Antibodies specific for the ctld7 domain of pla2r and uses thereof

Abstract

The application discloses a specific antibody for a CTLD7 domain of PLA2R and application thereof, and belongs to the technical field of bioengineering. The application provides a CTLD7 domain antibody of PLA2R or an antigen binding fragment thereof. The CTLD7 domain antibody or the antigen binding fragment thereof provided by the application can specifically recognize a corresponding PLA2R domain and has high affinity. In addition, the application provides a standard curve established by using the CTLD7 domain antibody of PLA2R as a standard product, and provides convenience for clinical detection of the concentration of IgG of anti-PLA2R CTLD7.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and relates to a specific antibody against the CTLD7 domain of human PLA2R, a detection kit containing the antibody and application thereof. BACKGROUND

[0002] Membranous nephropathy (MN) is a kidney-specific autoimmune disease, and the main pathological feature is the deposition of immune complexes on the glomerular basement membrane (GBM) and the significant thickening of the GBM. According to the different 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, has a large extracellular region, and is highly glycosylated and composed of multiple individual domains (a single cysteine-rich domain (CysR), a single fibronectin type II domain (FNII) and 8-10 C-type lectin domains (CTLD)). These individual domains can directly bind to circulating autoantibodies.

[0004] Studies have found the correlation between PLA2R-reactive epitopes and disease activity, and proposed the hypothesis of epitope spreading. “Epitope spreading” refers to the immune response to endogenous epitopes after the release of self-antigens during chronic autoimmune or inflammatory reactions, 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 spreading of antigenic determinants starts from the CysR domain and then gradually spreads to CTLD1, CTLD7 or CTLD8. In addition, it is believed that the analysis of PLA2R epitope characteristics and spreading is a powerful means of monitoring disease severity and stratifying patients by kidney prognosis.

[0005] Therefore, detecting anti-PLA2R autoantibodies is an important way to monitor IMN, and the main subtype of autoantibodies in most IMN patients is IgG4, and there is currently no quantitative detection method for antibodies against the PLA2R domain. It is crucial to seek a quantitative detection method for antibodies against the CTLD7 domain of PLA2R and the CTLD7 domain-specific IgG antibody level. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the present application provides a specific antibody against the PLA2R domain, i.e. a CTLD7 domain antibody Anti-hPLA2R CTLD7-rRmab (h4Fc). The rabbit-derived monoclonal antibody prepared by the single B cell technology of the present application has higher sensitivity and significantly shorter production cycle. The standard curve established by using the antibody can effectively calculate the content of the autoantibody of the CTLD7 domain of PLA2R in the clinical sample, and provide a tool for the auxiliary diagnosis of PLA2R-related diseases, and further provide the application of the kit comprising the antibody in 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 CTLD7 domain of PLA2R.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region CDR-H1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, and a heavy chain variable region CDR-H2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, and a heavy chain variable region CDR-H3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 3, and a light chain variable region CDR-L1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 4, and a light chain variable region CDR-L2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SAS, and a light chain variable region CDR-L3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 5.

[0009] In some embodiments, the antibody or antigen-binding fragment comprises: a CDR-H1 as set forth in SEQ ID NO: 1, and a CDR-H2 as set forth in SEQ ID NO: 2, and a CDR-H3 as set forth in SEQ ID NO: 3, and a CDR-L1 as set forth in SEQ ID NO: 4, and a CDR-L2 of the amino acid sequence SAS, and a CDR-L3 as set forth in SEQ ID NO: 5.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises a variable region of: (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 as 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 as 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.

[0011] In one embodiment, the antibody or antigen-binding fragment thereof of the present application comprises an alpha (a), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present application is of the IgGl, IgG2, IgG3, or IgG4 subclass. In one embodiment, the antibody or antigen-binding fragment thereof of the present application comprises a lambda (λ) light chain or a kappa (κ) light chain. In some embodiments, the "class" of an antibody refers to the type of constant domains or constant regions possessed by its heavy chains. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2, of which the heavy chain constant domains corresponding to the different classes are called α, δ, ε, γ, and μ, respectively. In some embodiments, the antibody is of the IgG4 isotype.

