a beaker of 10 mM PBS solution (pH7.4). Take the marker that has been dialyzed overnight, pass it through a dextran gel G-25 column, separate the free ALP, collect the labeled antibody and store it at 4°C.

[0116] (2) Composition of magnetic microparticle chemiluminescence detection reagent constructed using ALP-labeled antibodies hCV18 and CV18 In this experiment, the magnetic microparticle chemiluminescence detection reagent includes R1 reagent, magnetic separation reagent, calibration solution series and chemiluminescence substrate solution.

[0117] The components of the magnetic microparticle chemiluminescent detection reagent constructed using the ALP labeled antibody CV18 are as follows: Among them, R1 reagent includes: 1) R1 antibody: anti-Kim-1 monoclonal antibody CV18 labeled with alkaline phosphatase (ALP), concentration of 0.6 μg / ml; 2) Buffer: including Tris, concentration is 12.0 g / L; sodium azide, concentration is 1.98 g / L; sodium chloride, concentration is 5.9 g / L; magnesium chloride solution with a molar concentration of 1 M, 1.0 mL / L; zinc chloride solution with a molar concentration of 0.1 M, 1.0 mL; fish skin gelatin, concentration is 10 g / L; bovine serum albumin, 5 g / L; newborn calf serum, 30 g / L; the rest is deionized water. The pH value of the buffer of R1 reagent is 8.0.

[0118] The R1 reagent is prepared using the buffer solution (pH 8.0) described in 2) above as a solvent and the R1 antibody as a solute.

[0119] Wherein, the magnetic separation reagent includes: 1) Magnetic particles: Magnetic particles coated with anti-Kim-1 monoclonal antibody (abcam, #228973), concentration 1 mg / ml; 2) Buffer: including Tris, concentration is 11.08 g / L; sodium azide, concentration is 1.917 g / L; sodium chloride, concentration is 5.56 g / L; magnesium chloride solution with molar concentration of 1 M, 1.0 mL / L; zinc chloride solution with molar concentration of 0.1 M, 1.0 mL / L; bovine serum albumin, concentration is 4.81 g / L; special horse serum, concentration is 4.91 g / L, and the remaining components are deionized water. The pH value of the buffer of the magnetic separation reagent is 8.0.

[0120] The magnetic separation reagent is prepared with the buffer solution (pH 8.0) described in 2) as solvent and magnetic particles as solute.

[0121] Among them, the calibration solution series includes: 1) Kim-1 recombinant protein antigen; 2) Buffer: including Tris, concentration is 9.1 g / L; sodium chloride, concentration is 12.9 g / L; tetracycline hydrochloride, concentration is 0.01 g / L; neomycin sulfate, concentration is 0.01 g / L; sodium azide, concentration is 2.0 g / L; casein 10.0 g / L; Tween 20, concentration is 3.1 g / L. The pH value of the buffer of the calibrator solution series is 7.6. The calibrator series includes Kim-1 recombinant protein antigen calibrators with different concentrations (0 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL).

[0122] The chemiluminescent substrate solution is a chemiluminescent substrate solution containing 0.3 mg / mL of a dioxane compound (APCL) prepared by diluting a 0.2 M Tris-HCl buffer solution with a pH value of 9.3.

[0123] The components of the magnetic microparticle chemiluminescent detection reagent constructed using the ALP-labeled antibody hCV18 are as follows: except that the antibody CV18 in the R1 reagent is replaced by the antibody hCV18, the other components and preparation methods remain unchanged.

[0124] (3) Application of magnetic microparticle chemiluminescent detection reagent constructed by antibodies hCV18 and CV18 in the detection of Kim-1 in human urine The content of Kim-1 protein in human urine was detected using a magnetic particle chemiluminescent detection reagent based on the ALP-labeled antibody CV18. The steps are as follows (where the R1 reagent is based on the antibody CV18): 1) 50 μL of the calibrator solution series (Kim-1 recombinant protein antigen concentrations are 0 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, and 1000 pg / mL) was added to the reaction tubes in sequence with 50 μL of R1 reagent and 50 μL of magnetic separation reagent, and mixed and incubated at 37°C for 15 minutes; 2) The reagents obtained in step 1) were combined with 25 μL of magnetic separation reagent and incubated at 37°C for 5 minutes; 3) Wash three times with washing solution (0.1 M TrisHCl buffer, pH 8, containing 0.02% Tween 20 and 15w / w% sodium chloride) to remove unbound antibodies and impurities; 4) Add 150 μL of chemiluminescent substrate solution, and use a chemiluminescence detector to measure the relative luminescence intensity (RLU) after ALP catalyzes the substrate to emit light. The results are shown in Table 10 below:

[0125]

