Antibodies specific for the CysR domain of PLA2R and their applications

Abstract

This application discloses a specific antibody targeting the CysR domain of PLA2R and its application, belonging to the field of bioengineering technology. This application provides an antibody targeting the CysR domain of PLA2R or an antigen-binding fragment thereof. The CysR domain antibody or antigen-binding fragment provided herein can specifically recognize the corresponding PLA2R domain with high affinity. Furthermore, this application provides a method for establishing a standard curve using the CysR domain antibody of PLA2R as a standard, facilitating clinical testing of the concentration of IgG against different PLA2R epitopes.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and relates 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 characterized by the deposition of immune complexes on the glomerular basement membrane (GBM), accompanied by significant GBM thickening. Based on its pathogenesis and etiology, MN can be divided into idiopathic membranous nephropathy (IMN) and secondary MN.

[0003] The 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-rich domain, CysR), a single fibronectin type II domain (Fibronectin type II domain, FNII) and 8 to 10 C-type lectin-like domains (CTLD). These separate domains can directly bind to circulating autoantibodies.

[0004] Studies have shown an association between PLA2R-reactive epitopes and disease activity, leading to the hypothesis of epitope spreading. "Epitope spreading" refers to the immune response to endogenous epitopes following the release of autoantigens during chronic autoimmune or inflammatory processes, typically spreading from the primary immunodominant epitope to other sites within the molecule. In IMN, CysR is the immunodominant epitope of PLA2R, and the spreading of the antigenic determinant begins within the CysR domain and then gradually spreads to CTLD1, CTLD7, or CTLD8. Furthermore, some studies suggest that analysis of PLA2R epitope signatures and spread is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis.

[0005] Therefore, detecting anti-PLA2R autoantibodies is an important approach for monitoring IMN. In most IMN patients, the predominant autoantibody subtype is IgG4, and there are currently no quantitative assays for antibodies targeting the PLA2R domain. It is crucial to develop a quantitative assay for antibodies targeting the CysR domain of PLA2R and for detecting CysR domain-specific IgG antibody levels. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present application provides a specific antibody targeting the PLA2R domain, namely, a CysR domain antibody, Anti-hPLA2R CysR-rRmab (h4Fc). This rabbit-derived monoclonal antibody, prepared using single B cell technology, exhibits enhanced sensitivity and a significantly shortened production cycle. The standard curve established using this antibody can effectively calculate the level of autoimmune antibodies to the CysR domain of PLA2R in clinical samples, providing a tool for the auxiliary diagnosis of PLA2R-related diseases. Furthermore, a kit containing this antibody is provided for the auxiliary diagnosis of membranous nephropathy.

[0007] In one aspect, 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 set forth 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 set forth in SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable region VH as set forth in SEQ ID NO: 6 and a light chain variable region VL as set forth 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 the group consisting of 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 or 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 the antibodies or antigen-binding fragments described above. In some embodiments, the kit is used for non-diagnostic immunoassay detection of autoimmune antibodies against specific PLA2R domains. In some embodiments, the domain is a CysR domain. In some embodiments, the kit utilizes chemiluminescence, electrochemiluminescence, or ELISA. In some embodiments, the kit is an indirect ELISA kit, utilizing the antibodies or antigen-binding fragments described above to establish a standard curve, facilitating clinical detection of IgG antibodies against specific PLA2R epitopes. In some embodiments, the kit further comprises a coating plate, a diluent, an anti-human IgG antibody, a wash 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 described above in the preparation of a product for detecting or diagnosing membranous nephropathy.

[0018] Beneficial effects of this application

[0019] The present application provides antibodies or antigen-binding fragments thereof specific for PLA2R domains, namely, CysR domain antibodies or antigen-binding fragments thereof. The CysR domain antibodies or antigen-binding fragments provided herein are capable of specifically recognizing the corresponding PLA2R CysR domain with high affinity. Furthermore, the present application provides a method for establishing a standard curve using domain antibodies, facilitating clinical testing of IgG concentrations against different epitope determinants of PLA2R. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Development of cytometric 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 with reference to the embodiments, but the embodiments of the present disclosure are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present disclosure. For those skilled in the art, other similar embodiments obtained without creative work fall within the scope of protection 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 this disclosure pertains. All patents, patent applications, and publications cited throughout the disclosure are incorporated herein by reference in their entirety. If multiple definitions exist for a term herein, those 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 understood as limiting the scope of protection of the present disclosure.