[0012] In one aspect, the present application provides a polynucleotide encoding the aforementioned antibody or antigen-binding fragment. The nucleic acid molecule described herein can be effective to express the aforementioned antibody or antigen-binding fragment thereof.

[0013] In one aspect, the present application provides a vector comprising the polynucleotide of the present application. The expression vector described herein can be effective to express the aforementioned antibody or antigen-binding fragment thereof.

[0014] In some embodiments, the vector comprises a viral vector, an expression vector, a recombinant expression vector. In some embodiments, the expression vector can be any suitable recombinant expression vector, the vector can be selected from the group consisting of pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, La Jolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as lambda GT10, lambda GT11, Zap II (Stratagene), lambda EMBL4, and lambda NM1149 can also be used. In some embodiments, the expression vector is pcDNA3.1.

[0015] In one aspect, the present application provides a host cell comprising a polynucleotide or a vector of the present application.

[0016] In some embodiments, the host cell is a eukaryotic cell, a prokaryotic cell. In some embodiments, the host cell is a 293 cell.

[0017] In one aspect, the present application provides a method of producing an antibody or an antigen binding fragment thereof, comprising:

[0018] (a) culturing the host cell of the present application under conditions suitable for expression of the antibody or the antigen binding fragment thereof, and

[0019] (b) optionally, recovering the antibody or the antigen binding fragment thereof.

[0020] In one aspect, the present application provides use of the antibody or the antigen binding fragment as described above, the polynucleotide as described above, the expression vector as described above, or the host cell as described above in the manufacture of a PLA2R detection product.

[0021] In one aspect, the present application provides a kit for detecting autoantibodies against a domain of PLA2R.

[0022] In some embodiments, the kit comprises the antibody or antigen binding fragment as described above. In some embodiments, the kit is used for immunodetection of autoantibodies in specific domains of PLA2R for non-diagnostic purposes. In some embodiments, the domain is the CTLD7 domain. In some embodiments, the kit is a chemiluminescence method, electrochemiluminescence method, ELISA. In some embodiments, the kit is an indirect ELISA detection kit. In some embodiments, the kit further comprises a coated plate, diluent, anti-human IgG antibody, washing solution and termination solution. In some embodiments, the kit is used to establish a standard curve, which provides convenience for clinical detection of IgG antibodies against specific epitopes of PLA2R.

[0023] In one aspect, the present application provides use of the kit as described above in the preparation of a product for detection or diagnosis of membranous nephropathy.

[0024] Advantages of the present application

[0025] The present application provides specific antibodies or antigen binding fragments thereof against the PLA2R domain, i.e. CTLD7 domain antibodies or antigen binding fragments thereof. The CTLD7 domain antibodies or antigen binding fragments thereof provided by the present application can specifically recognize the corresponding PLA2R CTLD7 domain, and have high affinity. In addition, the present application provides a standard curve established by domain antibodies, which provides convenience for clinical detection of the concentration of IgG against different epitope determinants of PLA2R. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Cell sorter plot for CTLD7 domain antibodies;

[0027] Figure 2 Standard curve for CTLD7 domain antibodies. DETAILED DESCRIPTION

[0028] The present disclosure will be described in detail below with reference to the embodiments. However, the embodiments of the present disclosure are not limited thereto, and it is obvious that the embodiments described below are only part of the embodiments of the present disclosure, and other similar embodiments can be obtained by those skilled in the art without creative labor, which fall within the protection scope of the present disclosure.

[0029] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. If there is a plurality of definitions for terms herein, those in this section prevail.

[0030] The technical solutions provided by the present disclosure will be described in detail below in combination with embodiments, but they should not be understood as limiting the scope of protection of the present disclosure.

[0031] Example 1: Preparation of anti-human PLA2R1 domain-specific monoclonal antibody

[0032] 1. Preparation of antigen

[0033] This example is to develop a specific monoclonal antibody against the specific domain CTLD7 of human phospholipase A2 receptor (PLA2R) for the establishment of a standard curve to calculate the content of autoantibodies in the specific domain corresponding to the clinical samples to be tested.

