CysR structural domain specific antibody of PLA2R and application thereof

Abstract

The invention discloses a specific antibody aiming at a CysR structural domain of PLA2R and application of the specific antibody, and belongs to the technical field of biological engineering. The invention provides a CysR structural domain antibody of PLA2R or an antigen binding fragment of the CysR structural domain antibody. The CysR structural domain antibody or the antigen binding fragment thereof provided by the invention can specifically recognize the corresponding PLA2R structural domain and has relatively high affinity. Besides, the invention provides a method for establishing a standard curve by using a CysR structural domain antibody of PLA2R as a standard substance, so that convenience is provided for clinical detection of concentrations of IgG with different anti-PLA2R epitopes.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and to a specific antibody against the CysR domain of human PLA2R, a detection kit containing the antibody, and applications thereof. Background Art

[0002] Membranous nephropathy (MN) is a kidney-specific autoimmune disease, the main pathological feature of which is the deposition of immune complexes on the glomerular basement membrane (GBM), accompanied by significant thickening of the GBM. According to its pathogenesis and causes, it can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy.

[0003] M-type phospholipase A2 receptor (PLA2R) is the main target antigen in idiopathic membranous nephropathy. PLA2R is expressed on the plasma membrane of human podocytes and has a large extracellular region that is highly glycosylated and consists of multiple separate domains (a single N-terminal cysteine-rich domain (Cysteine-richdomain, CysR), a single type II fibronectin domain (FibronectintypeII domain, FNII) and 8 to 10 C-type lectin-like domains (C-typelectindomain, CTLD)). These separate domains can directly bind to circulating autoantibodies.

[0004] Studies have found an association between PLA2R reactive epitopes and disease activity, and proposed the hypothesis of epitope spreading. "Epitope spreading" refers to the immune response to endogenous epitopes following the release of self-antigens during chronic autoimmune or inflammatory responses, which usually spreads from the main immunodominant epitope to other sites within the molecule. In IMN, CysR is the immunodominant epitope of PLA2R, and the spread of antigenic determinants begins in the CysR domain and then gradually spreads to CTLD1, CTLD7 or CTLD8. In addition, some studies have suggested that the analysis of PLA2R epitope characteristics and spread is a powerful means to monitor disease severity and stratify patients by renal prognosis.

[0005] Therefore, detecting anti-PLA2R autoantibodies is an important way to monitor IMN. In most IMN patients, the main subtype of autoantibodies is IgG4, and there is currently no quantitative detection method for antibodies against the PLA2R domain. It is crucial to seek an antibody against the CysR domain of PLA2R and a quantitative detection method for the level of CysR domain-specific IgG antibodies. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present application provides a specific antibody against the PLA2R domain, that is, a CysR domain antibody Anti-hPLA2R CysR-rRmab (h4Fc). The rabbit-derived monoclonal antibody prepared by the present application using single B cell technology has higher sensitivity and significantly shortened production cycle. The standard curve established using the antibody can effectively calculate the content of autoimmune antibodies of the CysR domain of PLA2R in clinical samples, providing a tool for auxiliary diagnosis of diseases related to PLA2R, and further providing the use of a kit containing the antibody in auxiliary diagnosis of membranous nephropathy.

[0007] On the one hand, the present application provides an antibody or antigen-binding fragment that specifically binds to the CysR domain of PLA2R, comprising: CDR-H1 as shown in SEQ ID NO: 1, and CDR-H2 as shown in SEQ ID NO: 2, and CDR-H3 as shown in SEQ ID NO: 3, and CDR-L1 as shown in SEQ ID NO: 4, and CDR-L2 with an amino acid sequence of SAS, and CDR-L3 as shown in SEQ ID NO: 5.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises the following variable regions: (a) a heavy chain variable region VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6; (b) a light chain variable region VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable region VH as shown in SEQ ID NO: 6, and a light chain variable region VL as shown in SEQ ID NO: 7.

[0009] In one aspect, the present application provides a polynucleotide encoding the above-mentioned antibody or antigen-binding fragment.