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

[0026] 1. Antigen Preparation

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

[0028] The domain CysR has the following 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 has the following sequence:

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

[0034] The domain CTLD-8 has the following sequence:

[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 successful preparation of the human PLA2R1 domain protein, a rabbit IgG-Fc-tagged domain protein (hPLA2R1-CysR-rFc) was added to immunize five New Zealand rabbits. For the first round of immunization, 500 μg of the immunogen was mixed with an equal amount of Freund's adjuvant to form an emulsion and injected subcutaneously at multiple sites. Two weeks later, a second round of immunization was performed, with 250 μg of the immunogen mixed with an equal amount of Freund's adjuvant to form an emulsion and injected subcutaneously at multiple sites. Serum titers were measured after the third round of immunization.

[0042] A small blood sample was collected from the marginal ear vein and, after coagulation, centrifuged at 8000 g to prepare serum. Serum was coated with a 6× His-tagged domain protein (hPLA2R1-CysR-His). Serum titers were determined by indirect ELISA. Rabbits with high serum titers were boosted once with multiple subcutaneous injections of 250 μg of the immunogen, and spleens were harvested.

[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 example is based on the specific recognition of lymphoid B cell surface markers by flow cytometry antibodies, and uses flow cytometry sorting to obtain specific single B cells from a single cell suspension.

[0046] The original was sorted, hPLA2R1-CysR-His was selected, and FITC dye was coupled.

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

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

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

[0050] Cell labeling operation: rabbit lymphocyte suspension, centrifuge at 300g for 5 minutes, add 5ml buffer solution, mix by inverting, and centrifuge at 300g for 5 minutes. Discard the supernatant, repeat once, take 30ul cell suspension for cell counting, take 40ul cell suspension for blank control tube and single staining tube to be labeled. The remaining cell liquid is used as sample tube, centrifuge at 300g for 5 minutes, and resuspend in a small amount of PBS solution. The blank tube is not processed. For single staining tube, add PBS solution to 100ul, and add 2ul PE, 2ul FITC, and 2ul DAPI dye respectively. The sample tube is 1.5ul / 10 of PE dye. 6 cells, FITC dye 2ug / 10 6 Calculate the amount of cells to be added, add the appropriate antibody, and incubate at 4°C for 30 minutes in the dark. After the antibody incubation is complete, add 2 ml of buffer, gently mix, centrifuge at 300 g for 5 minutes, and repeat the wash three times. Resuspend in 1 ml of buffer, filter the cells, and prepare 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 was programmed to sort antigen-specific B cells into a 96-well plate, with only one cell per well. After sorting, the plate was immediately stored at low temperatures. In this example, a dry ice box was provided for temporary storage. Cell lysis buffer was added to the wells, and the sorted 96-well PCR plate was used directly for single B cell PCR experiments.

[0052] 4. Preparation of Rabbit Single B Cell cDNA

[0053] Single B cell cDNA library preparation is based on SMART 5' RACE technology. The reagents used are all commercially available N711 kits from Nanjing Vazyme Biotechnology Co., Ltd. For the amplification systems involved in the experiments in the examples, please refer to the N711 kit instructions.

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

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

[0056] Single B cell DNA double-strand synthesis: After the cDNA single-strand synthesis reaction is complete, 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 encoding gene amplification were all P515 kits from Nanjing Vazyme Biotechnology Co., Ltd. (Vazyme), which are commercially available. 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 cassette.

[0061] The downstream primer for retrieval of the antibody heavy chain encoding gene is located in the constant region and contains 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 to construct a linear expression frame.

[0063] For the gene amplification of the antibody heavy chain coding region, 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 positivity rate of amplified products encoding the antibody light and heavy chains in the same 96-well plate was over 80%. Clear bands were detected by agarose gel electrophoresis, demonstrating the effectiveness of both single B cell flow cytometry sorting and the coding gene amplification assay. The amplified products were used to construct recombinant expression plasmids.

[0073] 6. Construction and expression of antibody heavy 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 was determined to keep the rabbit Fab segment unchanged and transform the Fc segment into human IgG4-Fc.