[0034] Domain CTLD-7, sequence is:

[0035] PNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAIC (SEQ ID NO: 8);

[0036] Domain CysR, sequence is:

[0037] KGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGG (SEQ ID NO: 9);

[0038] Domain CTLD-1, sequence is:

[0039] CYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKK (SEQ ID NO: 10);

[0040] Domain CTLD-8, sequence is:

[0041] WIKFKSNCYSFSTVLDSMSFEAAHEFCKKEGSNLLTIKDEAENAFLLEELFAFGSSVQMVWLNAQFDGNNETIKWFDGTPTDQSNWGIRKPDTDYFKPHHCVALRIPEGLWQLSPCQEKKGFICK (SEQ ID NO: 11);

[0042] The human PLA2R1 extracellular domain protein has the sequence of:

[0043]

[0044] Expression of human PLA2R domain protein, select HEK293F mammalian cell expression.

[0045] When designing the expression of domain protein, signal peptide is added to the N-terminus of the sequence for secretory expression of the protein, and rabbit IgG-Fc (rFc) tag sequence and 6xHis (His) tag sequence are added to the C-terminus of the sequence, respectively.

[0046] 2. Preparation of animal immunization library

[0047] After the successful preparation of human PLA2R1 domain protein, five New Zealand rabbits were immunized with rabbit IgG-Fc tagged domain protein (hPLA2R1-CTLD7-rFc). In the first immunization, 500 ug of immunogen was mixed with an equal amount of Freund's adjuvant to prepare an emulsifier, which was injected subcutaneously in multiple points. Two weeks later, the second immunization was performed, and 250 ug of immunogen was mixed with an equal amount of Freund's adjuvant to prepare an emulsifier, which was injected subcutaneously in multiple points. The serum titer of the animals was determined after the third immunization.

[0048] A small amount of blood sample was collected from the auricular marginal vein and centrifuged at 8000g after clotting to prepare serum. The plate was coated with 6xHis tagged domain protein (hPLA2R1-CTLD7-His), and the serum titer was determined by indirect ELISA method. The rabbit with high serum titer was subcutaneously injected with 250 ug of immunogen in multiple points for one time of booster immunization, and the rabbit spleen was removed.

[0049] The rabbit spleen was physically ground and filtered through a porous filter to prepare a single cell suspension.

[0050] 3. Sorting of antigen-specific single B cells

[0051] This example is based on the specific recognition of lymphocyte B cell surface markers by flow cytometry, and the specific single B cells in the single cell suspension are obtained by flow cytometry sorting.

[0052] The sorting principle is to select hPLA2R1-CTLD7-His, which is coupled with FITC dye.

[0053] The anti-rabbit IgG-Fc specific secondary antibody is a self-developed antibody, which is coupled with PE dye.

[0054] When labeling cells, DAPI dye is added to distinguish dead / live cells (Dead / Live cells).

[0055] Lymphocyte B cell sorting scheme: Dead / Live- / IgG+ / Antigen+.

[0056] Cell labeling operation: rabbit lymphocyte suspension, 300g centrifugal 5min, add 5ml buffer liquid, upside down mix, 300g centrifugal 5min. Discard the supernatant, repeat 1 time, take 30ul cell suspension for cell counting, take 40ul cell suspension for blank control tube and single dye tube to be labeled. The remaining cell solution is used as a sample tube, 300g centrifugal 5min, resuspended with a small amount of PBS solution. The blank tube is not treated. Single dye tube, supplement PBS solution to 100ul, add 2ul PE, 2ul FITC, 2ul DAPI dye respectively. The sample tube is added with PE dye 1.5ul / 10 6 Cells, FITC dye 2ug / 10 6 Cells, calculate the actual amount of addition, add the corresponding antibody in the dark, 4℃ for 30min. After incubation of the antibody, add 2ml buffer solution, mix gently, 300g centrifugal 5min, repeat 3 times. Resuspend with 1ml buffer solution, filter the cells, and wait for machine sorting.

[0057] After completing the fluorescence compensation adjustment, the viable cell population, PE and FITC double positive signal cell population are sequentially circled (sorting diagram as Figure 1 ). Set the program of flow cytometry, sort the antigen-specific B cells into 96-well plates, each well containing only one cell, and store the plate immediately after sorting at low temperature. Dry ice box is prepared for short-term storage in this example. The well contains cell lysis solution, and the 96-well PCR plate is directly used for single B cell PCR experiment.

