CTLD8 structural domain specific antibody of PLA2R and application thereof

Abstract

The invention discloses a CTLD8 structural domain specific antibody aiming at PLA2R and application of the CTLD8 structural domain specific antibody, and belongs to the technical field of biological engineering. The invention provides a CTLD8 structural domain antibody of PLA2R or an antigen binding fragment of the CTLD8 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 CTLD8 structural domain antibody of PLA2R as a standard substance, so that convenience is provided for clinical detection of concentrations of IgG resisting different epitopes of PLA2R.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, specifically to a specific antibody against the CTLD8 domain of human PLA2R, a detection kit containing the antibody, and its application. Background Technology

[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 thickening of the GBM. Based on its pathogenesis and etiology, it can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy.

[0003] The M-type phospholipase A2 receptor (PLA2R) is a major 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 individual domains (a single N-terminal cysteine-rich domain (CysR), a single type II fibronectin domain (FNII), and 8 to 10 type C lectin domains (CTLD)). These individual domains can directly bind to circulating autoantibodies.

[0004] Studies have identified an association between PLA2R reactive epitopes and disease activity, proposing the epitope diffusion hypothesis. Epitope diffusion refers to the immune response to endogenous epitopes following the release of autoantigens during chronic autoimmune or inflammatory responses, typically spreading from the dominant immunodominant epitope to other sites within the molecule. In the IMN, CysR is the immunodominant epitope of PLA2R, and the diffusion of antigenic determinants begins in the CysR domain, then gradually spreads to CTLD1, CTLD7, or CTLD8. Furthermore, some studies suggest that analyzing PLA2R epitope characteristics and diffusion provides a powerful tool for monitoring disease severity and stratifying patients based on 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 currently there is no quantitative detection method for antibodies targeting the PLA2R domain. Developing a quantitative detection method for antibodies targeting the PLA2R CTLD8 domain and for CTLD8 domain-specific IgG antibodies is crucial. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this application provides a specific antibody targeting the PLA2R domain, specifically a CTLD8 domain antibody, Anti-hPLA2R CTLD8-rRmab (h4Fc). The rabbit-derived monoclonal antibody prepared using single-B cell technology in this application exhibits higher sensitivity and a significantly shorter production cycle. A standard curve established using this antibody can effectively calculate the content of autoantibodies against the CTLD8 domain of PLA2R in clinical samples, providing a tool for the auxiliary diagnosis of PLA2R-related diseases. Furthermore, this application provides a kit containing this antibody for the auxiliary diagnosis of membranous nephropathy.

[0007] On the one hand, this application provides an antibody or antigen-binding fragment that specifically binds to the CTLD8 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 with the amino acid sequence shown in SEQ ID NO: 1; a heavy chain variable region CDR-H2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 3 ...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 with the amino acid sequence shown in SEQ ID NO: 1; and a heavy chain variable region CDR-H3 having at least 90%, 91%, 92%, 93%, 94%, 95 The light chain variable region CDR-L1, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in NO:4, and the light chain variable region CDR-L2, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region CDR-L3, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

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

[0010] 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 with the amino acid sequence shown in SEQ ID NO: 6; and (b) a light chain variable region VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the antibody comprises the heavy chain variable region VH as shown in SEQ ID NO: 6 and the light chain variable region VL as shown in SEQ ID NO: 7.

[0011] In one embodiment, the antibody or its antigen-binding fragment of the present invention comprises an α heavy chain, a δ heavy chain, an ε heavy chain, a γ heavy chain, or a μ heavy chain. In one embodiment, the antibody or its antigen-binding fragment of the present invention belongs to a subclass of IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody or its antigen-binding fragment of the present invention comprises a λ light chain or a κ light chain. In some embodiments, the “class” of the antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, wherein the constant domains of the heavy chains corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. In some embodiments, the antibody is an IgG4 isotype.

[0012] On the one hand, this application provides a polynucleotide encoding the aforementioned antibody or antigen-binding fragment. The nucleic acid molecule described in this application can effectively express the aforementioned antibody or its antigen-binding fragment.