[0126] 5) According to the values ​​in Table 10, we can obtain Figure 6 Kim-1 protein concentration-luminescence value standard curve shown; 6) Add 50 μL of the sample to be tested (urine samples of different disease types, negative samples are physical examination samples, and positive samples are acute kidney injury and diabetic nephropathy samples) and 50 μL of R1 reagent to the reaction tube in sequence, mix and incubate at 37°C for 15 minutes; 7) The reagent obtained in step 6) above was combined with 25 μL of magnetic separation reagent and incubated at 37°C for 5 minutes; 8) Wash three times with washing solution (0.1 M TrisHCl buffer, pH 8, containing 0.02% Tween 20 and 15w / w% sodium chloride) to remove unbound antibodies and impurities; 9) 150 μL of luminescent substrate solution. After ALP catalyzes the substrate to emit light, a chemiluminescence detector is used to measure the relative luminescence intensity (RLU). 10) Calculate the Kim-1 protein concentration value corresponding to the sample RLU based on the Kim-1 recombinant protein concentration-luminescence value standard curve obtained in step 5).

[0127] The steps of detecting the content of Kim-1 protein in human urine using a magnetic microparticle chemiluminescence detection reagent constructed based on the ALP-labeled antibody hCV18 are as follows: except for using the R1 reagent based on the antibody hCV18, the steps are the same as the above steps 1)-10).

[0128] Magnetic particle chemiluminescence detection reagents were constructed using antibodies hCV18 and CV18, respectively, and used to detect the content of Kim-1 protein in human urine. The difference between the two was compared. The results are shown in Table 11 (the data in the table are the content of Kim-1 protein, in pg / ml).

[0129]

[0130] As shown in Table 11, both antibody hCV18 and antibody CV18 can clearly detect Kim-1 protein in human urine by magnetic particle chemiluminescence detection method, and the specificity of hCV18 antibody is significantly higher than that of CV18 antibody. It can be judged that hCV18 antibody has a great advantage in magnetic particle chemiluminescence detection in specific recognition of Kim-1 protein in human urine.

[0131] The present invention provides anti-Kim-1 monoclonal antibodies CV18 and / or hCV18, which have the characteristics of high affinity and specificity, and also provides a method for detecting Kim-1 in human urine by a magnetic particle chemiluminescence method based on the hCV18 antibody. The present invention solves the problem of relative shortage of antibody raw materials for Kim-1 detection projects in the current market.

[0132] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An anti-Kim-1 antibody or an antigen-binding fragment thereof, characterized in that The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR1 as shown in positions 31-35 of SEQ ID NO:1, a CDR2 as shown in positions 50-66 of SEQ ID NO:1, and a CDR3 as shown in positions 99-109 of SEQ ID NO:1; the light chain variable region comprises a CDR1 as shown in positions 24-39 of SEQ ID NO:2, a CDR2 as shown in positions 55-61 of SEQ ID NO:2, and a CDR3 as shown in positions 94-105 of SEQ ID NO:

2.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown at positions 1-120 of SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown at positions 1-115 of SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region as shown in A1) or A2) below: A1) the amino acid sequence of the heavy chain constant region is as shown in positions 121-444 of SEQ ID NO: 1, and the amino acid sequence of the light chain constant region is as shown in positions 116-222 of SEQ ID NO: 2; A2) The amino acid sequence of the heavy chain constant region is shown at positions 121-450 of SEQ ID NO:5, and the amino acid sequence of the light chain constant region is shown at positions 116-222 of SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises the heavy chain and light chain shown in B1) or B2) below: B1) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2; B2) The amino acid sequence of the heavy chain is shown in SEQ ID NO:5, and the amino acid sequence of the light chain is shown in SEQ ID NO:

6.

5. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; C2) a nucleic acid molecule encoding the heavy chain and light chain of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; C3) an expression cassette containing the nucleic acid molecule described in C1) or C2); C4) a recombinant vector containing the nucleic acid molecule described in C1) or C2); C5) a recombinant microorganism containing the nucleic acid molecule described in C1) or C2); C6) A recombinant host cell containing the nucleic acid molecule described in C1) or C2).

6. The biomaterial according to claim 5, characterized in that The nucleic acid molecule is any of the following: D1) a DNA molecule encoding a sequence as shown in positions 1 to 360 of SEQ ID NO: 3 and positions 1 to 345 of SEQ ID NO: 4; D2) a DNA molecule with coding sequences as shown in SEQ ID NO:3 and SEQ ID NO:4; D3) A DNA molecule encoding the sequences shown in SEQ ID NO:7 and SEQ ID NO:

8.

7. An antibody conjugate comprising an antibody portion and a conjugate portion, characterized in that: The antibody portion comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the biomaterial according to claim 5 or 6, or the antibody conjugate according to claim 7 in any of the following: E1) Use in preparing a product for detecting Kim-1 protein; E2) Use in the preparation of products for diagnosing or screening Kim-1 target related diseases.

9. A reagent or a kit, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the antibody conjugate according to claim 7.

10. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The preparation method comprises expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in a host cell, and recovering or isolating the antibody or antigen-binding fragment thereof.

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