[0058] 4. Rabbit single B cell cDNA preparation

[0059] Single B cell cDNA library preparation is based on SMART 5'RACE technology, and all reagents are Nanjing Vazyme Biotech Co., Ltd. (Vazyme) N711 kit, which can be purchased in the market. The amplification system involved in the experiment in the example can refer to the N711 kit instruction manual.

[0060] Single B cell RNA reverse transcription: after thawing, the 96-well plate is placed in the PCR instrument to run the program, and after the program is completed, it is placed on ice for 2min.

[0061] Single B cell cDNA single strand synthesis: after the reverse transcription reaction program is completed, the single strand synthesis system can be added. After the system is added, the well plate is mixed gently, placed in the PCR instrument to run the program, and after the program is completed, the well plate sample is placed on ice for 2min.

[0062] 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 well plate is mixed gently, centrifuged and placed in the PCR instrument to run the program, and after the program is completed, the well plate sample is placed on ice.

[0063] 5. Rabbit single B cell PCR technology to amplify antibody encoding genes

[0064] Single B cell cDNA library, which can be used to extract naturally paired antibody heavy and light chain encoding genes.

[0065] The reagents used for amplification of the encoding genes are P515 kits from Nanjing Vazyme Biotech Co., Ltd. (Vazyme), which can be purchased in the market. The amplification system involved in the experiments in the examples can be referred to the P515 kit instructions.

[0066] The upstream primer contains a homologous arm that overlaps with the 3' end of the CMV promoter gene sequence, so that the antibody encoding gene can be directly used for the construction of the recombinant expression frame after extraction.

[0067] The downstream primer for the extraction of the antibody heavy chain encoding gene is located in the constant region, which contains a homologous arm that overlaps with the BGH-polyA gene sequence.

[0068] The downstream primer for the extraction of the light chain encoding gene is located in the constant region, which contains a homologous arm that overlaps with the BGH-polyA gene sequence, so that the antibody encoding gene can be directly used for the construction of the recombinant expression frame after extraction.

[0069] The forward primer sequence for amplification of the antibody heavy chain encoding region is:

[0070] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 13).

[0071] The reverse primer sequence for amplification of the antibody heavy chain encoding region is:

[0072] tagtggatccgagctcggtacctcatttacccggagagcg (SEQ ID NO: 14).

[0073] The forward primer sequence for amplification of the antibody light chain encoding region is:

[0074] caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 13).

[0075] The reverse primer sequence for amplification of the antibody light chain encoding region is:

[0076] tagtggatccgagctcggtacctcaacagtcacccctattg (SEQ ID NO: 15).

[0077] Antibody light chain, heavy chain encoding gene retrieval: according to the instructions to add PCR amplification system, hole plate light mixing, placed in PCR instrument running program, program end hole plate sample on ice, static.

[0078] In this embodiment, the antibody light chain, heavy chain encoding gene in the same 96 well plate amplification product pairing positive rate is more than 80%, with agarose gel electrophoresis detection clear band, shows that single B cell flow cytometry and encoding gene amplification experiment are effective. Amplification product, for the construction of recombinant expression plasmid.

[0079] 6. Construction and expression of antibody heavy chain and light chain recombinant expression plasmid

[0080] The development of domain specific antibodies is used to calculate the content of autoantibodies corresponding to the domain in human clinical samples, so the antibody modification scheme is determined as follows: the rabbit Fab segment remains unchanged, and the Fc segment is modified to human IgG4-Fc.

[0081] Recombinant expression vector, select pcDNA3.1 (Invitrogen), purchased on ThermoFisher SCIENTIFIC website. Before recombinant construction, select Hind III restriction enzyme for single enzyme digestion to realize linearization of expression vector, and the restriction enzyme is purchased on New England Biolabs website.

[0082] High efficiency recombination of vector and encoding gene, select seamless cloning kit, C115# kit is purchased on Vazyme website.

[0083] Construction of recombinant expression plasmid: the amplification product of antibody heavy chain and light chain encoding is connected with pcDNA3.1 linearized vector by seamless cloning technology, and then transformed into E. coli DH5α competent cells, coated with LB fixed medium plate, and the plate was inverted and cultured at 37℃ overnight.