[0013] On the one hand, this application provides a vector containing the polynucleotide of this application. The expression vector described in this application can effectively express the aforementioned antibody or its antigen-binding fragment.

[0014] In some embodiments, the vector includes a viral vector, an expression vector, or a recombinant expression vector. In some embodiments, the expression vector can be any suitable recombinant expression vector selected from the pUC series (Fermentas Life Sciences, Glen Burnie, 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.

[0015] On the one hand, this application provides a host cell containing the polynucleotide or vector of this application.

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

[0017] On one hand, the present invention provides a method for generating an antibody or an antigen-binding fragment thereof, comprising: (a) The host cells of the present invention are cultured under conditions suitable for expressing the antibody or its antigen-binding fragment, and (b) Optionally, the antibody or its antigen-binding fragment may be recovered.

[0018] On the one hand, this application provides the use of the antibody or antigen-binding fragment, the polynucleotide, the expression vector, or the host cell described above in the preparation of PLA2R detection products.

[0019] On the one hand, this application provides a kit for detecting autoantibodies of the PLA2R domain.

[0020] In some embodiments, the kit comprises an antibody or antigen-binding fragment as described above. In some embodiments, the kit is used for non-diagnostic immunoassay to detect autoantibodies in a specific domain of PLA2R. In some embodiments, the domain is the CTLD8 domain. In some embodiments, the kit is a chemiluminescence, electrochemiluminescence, or ELISA assay. In some embodiments, the kit is an indirect ELISA assay kit. In some embodiments, the kit further includes a coating plate, diluent, anti-human IgG antibody, washing buffer, and stop solution. In some embodiments, the kit is used to establish a standard curve, facilitating the clinical detection of IgG antibodies against specific epitopes of PLA2R.

[0021] On the one hand, this application provides the use of the kit described above in the preparation of products for the detection or diagnosis of membranous nephropathy.

[0022] Beneficial effects of this application This application provides a specific antibody or antigen-binding fragment thereof targeting the PLA2R domain, specifically an antibody or antigen-binding fragment thereof targeting the CTLD8 domain. The CTLD8 domain antibody or antigen-binding fragment provided in this application can specifically recognize the corresponding PLA2R CTLD8 domain and exhibits high affinity. Furthermore, this application provides a method for establishing a standard curve using domain antibodies, facilitating the clinical detection of the concentration of anti-PLA2R IgG with different epitope determinants. Attached Figure Description

[0023] Figure 1 Cell sorting diagram for developing antibodies targeting the CTLD8 domain; Figure 2 The standard curve for the CTLD8 domain antibody is shown. Detailed Implementation

[0024] The present disclosure will be described in detail below with reference to the embodiments. However, the implementation of the present disclosure is 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 can be obtained without creative effort and all fall within the protection scope of the present disclosure.

[0025] 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 pertains. All patents, patent applications, and publications referenced throughout this disclosure are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, those definitions in this section shall prevail.

[0026] The technical solutions provided in this disclosure will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this disclosure.

[0027] Example 1: Preparation of anti-human PLA2R1 domain-specific monoclonal antibody 1. Antigen preparation This embodiment describes the development of a specific monoclonal antibody targeting the CTLD8 domain of the human phospholipase A2 receptor (PLA2R) for the establishment of a standard curve to calculate the content of autoimmune antibodies in the corresponding specific domain of the clinical sample to be tested.

[0028] The CTLD-8 domain has the following sequence: WIKFKSNCYSFSTVLDSMSFEAAHEFCKKEGSNLLTIKDEAENAFLLEELFAFGSSVQMVWLNAQFDGNNETIKWFDGTPTDQSNWGIRKPDTDYFKPHHCVALRIPEGLWQLSPCQEKKGFICK (SEQ ID NO: 8); The CysR domain has the following sequence: KGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGG (SEQ ID NO: 9); The CTLD-1 domain has the following sequence: CYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKK (SEQ ID NO: 10); Domain CTLD-7, sequence: PNTLEYGNRTYKIINANMTWYAAIKTCLMHKAQLVSITDQYHQSFLTVVLNRLGYAHWIGLFTTDNGLNFDWSDGTKSSFTFWKDEESSLLGDCVFADSNGRWHSTACESFLQGAIC(SEQ ID NO: 11) The sequence of the human PLA2R1 extracellular domain protein is as follows:

[0029] The expression of the human PLA2R domain protein was performed in HEK293F mammalian cells.