[0010] In one aspect, the present application provides a vector comprising the polynucleotide of the present application.

[0011] In some embodiments, the vector includes a viral vector, an expression vector, a recombinant expression vector. In some embodiments, the expression vector can be any suitable recombinant expression vector, and the vector can be selected from pUC series (Fermentas Life Sciences, GlenBurnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, ZapII (Stratagene), λEMBL4 and λNM1149 can also be used. In some embodiments, the expression vector is pcDNA3.1.

[0012] In one aspect, the present application provides a host cell comprising the polynucleotide or vector of the present application.

[0013] In some embodiments, the host cell is a eukaryotic cell, a prokaryotic cell. In some embodiments, the host cell is a 293 cell.

[0014] In one aspect, the present application provides a kit for detecting autoimmune antibodies against the PLA2R domain.

[0015] In some embodiments, the kit comprises an antibody or antigen binding fragment as described above. In some embodiments, the kit is for non-diagnostic immunodetection of autoimmune antibodies in a specific domain of PLA2R. In some embodiments, the domain is a CysR domain. In some embodiments, the kit is a chemiluminescence method, an electrochemiluminescence method, or an ELISA. In some embodiments, the kit is an indirect ELISA detection kit, and a standard curve is established using the above-mentioned antibody or antigen binding fragment, which provides convenience for clinical detection of IgG antibodies to specific epitopes of PLA2R. In some embodiments, the kit also includes a coating plate, a diluent, an anti-human IgG antibody, a cleaning solution, and a stop solution.

[0016] In one aspect, the present application provides the use of the above-mentioned antibody or antigen-binding fragment in the preparation of a PLA2R detection product.

[0017] In one aspect, the present application provides the use of the kit as described above in the preparation of a membranous nephropathy detection or diagnosis product.

[0018] Beneficial effects of this application

[0019] The present application provides a specific antibody or an antigen-binding fragment thereof for the PLA2R domain, that is, a CysR domain antibody or an antigen-binding fragment thereof. The CysR domain antibody or an antigen-binding fragment thereof provided by the present application can specifically recognize the CysR domain of the corresponding PLA2R and has a high affinity. In addition, the present application provides a method for establishing a standard curve using a domain antibody, which facilitates the clinical detection of the concentration of anti-PLA2R different epitope determinant cluster IgG. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Development of cytometry sorting maps for CysR domain antibodies;

[0021] Figure 2 is the standard curve of CysR domain antibody; DETAILED DESCRIPTION

[0022] The present disclosure is described in detail below in conjunction with embodiments, but the implementation methods of the present disclosure are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present disclosure. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present disclosure.

[0023] Unless otherwise defined, all terms used herein have the same meaning as those generally understood by those skilled in the art to which the present disclosure belongs. All patents, patent applications and publications cited throughout the disclosure are incorporated herein by reference in their entirety. If there are multiple definitions for a term herein, those definitions in this section shall prevail.

[0024] The technical solutions provided by the present disclosure are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present disclosure.

[0025] Example 1: Preparation of anti-human PLA2R1 domain-specific monoclonal antibodies

[0026] 1. Antigen Preparation

[0027] This example is to develop a specific monoclonal antibody against the CysR domain of human PLA2R domain for use in establishing a standard curve to calculate the content of autoimmune antibodies in the corresponding specific domain of the clinical sample to be tested.

[0028] Domain CysR, sequence:

[0029] KGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNF SAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGG G(SEQ ID NO: 8)

[0030] The domain CTLD-1, the sequence is:

[0031] CYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLD EHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKK(SEQ ID NO: 9)

[0032] The domain CTLD-7, the sequence is:

[0033] PNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAH WIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAIC(SEQ ID NO: 10)

[0034] The domain CTLD-8, the sequence is:

[0035] WIKFKSNCYSFSTVLDSMSFEAAHEFCKKEGSNLLTIKDEAENAFLLEELFAFGSSVQ MVWLNAQFDGNNETIKWFDGTPTDQSNWGIRKPDTDYFKPHHCVALRIPEGLWQLSPCQE KKGFICK (SEQ ID NO: 11)

[0036] Human PLA2R1 extracellular domain protein, sequence:

[0037]

[0038] HEK293 mammalian cells were selected for expression of human PLA2R domain protein.