[0084] Recombinant positive clone selection: the heavy chain and light chain of the primary screening antibody were picked up respectively, and the positive rate of the colonies was determined after PCR bacteria detection. If the bacteria detection positive rate is low, single colony can be picked up for further bacteria detection.

[0085] Bacterial detection PCR of recombinant plasmid: the upstream primer sequence located in the vector is: caagctggctagcgtttaaactt (SEQ ID NO: 16).

[0086] The downstream primer sequence of antibody heavy chain bacteria detection PCR is: ctcatttacccggagagcg (SEQ ID NO: 17).

[0087] The sequence of the downstream primer of the antibody light chain PCR is acctcaacagtcacccctattg (SEQ ID NO: 18).

[0088] Recombinant positive clone sequencing: 5 clones of each of the antibody heavy chain and light chain were selected for sequencing in Shanghai Shengong Biotechnology Co., Ltd.

[0089] Rabbit antibody gene sequence analysis: The V region gene of the antibody sequence was determined using the IMGT database, and the CDR1 / CDR2 / CDR3 regions of the antibody heavy chain and light chain were analyzed. The correct sequence number of the positive clone of the PCR was determined.

[0090] Small-scale expression of recombinant expression plasmid: The antibody light chain and heavy chain plasmids were obtained by small-scale expression of the correct sequence clone in the bacterial solution. The mixed plasmids were co-transfected into HEK293 mammalian cells. After 10 days of cell transfection, the cell supernatant was collected by centrifugation. The supernatant was subjected to antigen-specificity evaluation, and the cell supernatant was purified after ELISA primary screening results.

[0091] In this embodiment, 100 plasmids were transfected in each round, i.e., 100 monoclonal antibodies were obtained in each round of transfection. A total of 3 rounds of transfection experiments were performed.

[0092] 7. Recombinant expression supernatant antigen specificity evaluation

[0093] The original selection was human PLA2R1 domain protein and extracellular domain protein, with His tag, named hPLA2R1-CTLD7-His, hPLA2R1-CysR-His, hPLA2R1-CTLD1-His, hPLA2R1-CTLD8-His, and hPLA2R1-total-His, respectively.

[0094] Antibody screening scheme for human PLA2R1 specific domain protein: The antibody to be tested specifically binds to a single domain while also binding to the extracellular domain protein, i.e., it is preliminarily determined as a specific antibody for the domain protein.

[0095] Sandwich ELISA was used to detect the cell supernatant, and the self-produced human secondary antibody Mouse Anti-Human IgG4-Fc mAb (MAHG4 Fc-Ab) was used to coat the plate, and the goat anti-rabbit secondary antibody coupled with HRP was purchased to detect. The addition of cell supernatant with a detection OD > 1.5 indicates normal expression of the recombinant plasmid.

[0096] Indirect ELISA was used to detect the cell supernatant, and the reactivity of the 5 antigens coated on the plate was evaluated, and the primary screening results of the supernatant of the well plate were obtained (only the detection data of 50 cell supernatants are shown, and Table 1 shows the detection data of the CTLD7 domain antibody).

[0097] Table 1: Affinity data table of CTLD7 domain antigen-antibody (partial)

[0098]

[0099] CTLD7 domain indirect ELISA detection results: a total of 300 monoclonal antibodies were screened, which reacted with hPLA2R1-CTLD7-His and total-His (OD>1.0) and did not react with CysR-His, CTLD1-His and CTLD8-His (OD<0.2), a total of 220 positive antibodies were screened, and 50 specific monoclonal antibodies were selected.

[0100] The cell supernatant determined by preliminary screening was purified by protein A to obtain a small amount of monoclonal antibody, with an average of 1-3 mg per strain.

[0101] Example 2: Further evaluation and screening of domain-specific antibodies on the ELISA platform

[0102] Further evaluation and screening scheme of the selected domain antibodies:

[0103] The concentration of the fixed domain protein was gradiently diluted, and the purified antibodies were screened by indirect ELISA method to evaluate and screen the specific antibodies of each domain of human PLA2R7 with strong antigen binding reactivity, wide detection linear range and high accuracy of calculation.