[0030] When designing the expression of domain proteins, a signal peptide is added to the N-terminus of the sequence for secretory protein expression, and a rabbit IgG-Fc (rFc) tag sequence and a 6×His (His) tag sequence are added to the C-terminus of the sequence, respectively.

[0031] 2. Preparation of animal immune libraries After successful preparation of the human PLA2R1 domain protein, five New Zealand rabbits were immunized with the rabbit IgG-Fc-tagged domain protein (hPLA2R1-CTLD8-rFc). For the first immunization, 500 μg of immunogen was mixed with an equal volume of Freund's adjuvant to prepare an emulsion, which was then injected subcutaneously at multiple sites. Two weeks later, a second immunization was performed, with 250 μg of immunogen mixed with an equal volume of Freund's adjuvant to prepare an emulsion, which was also injected subcutaneously at multiple sites. Serum titers were measured after the third immunization.

[0032] A small blood sample was collected from the marginal ear vein and, after clotting, centrifuged at 8000g to prepare serum. A 6×His-tagged domain protein (hPLA2R1-CTLD8-His) was added to the plate, and serum titer was determined using an indirect ELISA method. Rabbits with high serum titers were selected, and a booster immunization was performed by subcutaneous injection of 250ug immunogen at multiple sites. The spleens of these rabbits were then harvested.

[0033] Rabbit spleens were physically ground and filtered through a porous mesh to prepare a single-cell suspension.

[0034] 3. Antigen-specific single B cell sorting This embodiment is based on the specific recognition of surface markers of lymphocyte B cells by flow cytometry antibodies, and uses flow cytometry cell sorting to obtain specific single B cells from single-cell suspensions.

[0035] The raw material was sorted, and hPLA2R1-CTLD8-His was selected and coupled with FITC dye.

[0036] The anti-rabbit IgG-Fc specific secondary antibody is a self-developed antibody conjugated with PE dye.

[0037] During cell labeling, DAPI dye is added to distinguish between dead and live cells.

[0038] B-cell sorting scheme: Dead / Live- / IgG+ / Antigen+.

[0039] Cell labeling procedure: Rabbit lymphocyte suspension, centrifuged at 300g for 5 min, added 5 ml of buffer, mixed by inverting, centrifuged at 300g for 5 min. Discard the supernatant, repeat once, take 30 μL of cell suspension for cell counting, and take 40 μL of cell suspension for blank control tubes and single-staining tubes to be labeled. The remaining cell suspension is used as sample tubes, centrifuged at 300g for 5 min, and resuspended with a small amount of PBS. Blank tubes are left untreated. For single-staining tubes, add PBS to 100 μL, and add 2 μL of PE, 2 μL of FITC, and 2 μL of DAPI dye respectively. Sample tubes are treated with PE dye at a ratio of 1.5 μL / 10 μL. 6 Cells, FITC dye 2ug / 10 6 Calculate the actual amount of cells added, add the corresponding amount of antibody in the dark, and incubate at 4°C for 30 minutes. After antibody incubation, add 2 ml of buffer, mix gently, centrifuge at 300 g for 5 minutes, and repeat the washing three times. Resuspend the cells in 1 ml of buffer, filter the cells, and wait for sorting.

[0040] After completing fluorescence compensation adjustment, the viable cell population, PE and FITC double-positive signal cell populations were sequentially delineated (sorting diagram as shown in the figure). Figure 1 The flow cytometer was programmed to sort antigen-specific B cells into 96-well plates, with only one cell per well. After sorting, the plates needed to be immediately stored at low temperature; dry ice boxes were provided in this example for short-term storage. The wells contained cell lysis buffer, and the sorted 96-well PCR plates were directly used for single-B-cell PCR experiments.