[0039] When designing the expression of the domain protein, a signal peptide is added to the N-terminus of the sequence for secretory expression of the protein, and a rabbit IgG-Fc (rFc) tag sequence and a 6×His (His) tag sequence are added to the C-terminus of the sequence.

[0040] 2. Preparation of Animal Immune Library

[0041] After the human PLA2R1 domain protein was successfully prepared, the domain protein (hPLA2R1-CysR-rFc) with rabbit IgG-Fc tag was added to immunize 5 New Zealand rabbits. In the first round of immunization, 500ug of immunogen was mixed with an equal amount of Freund's adjuvant to make an emulsion, which was injected subcutaneously at multiple points; the second immunization was performed 2 weeks later, 250ug of immunogen was mixed with an equal amount of Freund's adjuvant to make an emulsion, which was injected subcutaneously at multiple points, and the serum titer of the animals was measured after the third immunization.

[0042] A small amount of blood was collected from the ear vein and centrifuged at 8000g to prepare serum after coagulation. The plate was coated with a 6×His-tagged domain protein (hPLA2R1-CysR-His) and the serum titer was determined by indirect ELISA. Rabbits with high serum titer were selected and 250ug of immunogen was injected subcutaneously at multiple points for booster immunization, and the spleen of the rabbit was taken.

[0043] Rabbit spleen was physically ground and filtered through a porous filter to prepare a single-cell suspension.

[0044] 3. Antigen-specific single B cell isolation

[0045] This embodiment is based on the specific recognition of lymphoid B cell surface markers by flow cytometry antibodies, and uses flow cytometry sorting to complete the acquisition of specific single B cells in a single cell suspension.

[0046] Sorted the original, selected hPLA2R1-CysR-His, and coupled it with FITC dye.

[0047] Anti-rabbit IgG-Fc specific secondary antibody is a self-developed antibody coupled with PE dye.

[0048] When labeling cells, DAPI dye is added to distinguish dead / live cells.

[0049] Lymphocyte B cell sorting scheme: Dead / Live- / IgG+ / Antigen+.

[0050] Cell labeling operation: Rabbit lymphocyte suspension, centrifuge at 300g for 5min, add 5ml buffer, mix by inverting, and centrifuge at 300g for 5min. Discard the supernatant, repeat once, take 30ul of cell suspension for cell counting, take 40ul of cell suspension for blank control tube and single stain tube to be labeled. The remaining cell liquid is used as sample tube, centrifuge at 300g for 5min, and resuspend in a small amount of PBS. The blank tube is not treated. For single stain tube, add PBS to 100ul, and add 2ul PE, 2ul FITC, and 2ul DAPI dyes respectively. Sample tubes are PE dye 1.5ul / 10 6 Cells, FITC dye 2ug / 10 6 Cells, calculate the actual amount to be added, add the corresponding antibodies in a dark place, and place at 4°C for 30 minutes. After the antibody incubation is completed, add 2 ml of buffer solution, mix gently, centrifuge at 300g for 5 minutes, and repeat the wash 3 times. Resuspend with 1 ml of buffer solution, filter the cells, and wait for sorting.

[0051] After completing the fluorescence compensation adjustment, the living cell population and the PE and FITC double positive signal cell population were circled in turn (the sorting diagram is shown in Figure 2). Figure 1 ). The flow cytometer is programmed to sort antigen-specific B cells into a 96-well plate, with only one cell in each well. After sorting, the well plate needs to be stored at low temperature immediately. In this embodiment, a dry ice box is provided for short-term storage. Cell lysis solution is contained in the wells, and the sorted 96-well PCR plate is directly used for single B cell PCR experiments.

[0052] 4. Preparation of rabbit single B cell cDNA

[0053] The single B cell cDNA library preparation is based on SMART 5'RACE technology, and the reagents used are all N711 kits from Nanjing Vazyme Biotechnology Co., Ltd. (Vazyme), which can be purchased on the market. The amplification systems involved in the experiments in the examples can all be referred to the N711 kit instructions.