[0104] The operation of the indirect ELISA platform is as follows:

[0105] Coating: Take the corresponding domain protein, dilute the coating solution to 2 ug / ml, add 100 ul of coating solution to each well, and incubate at 37°C for 1 h, then incubate at 4°C overnight.

[0106] Blocking: The next day, take out the enzyme-labeled plate and wash it with PBST for 3 times. Add 200 ul of blocking solution (1% BSA) to each well, incubate at 37°C for 2 h, and wash with PBST for 3 times.

[0107] Add primary antibody-domain antibody: Dilute the corresponding primary antibody by 2 times gradient, add 100 ul of dilution liquid to each well. Set up negative control and blank control, incubate at 37°C for 1 h, and wash with PBST for 3 times.

[0108] Add self-produced anti-human IgG4-Fc secondary antibody: Dilute the secondary antibody by 1:5000, add 100 ul to each well, incubate at 37°C for 1 h, and wash with PBST for 3 times.

[0109] Add substrate: Add 100 ul of substrate reaction solution (freshly prepared, avoid light) to each well, incubate at 37°C for 30 min, then add stop solution. Add substrate for color development, and measure A450 value on the enzyme-labeled instrument.

[0110] CTLD7 domain protein screening out 50 specific antibodies, using hPLA2R1-CTLD7-His and hPLA2R1-CTLD7-rFc protein coated enzyme labeled plate, indirect ELISA experiment detection and screening out 1 better antibody, selected named Anti-hPLA2R CTLD7-rRmab (h4Fc). The experimental results show that hPLA2R1-CTLD7-His protein plate, the detection range of selected antibody is 15.62-2000.00 ng / ml, see Figure 2 , which can be used for the detection of clinical samples.

[0111] CTLD7 domain antibody (Anti-hPLA2R CTLD7-rRmab (h4Fc)), the heavy chain variable region sequence is:

[0112] QSLEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAPGEGLEWIGTVGVSGSIWYASWVRGRFTISRTSTTVDLRITSPTSEDTTTYFCVRFYSNFWGPGTLVTVSS (SEQ ID NO: 6);

[0113] CDR1: GFSLSNYA (SEQ ID NO: 1); CDR2: VGVSGSI (SEQ ID NO: 2); CDR3: VRFYSNF (SEQ ID NO: 3).

[0114] The light chain variable region sequence is:

[0115] AQVLTQTPSSVSAAVGGTVTINCQSTQNVYAKNRLAWYQQKPGQPPKRLIDSASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYFCLGSYDCNSASCHAFGGGTEVVVN (SEQ ID NO: 7);

[0116] CDR1: QNVYAKNR (SEQ ID NO: 4); CDR2: SAS; CDR3: LGSYDCNSASCHA (SEQ ID NO: 5)

[0117] Although the embodiments of the present application have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the CTLD7 domain of PLA2R, characterized in that, The amino acid sequence of the heavy chain CDRH1 of the antibody or antigen binding fragment thereof is shown as SEQ ID NO: 1, the amino acid sequence of the heavy chain CDRH2 is shown as SEQ ID NO: 2, the amino acid sequence of the heavy chain CDRH3 is shown as SEQ ID NO: 3, the amino acid sequence of the light chain CDRL1 is shown as SEQ ID NO: 4, the amino acid sequence of the light chain CDRL2 is SAS, and the amino acid sequence of the light chain CDRL3 is shown as SEQ ID NO:

5.

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

7.

3. A polynucleotide, comprising, The polynucleotide encodes the antibody or antigen binding fragment thereof of claim 1.

4. A vector, characterized by, The vector comprises the polynucleotide of claim 3.

5. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 3 or the vector of claim 4.

6. A method of producing an antibody or antigen binding fragment thereof, characterized in that, The method comprises: (a) culturing the host cell of claim 5 under conditions suitable for expression of the antibody or antigen binding fragment thereof, and (b) optionally, recovering the antibody or antigen binding fragment thereof.

7. A kit for detecting PLA2R autoantibodies, characterized in that The kit comprises the antibody or antigen binding fragment thereof of any one of claims 1-2.

8. The kit of claim 7, wherein The kit further comprises a coated plate, diluent, anti-human IgG antibody, washing solution and termination solution.

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

10. Use of the kit of any one of claims 7-8 in the preparation of a membranous nephropathy detection or diagnosis product.

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