[0041] 4. Preparation of cDNA from rabbit single B cells The preparation of single-cell cDNA libraries was based on SMART 5'RACE technology, and all reagents used were the Vazyme N711 kit from Nanjing Novizan Biotechnology Co., Ltd., which is commercially available. The amplification systems involved in the experiments described in the examples can be found in the N711 kit instructions.

[0042] Single B cell RNA reverse transcription: After sorting, thaw the 96-well plates and place them in a PCR instrument to run the program. After the program is completed, let them stand on ice for 2 minutes.

[0043] Single-stranded cDNA synthesis in B cells: After the reverse transcription reaction is complete, the single-stranded synthesis system can be added. After adding the system, gently mix the wells and place them in a PCR instrument to run the program. After the program is complete, incubate the samples on ice for 2 minutes.

[0044] Single-cell B-cell DNA double-strand synthesis: After the synthesis reaction of the cDNA single-strand product is completed, the double-strand synthesis system can be added. After adding the system, gently mix the well plate, centrifuge, and then place it in a PCR instrument to run the program. After the program is completed, incubate the well plate samples on ice.

[0045] 5. Amplification of antibody-encoding genes using rabbit single-cell B-cell PCR technology A single B-cell cDNA library can be used to retrieve genes encoding naturally paired antibody heavy and light chains.

[0046] All reagents used for gene amplification were the Vazyme P515 kit from Nanjing Novizan Biotechnology Co., Ltd., which are commercially available. The amplification systems used in the experiments described in this example can be found in the P515 kit instructions.

[0047] The upstream primer contains a homologous arm that interlocks with the 3' end of the promoter CMV gene sequence, so the antibody-encoding gene can be directly used to construct the recombinant expression cassette after retrieval.

[0048] The downstream primer for the antibody heavy chain encoding gene is located in a constant region and contains a homologous arm that interacts with the BGH-polyA gene sequence.

[0049] The downstream primer for the light chain encoding gene is located in a constant region and contains a homologous arm complementary to the BGH-polyA gene sequence. Therefore, after the antibody encoding gene is retrieved, it can be directly used to construct a recombinant expression cassette.

[0050] Amplification of the antibody heavy chain coding region gene; forward primer sequence is: caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 13).

[0051] Antibody heavy chain coding region gene amplification, reverse primer sequence is: tagtggatccgagctcggtacctcatttacccggagagcg (SEQ ID NO: 14).

[0052] The forward primer sequence for amplifying the antibody light chain coding region gene is as follows: caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc (SEQ ID NO: 13).

[0053] Antibody light chain coding region gene amplification, reverse primer sequence is: tagtggatccgagctcggtacctcaacagtcacccctattg (SEQ ID NO: 15).

[0054] Extraction of antibody light and heavy chain encoding genes: Add to the PCR amplification system according to the instructions, mix gently in the well plate, place in the PCR instrument and run the program. After the program is completed, place the well plate samples on ice.

[0055] In this embodiment, the pairing positivity rate of the amplification products encoding the antibody light and heavy chains in the same 96-well plate was over 80%, and the bands were clear as detected by agarose gel electrophoresis, indicating that both the single-cell flow cytometry sorting and the encoding gene amplification experiment were effective. The amplification products were used for the construction of recombinant expression plasmids.

[0056] 6. Construction and expression of antibody heavy and light chain recombinant expression plasmids The development of domain-specific antibodies is intended to calculate the content of autoantibodies corresponding to the domain in human clinical samples. Therefore, the antibody modification scheme was determined to keep the rabbit Fab segment unchanged and modify the Fc segment to human IgG4-Fc.

[0057] The recombinant expression vector, pcDNA3.1 (Invitrogen), was purchased from the ThermoFisher SCIENTIFIC website. Before recombinant construction, the expression vector was linearized by single digestion with HindIII restriction enzyme, which was purchased from the New England Biolabs website.

[0058] For efficient recombination of vectors and coding genes, choose the seamless cloning kit, and purchase the C115# kit from the Vazyme website.