[0054] Single B cell RNA reverse transcription: After the sorting is completed, the 96-well plate is thawed and placed in a PCR instrument to run the program. After the program is completed, it is placed on ice for 2 minutes.

[0055] Single-stranded cDNA synthesis of single B cells: After the reverse transcription reaction program is completed, the single-stranded synthesis system can be added. After the system is added, the plate is gently mixed and placed in the PCR instrument to run the program. After the program is completed, the plate samples are placed on ice for 2 minutes.

[0056] Single B cell DNA double-strand synthesis: After the synthesis reaction of cDNA single-strand product is completed, the double-strand synthesis system can be added. After the system is added, the plate is gently mixed, centrifuged and placed in a PCR instrument to run the program. After the program is completed, the plate samples are placed on ice.

[0057] 5. Rabbit single B cell PCR technology to amplify antibody encoding genes

[0058] A single B cell cDNA library can retrieve naturally paired antibody heavy and light chain encoding genes.

[0059] The reagents used for the amplification of the coding gene are all P515 kits produced by Nanjing Vazyme Biotechnology Co., Ltd. (Vazyme), which can be purchased on the market. The amplification systems involved in the experiments in the examples can all be referred to the instructions of the P515 kit.

[0060] The upstream primer contains a homology arm that overlaps with the 3' end of the promoter CMV gene sequence, so after the antibody encoding gene is retrieved, it can be directly used to construct a linear expression frame.

[0061] The downstream primer for extracting the antibody heavy chain encoding gene is located in the constant region and includes a homology arm that overlaps with the BGH-polyA gene sequence.

[0062] The downstream primer for extracting the light chain encoding gene is located in the constant region and contains a homology arm complementary to the BGH-polyA gene sequence. Therefore, after the antibody encoding gene is extracted, it can be directly used for the construction of a linear expression frame.

[0063] Antibody heavy chain coding region gene amplification, the forward primer sequence is:

[0064] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 13).

[0065] Antibody heavy chain coding region gene amplification, reverse primer sequence is:

[0066] tagtggatccgagctcggtacctcatttacccggagagcg (SEQ ID NO: 14).

[0067] Antibody light chain coding region gene amplification, the forward primer sequence is:

[0068] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 13).

[0069] Antibody light chain coding region gene amplification, reverse primer sequence is:

[0070] tagtggatccgagctcggtacctcaacagtcacccctattg (SEQ ID NO: 15).

[0071] Retrieve the antibody light chain and heavy chain encoding genes: Add the PCR amplification system according to the instructions, gently mix the well plate, place it in the PCR instrument to run the program, and after the program is completed, let the well plate samples stand on ice.

[0072] In this example, the pairing positive rate of the amplified products of the antibody light chain and heavy chain encoding genes in the same 96-well plate was above 80%, and the bands were clear by agarose gel electrophoresis, indicating that the single B cell flow sorting and encoding gene amplification experiments were effective. The amplified products were used to construct recombinant expression plasmids.

[0073] 6. Construction and expression of antibody heavy chain and light chain recombinant expression plasmids

[0074] The development of domain-specific antibodies is used to calculate the content of autoantibodies to the corresponding domains in human clinical samples. Therefore, the antibody modification plan is to keep the rabbit Fab segment unchanged and transform the Fc segment into human IgG4-Fc.

[0075] The recombinant expression vector was pcDNA3.1 (Invitrogen), which was purchased from the official website of ThermoFisher SCIENTIFIC. Before the recombinant construction, the expression vector was linearized by single digestion with HindⅢ restriction enzyme, which was purchased from the official website of New England Biolabs.

[0076] For efficient recombination between vector and coding gene, choose seamless cloning kit and purchase C115# kit on Vazyme official website. Construction of recombinant expression plasmid: The coding amplification products of antibody heavy chain and light chain are circularly connected with pcDNA3.1 linearized vector using seamless cloning technology, and then transformed into Escherichia coli DH5α competent cells, coated with LB fixed medium plates, and inverted at 37°C overnight.