[0075] The recombinant expression vector, pcDNA3.1 (Invitrogen), was purchased from the ThermoFisher SCIENTIFIC website. Prior to recombinant construction, the expression vector was linearized using a single restriction enzyme, Hind III, purchased from the New England Biolabs website.

[0076] For efficient recombination between the vector and the coding gene, use the seamless cloning kit (C115#) available on the Vazyme website. Construction of the recombinant expression plasmid: The antibody heavy and light chain encoding amplification products were circularly linked to the linearized pcDNA3.1 vector using seamless cloning technology. The cells were then transformed into competent E. coli DH5α cells, plated with LB fixed medium, and incubated inverted at 37°C overnight.

[0077] Selection of recombinant positive clones: Initially screen the heavy and light chains of the antibody, picking eight single colonies for each. After PCR testing, determine the colony positivity rate. If the positive rate is low, single colonies can be picked for further 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 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 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 heavy and light chain CDR1 / CDR2 / CDR3 regions were analyzed to derive and determine the correct sequence number of the PCR-positive clone.

[0083] Small-scale expression of recombinant expression plasmids: After cloning the correct sequence, small-scale extraction of the antibody light and heavy chain plasmids is performed from the bacterial culture. The plasmids are mixed and co-transfected into HEK293 mammalian cells. Ten days after transfection, the cell supernatant is collected by centrifugation. The supernatant is then assayed for antigen specificity and purified after initial ELISA screening results are obtained.

[0084] In this example, 100 plasmids were transfected per round, i.e., 100 monoclonal antibodies were obtained per round of transfection. A total of three rounds of transfection experiments were performed.

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

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

[0087] Antibody screening plan for human PLA2R1 specific domain protein: The antibody to be tested specifically binds 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 assayed by sandwich ELISA. The plate was coated with a self-produced rabbit secondary antibody, Mouse Anti-Rabbit IgG-Fc mAb (MARIgG Fc-Ab), and detection was performed with a purchased goat anti-rabbit secondary antibody conjugated to HRP. When the cell supernatant was added, an OD > 1.5 was detected, indicating normal expression of the recombinant plasmid.

[0089] The cell supernatant was tested by indirect ELISA, and the reactivity of the 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 are shown as an example, and Table 1 is the test data of CysR domain antibodies).

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

[0091]

[0092]

[0093] Results of indirect ELISA test for CysR domain: A total of 300 monoclonal antibodies were initially screened and transfected, and they reacted simultaneously with hPLA2R1-CysR-His and total-His (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 supernatant identified in the preliminary screening was 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 purified antibodies from the initial screening were serially diluted at a fixed domain protein concentration. The indirect ELISA method was used to evaluate and screen specific antibodies for each domain of human PLA2R1 with strong antigen-binding reactivity, a wide linear range of detection, 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 of coating solution to each well, incubate at 37℃ for 1h, and then incubate at 4℃ overnight.

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

[0101] Add primary antibody-domain antibody: dilute the corresponding primary antibody by 2×, adding 100 μl of diluent to each well. Set up negative and blank controls, incubate at 37°C for 1 hour, and wash three times with PBST.

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

[0103] Add substrate: Add 100 μl of substrate reaction solution (freshly prepared and protected from light) to each well and incubate at 37°C for 30 minutes. Then remove and add stop solution. Add substrate for color development and measure the A450 value with a microplate reader.

[0104] Fifty specific antibodies were initially screened for the CysR domain protein. Both hPLA2R1-CysR-His and hPLA2R1-CysR-rFc proteins were used to coat ELISA plates. One superior antibody was selected and named Anti-hPLA2R CysR-rRmab (h4Fc) after indirect ELISA testing. The results showed that the detection range of the selected antibody for the hPLA2R-CysR-His protein-coated plate was 3.9-250.0 ng / 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] While 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 invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the CysR domain of PLA2R, wherein the amino acid sequences of its heavy chains CDRH1 to CDRH3 are shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively, the amino acid sequence of its light chain CDRL1 is shown in 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 in 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. A host cell comprising the polynucleotide according to claim 3 or the vector according to claim 4.

6. A kit for detecting PLA2R autoantibodies, 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 preparing a product for detecting or diagnosing membranous nephropathy.

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