[0059] Construction of recombinant expression plasmid: The amplified products encoding the antibody heavy and light chains were circularized with the pcDNA3.1 linearized vector using seamless cloning technology, and then transformed into E. coli DH5α competent cells. The plasmids were then plated on LB fixation medium plates and incubated overnight at 37°C with the plates inverted.

[0060] Selection of recombinant positive clones: For the initial screening of heavy and light chains of antibodies, 8 single colonies are picked from each chain, and the colony positivity rate is determined by PCR testing. If the positivity rate is low, single colonies can be picked for further testing.

[0061] The bacterial detection PCR of the recombinant plasmid uses the upstream primer sequence of caagctggctagcgtttaaactt (SEQ ID NO: 16).

[0062] The downstream primer sequence for antibody heavy chain bacterial detection PCR is: ctcatttacccggagagcg (SEQ ID NO: 17).

[0063] The downstream primer sequence for antibody light chain bacterial detection PCR is: acctcaacagtcacccctattg (SEQ ID NO: 18).

[0064] Recombinant positive clones were sent for testing: five clones of the heavy chain and five clones of the antibody light chain were selected from the PCR positive clones and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0065] Rabbit antibody gene sequence analysis: The V region of the antibody sequence was determined using the IMGT database, and the antibody sequence was analyzed for the CDR1 / CDR2 / CDR3 regions of the heavy chain and light chain. The correct sequence number of the PCR-positive clones was then derived and determined.

[0066] Small-scale expression of recombinant expression plasmids: Cloning of the correct sequence and small-scale extraction of antibody light and heavy chain plasmids from bacterial culture. The plasmids were then co-transfected into HEK293 mammalian cells. Ten days after transfection, the cell supernatant was collected by centrifugation. The supernatant was used for antigen specificity assessment, and cell supernatant purification was performed after the initial ELISA screening results were available.

[0067] In this embodiment, 100 plasmids were transfected in each round, meaning that 100 monoclonal antibodies could be obtained in each round of transfection. A total of 3 rounds of transfection experiments were performed.

[0068] 7. Evaluation of antigen specificity in recombinant expression supernatant The original selected human PLA2R1 domain proteins and extracellular domain proteins were screened and tagged with His, and named as hPLA2R1-CysR-His, hPLA2R1-CTLD1-His, hPLA2R1-CTLD7-His, hPLA2R1-CTLD8-His, and hPLA2R1-total-His, respectively.

[0069] Antibody screening scheme for specific domain proteins of human PLA2R1: The antibody to be tested can bind to a single domain while also binding to extracellular domain proteins, i.e., it is initially identified as a specific antibody for the domain protein.

[0070] The cell supernatant was detected by sandwich ELISA. The cell supernatant was coated with our own human secondary antibody, Mouse Anti-Human IgG4-Fc mAb (MAHG4 Fc-Ab), and HRP was conjugated with a purchased goat anti-rabbit secondary antibody. The addition of cell supernatant and an OD > 1.5 indicated normal expression of the recombinant plasmid.

[0071] Indirect ELISA was used to detect cell supernatants. The reactivity of the plates coated with five antigens was evaluated to obtain the initial screening results of the supernatants of the well plates (only the detection data of the supernatants of 50 cell lines are shown as an example, and Table 1 shows the detection data of CTLD8 domain antibody).

[0072] Table 1: Partial Affinity Data for CTLD8 Domain Antigen-Antibody

[0073] Indirect ELISA detection results of the CTLD8 domain: 300 monoclonal antibodies were initially screened and co-transfected. They reacted simultaneously with hPLA2R1-CTLD8-His and total-His (OD>1.0) and did not react with CysR-His, CTLD1-His, and CTLD7-His (OD<0.2), resulting in 200 positive antibodies in the initial screening. Among them, 50 specific monoclonal antibodies were selected.

[0074] The cell supernatant identified in the initial screening was purified by protein A to obtain a small amount of monoclonal antibody, with an average of 1-3 mg per strain.