[0077] Selection of recombinant positive clones: Initially screen the heavy and light chains of the antibody, pick 8 single colonies respectively, and determine the colony positive rate after PCR bacterial test. If the bacterial test positive rate is low, single colonies can be picked for further bacterial testing.

[0078] The bacterial detection PCR of the recombinant plasmid, the upstream primer sequence of the vector is: caagctggctagcgtttaaactt (SEQ ID NO: 16).

[0079] The sequence of the downstream primer for antibody heavy chain detection PCR is: ctcatttacccggagagcg (SEQ ID NO: 17).

[0080] The sequence of the downstream primer for antibody light chain bacterial detection PCR is: acctcaacagtcacccctattg (SEQ ID NO: 18).

[0081] Recombinant positive clones were sent for testing: 5 clones of the antibody heavy chain and light chain were selected from the PCR-positive clones for bacterial testing and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0082] Rabbit antibody gene sequence analysis: The IMGT database was used to determine the V region gene of the antibody sequence, and the antibody sequence was analyzed for the antibody heavy chain and light chain CDR1 / CDR2 / CDR3 regions, and the correct sequence number of the bacterial test PCR positive clone was derived and determined.

[0083] Small-scale expression of recombinant expression plasmids: clone the correct sequence, extract the antibody light chain and heavy chain plasmids from the bacterial solution. Mix the plasmids and co-transfect them into HEK293 mammalian cells. 10 days after cell transfection, collect the cell supernatant by centrifugation. The supernatant is evaluated for antigen specificity, and the cell supernatant is purified after the initial ELISA screening results.

[0084] In this example, 100 plasmids were transfected in each round, that is, 100 monoclonal antibodies were obtained in each round of transfection. A total of 3 rounds of transfection experiments were performed.

[0085] 7. Evaluation of Antigen Specificity of Recombinant Expression Supernatant

[0086] The original human PLA2R1 domain protein and extracellular domain protein were selected and tagged with His, and were named hPLA2R1-CysR-His, hPLA2R1-CTLD1-His, hPLA2R1-CTLD7-His, hPLA2R1-CTLD8-His, and hPLA2R1-total-His, respectively.

[0087] Antibody screening program for specific domain proteins of human PLA2R1: The antibody to be tested can bind specifically to a single domain and can also bind to the extracellular domain protein, that is, it is initially determined to be a specific antibody for the domain protein.

[0088] The cell supernatant was detected by sandwich ELISA, and the self-produced rabbit secondary antibody MouseAnti-Rabbit IgG-Fc mAb (MARIgG Fc-Ab) was used to coat the plate, and the purchased goat anti-rabbit secondary antibody was coupled to HRP for detection. The addition of cell supernatant and the detection OD>1.5 indicated the normal expression of the recombinant plasmid.

[0089] Indirect ELISA was used to detect the cell supernatant, and the reactivity of 5 antigen-coated plates was evaluated to obtain the initial screening test results of the plate supernatant (only the test data of 50 cell supernatants were shown as an example, and Table 1 is the test data of CysR domain antibody).

[0090] Table 1: CysR domain antigen-antibody affinity data table (partial)

[0091]

[0092]

[0093] Results of indirect ELISA test of CysR domain: 300 monoclonal antibodies were initially screened and transfected, which reacted with hPLA2R1-CysR-His and total-His simultaneously (OD>1.0), and did not react with CTLD1-His, CTLD7-His, and CTLD8-His (OD<0.5). A total of 220 positive antibodies were initially screened, of which 50 specific monoclonal antibodies were selected.

[0094] The cell supernatants identified in the preliminary screening were purified by protein A to obtain a small amount of monoclonal antibodies, with an average of 1-3 mg per strain.

[0095] Example 2: Further evaluation and screening of domain-specific antibodies on the ELISA platform

[0096] Further evaluation and screening plan for the optimal domain antibodies in the initial screening:

[0097] The concentration of domain proteins was fixed, the purified antibodies from the initial screening were graded diluted, and the indirect ELISA method was used to evaluate and screen out specific antibodies for each domain of human PLA2R1 with strong antigen binding reactivity, wide detection linear range, and high back-calculation accuracy.