[0075] Example 2: Further evaluation and screening of domain-specific antibodies on the ELISA platform Further evaluation and screening protocols for domain antibodies selected from the initial screening: By fixing the concentration of domain proteins, serially diluting the initially screened purified antibodies, and using the indirect ELISA method, specific antibodies for each domain of human PLA2R1 with strong antigen-binding reactivity, a wide detection linear range, and high backtesting accuracy were evaluated and screened.

[0076] The operation of the indirect ELISA platform is as follows: Coating: Take the corresponding domain protein, dilute the coating buffer to 2ug / ml, add 100ul of coating buffer to each well, incubate at 37℃ for 1h and then at 4℃ overnight.

[0077] Blocking: The next day, remove the microplate and wash three times with PBST. Add 200 μL of blocking buffer (1% BSA) to each well, incubate at 37°C for 2 hours, and wash three times with PBST.

[0078] Add primary antibody-domain antibody: Dilute the corresponding primary antibody serially by 2×, adding 100 μL of dilution buffer to each well. Set up negative control and blank control, incubate at 37°C for 1 h, and wash 3 times with PBST.

[0079] Add self-produced anti-human IgG4-Fc secondary antibody: dilute the secondary antibody 1:5000, 100ul per well, incubate at 37℃ for 1h, and wash 3 times with PBST.

[0080] Add substrate: Add 100 μL of substrate reaction solution (freshly prepared and protected from light) to each well, incubate at 37°C for 30 min, then remove and add stop solution. After substrate addition and color development, measure the A450 value using a microplate reader.

[0081] Fifty specific antibodies were initially screened from the CTLD8 domain protein. HPLA2R1-CTLD8-His and HPLA2R1-CTLD8-rFc proteins were then coated onto ELISA plates. One superior antibody was selected and named Anti-hPLA2R CTLD8-rRmab (h4Fc) after indirect ELISA detection. Experimental results showed that the detection range of the selected antibody was 48.82-6250.00 ng / ml after coating with hPLA2R1-CTLD8-His protein. Figure 2 It can be used for the testing of clinical samples.

[0082] The CTLD8 domain antibody (Anti-hPLA2R CTLD8-rRmab (h4Fc)) has the following heavy chain variable region sequence: QSVEESGGRLVTPGTPLTLTCTVSGIDVSRNAMSWVRQAPGEGLEWIGTIGRSGSTYYATWAKGRFTVSKTSSTTVDLKMTSPTTVDTATYFCGRDDFNIWGPGTLVTVSS(SEQ ID NO: 6); CDR1: GIDVSRNA (SEQ ID NO: 1); CDR2: IGRSGST (SEQ ID NO: 2); CDR3: GRDDFNI (SEQ ID NO: 3).

[0083] The light chain variable region sequence is as follows: AQVLTQTASSVSAAVGGTVTISCQSSQSVWDNKWLVWFQQKPGQPPKQLIYAASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGGYIGTTYTFGGGTEVVVK (SEQ ID NO: 7); CDR1:QSVWDNKW(SEQ ID NO:4); CDR2:AAS; CDR3:LGGYIGTTYT(SEQ ID NO:5) Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the CTLD8 domain of PLA2R, characterized in that, The amino acid sequence of the heavy chain CDRH1 of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain CDRH2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain CDRH3 is shown in SEQ ID NO: 3, the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO: 4, the amino acid sequence of the light chain CDRL2 is AAS, and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO:

5.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment 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.

3. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or its antigen-binding fragment as described in claim 1.

4. A carrier, characterized in that, 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 for generating an antibody or an antigen-binding fragment thereof, characterized in that, The method includes: (a) Culture the host cells of claim 5 under conditions suitable for expressing the antibody or its antigen-binding fragment, and (b) Optionally, the antibody or its antigen-binding fragment may be recovered.

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

8. The reagent kit according to claim 7, characterized in that, The kit also includes a coating plate, diluent, anti-human IgG antibody, washing solution, and stop solution.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2 in the preparation of PLA2R detection products.

10. The use of the kit according to any one of claims 7-8 in the preparation of products for the detection or diagnosis of membranous nephropathy.

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