[0098] The operation of the indirect ELISA platform is as follows:

[0099] Coating: Take the corresponding domain protein, dilute the coating solution to 2ug / ml, add 100ul coating solution to each well, incubate at 37℃ for 1h and then at 4℃ overnight.

[0100] Blocking: The next day, remove the ELISA plate and wash it three times with PBST. Add 200ul of blocking solution (1% BSA) to each well, incubate at 37°C for 2h, and wash it three times with PBST.

[0101] Add primary antibody-domain antibody: dilute the corresponding primary antibody by 2×, add 100ul diluent to each well. Set up negative control and blank control, act at 37℃ for 1h, and wash 3 times with PBST.

[0102] Add self-produced anti-human IgG4-Fc secondary antibody: dilute the secondary antibody at 1:5000, 100ul per well, act at 37℃ for 1h, and wash 3 times with PBST.

[0103] Add substrate: add 100ul of substrate reaction solution (freshly prepared and used, protected from light) to each well, incubate at 37℃ for 30min, then remove and add stop solution. Add substrate for color development, and measure A450 value with an enzyme-labeled instrument.

[0104] CysR domain protein screened 50 specific antibodies, and hPLA2R1-CysR-His and hPLA2R1-CysR-rFc proteins were used to coat ELISA plates, respectively. After indirect ELISA test, a better antibody was screened and named Anti-hPLA2R CysR-rRmab (h4Fc). The experimental results showed that the detection range of the selected antibody for the hPLA2R-CysR-His protein coated plate was 3.9-250.0ng / ml. Figure 2 , which can be used for the detection of clinical samples.

[0105] CysR domain antibody (Anti-hPLA2R CysR-rRmab (h4Fc)), the heavy chain variable region sequence is:

[0106] QSLEESGGRLVTPGTPLTLTCTVSGIDLSSNAMGWVRQAPGKGLEYIGIIGGVGSAYYA SWAKGRFTISKTSSTTVDLKITSPTTEDTATYFCARGGDGAVISYFALWGQGTLVTVSS (SEQ ID NO: 6);

[0107] CDR1: GIDLSSNA (SEQ ID NO: 1); CDR2: IGGVGSA (SEQ ID NO: 2);

[0108] CDR3:ARGGDGAVISYFAL(SEQ ID NO: 3)

[0109] The light chain variable region sequence is:

[0110] AQVLTQTPSPVSAAVGGTVTINCQASQSVYNNDRLAWYQQKPGQPPKLLIYSASTLAS GVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGYYPVGINAFGGGTEVVVK (SEQ ID NO: 7);

[0111] CDR1: QSVYNNDR (SEQ ID NO: 4); CDR2: SAS; CDR3: QGYYPVGINA (SEQ ID NO: 5)

[0112] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to the CysR domain of PLA2R, wherein the amino acid sequences of its heavy chains CDRH1-CDRH3 are shown as SEQ ID NO: 1-SEQ ID NO: 3, respectively, the amino acid sequence of its light chain CDRL1 is shown as SEQ ID NO: 4, the amino acid sequence of its light chain CDRL2 is SAS, and the amino acid sequence of its light chain CDRL3 is shown as SEQ ID NO:

5.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain variable region VH as shown in SEQ ID NO: 6 and a light chain variable region VL as shown in SEQ ID NO:

7.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

4. A carrier, characterized in that It comprises the polynucleotide according to claim 3.

5. A host cell comprising the polynucleotide or vector of the present application.

6. A kit for detecting PLA2R autoimmune antibodies, characterized in that: The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

7. The kit according to claim 6, further comprising a coating plate, a diluent, an anti-human IgG antibody, a cleaning solution and a stop solution.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 in the preparation of a PLA2R detection product.

9. Use of the kit according to any one of claims 6 to 7 in the preparation of a product for the detection or diagnosis of membranous nephropathy.

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