A high-affinity monoclonal antibody against BP230 and its application

Abstract

The present invention provides a high-affinity monoclonal antibody against BP230 and its application. The method uses BP230 antigen protein to immunize experimental rabbits multiple times with BP230 protein, followed by serum titer testing. Splenic tissue and PBMCs from the rabbits with high titers are then isolated and cultured for single B cell sorting. RNA is extracted from positive clones and reverse-transcribed into cDNA, whereupon all antibody VH and VL gene segments are amplified. The in vitro amplified VH and VL gene segments are cloned into an expression vector containing human IgG constant regions for recombinant expression. The recombinant antibodies are functionally verified by ELISA, and humanized monoclonal antibodies with good specificity and strong affinity are screened.

Description

[0001] This application is a divisional application of patent application No. 2024105515440 (the application date of the original application is 2024 / 5 / 7, and the name of the invention is a high-affinity monoclonal antibody against BP230 and its application). Technical Field

[0002] The present invention belongs to the technical field of biomedicine, and particularly relates to an anti-BP230 high-affinity monoclonal antibody and applications thereof. Background Art

[0003] Bullous pemphigoid (BP) is an autoimmune disease that develops when the immune system attacks the skin, causing blisters. It most commonly affects people aged 60 to 80 years. Characteristic clinical manifestations are tense blisters and severe, widespread pruritus. Histopathologically, hematoxylin and eosin (HE) staining reveals subepidermal fissures with eosinophil infiltration. Direct immunofluorescence (DIF) reveals linear deposits of autoantibodies and / or complement along the basement membrane zone, while salt-split indirect immunofluorescence (ssIIF) reveals deposits of autoantibodies and / or complement on the epidermal side.

[0004] The target antigens of autoantibodies in patient sera are BP180 and BP230, also known as BPAG1 and BPAG2, with molecular weights of 180 kDa and 230 kDa, respectively. BP230 is a cytoplasmic hemidesmosome glycoprotein with three main isoforms: the skin isoform (BPAG1-e), the neural isoforms (BPAG1-n and BPAG1-a), and the muscle isoform (BPAG1-b). These isoforms share certain structural homology, each with tissue specificity and distinct epitopes. Most sera with BP show strong positive reactions with different regions of BP230, particularly the C-terminus. The N-terminus of BP230 is crucial for its binding to hemidesmosomes and its interaction with BP180 and the β4 subunit of the α6β4 integrin. Anti-BP230 antibodies are considered secondary products that can exacerbate the inflammatory response. Therefore, measuring anti-BP230 antibody levels in BP patients is crucial for assisting clinical diagnosis.

[0005] Monoclonal antibodies are widely used in therapy, diagnosis, and biology due to their high specificity and affinity. Currently, most monoclonal antibodies against human antigens are produced in mice, but many human immunogens fail to stimulate antibody responses in mice. Compared to other animal models such as mice and rats, rabbits can respond to a wider range of antigens. The rabbit immune system produces antibody diversity and optimizes affinity through mechanisms that differ from those of mice and other rodents. Therefore, rabbits are excellent hosts for monoclonal antibody production. In addition, rabbits are larger in size and have larger spleens, which can produce more antibodies. The preparation of rabbit monoclonal antibodies is an important research task and can be used in a variety of applications, including immunofluorescence, immunohistochemistry, flow cytometry, protein immunoblotting, and ELISA. Currently, no anti-BP230 monoclonal antibodies have been prepared in the prior art.

[0006] BP is a common and intractable acquired autoimmune disease. Patients present with autoantibodies to hemidesmosomal proteins, including anti-BP180 and anti-BP230, in their sera. Diagnostic criteria for BP include clinical manifestations, histopathology, direct immunofluorescence, indirect immunofluorescence, and specific antibody testing. Treatment varies according to the severity of the disease, primarily relying on glucocorticoids, antibiotics, and immunosuppressants. Currently, glucocorticoids are recognized as first-line treatments for BP, with significant clinical efficacy. However, long-term use can cause numerous complications, some of which are so severe that they prevent their use in some patients. Classic hybridoma technology requires significant time and effort to obtain high-affinity antibodies, and subsequent humanization is required, making it difficult to obtain human monoclonal antibodies and making it impossible to achieve absolute quantification of antibody levels. Consequently, these treatments lack specificity, result in poor efficacy, and require prolonged treatment.

[0007] Furthermore, immunotherapy in cancer patients can also trigger BP. PD-1 / PD-L1 checkpoint inhibitors, used to treat various solid and hematologic malignancies, are a class of drugs with a high incidence of BP-inducing diseases in recent years. Most cases develop blisters or bullae within 6-8 months of initiating PD-1 / PD-L1 inhibitor treatment, with a minority experiencing mucosal involvement. With the widespread adoption of PD-1 immunotherapy, the demand for BP testing will increase. However, there is currently a lack of effective diagnostic methods for the rapid and accurate diagnosis of these conditions. Therefore, developing a method for detecting these conditions is of great value. Summary of the Invention

[0008] In response to the shortcomings of the prior art, the present invention aims to provide a high-affinity monoclonal antibody against BP230 and its use. This high-affinity monoclonal antibody exhibits high activity, good stability, and strong specificity, and can serve as a reference standard for qualitative detection of BP230 positivity and quantitative detection of anti-BP230 autoantibody levels in BP patients.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a high-affinity monoclonal antibody against BP230, wherein the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is shown in SEQ ID NO.10, and the amino acid sequence of CDR3 is shown in SEQ ID NO.11; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.12, the amino acid sequence of CDR2 is RAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.13.

[0011] The present invention immunizes New Zealand white rabbits, extracts PBMC, and performs single B cell sorting and culture. Sequence analysis of positive clones is followed by recombinant expression. The resulting high-affinity rabbit monoclonal antibody can quantitatively measure anti-BP230 autoantibody levels in BP patients, laying an experimental foundation for studying the pathogenic mechanism of BP and achieving the goal of diagnosing or treating autoimmune bullous diseases.

[0012] Specifically, the present invention performs multiple immunizations of experimental rabbits with BP230 protein, and then performs serum titer testing after immunization. The spleen tissue and peripheral blood PBMC of rabbits with high titer are separated, and single B cells are sorted and cultured. RNA is extracted from positive clones and reverse transcribed into cDNA, and all the antibody VH and VL gene segments therein are amplified. The in vitro amplified VH and VL gene segments are cloned into an expression vector containing a human IgG constant region and recombinantly expressed. The recombinant antibodies are functionally verified by the ELISA method, and finally humanized monoclonal antibodies with good specificity and strong affinity are screened therefrom.

[0013] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody comprises SEQ ID NO. 14, or an amino acid sequence that is at least 80%, 85%, 90% or 95% homologous to SEQ ID NO. 14 and has the same or similar functions.

[0014] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody comprises SEQ ID NO. 16, or an amino acid sequence that is at least 80%, 85%, 90% or 95% homologous to SEQ ID NO. 16 and has the same or similar functions.

[0015] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.14; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.16.

[0016] Preferably, the heavy chain amino acid sequence of the monoclonal antibody comprises SEQ ID NO. 15, or an amino acid sequence that is at least 80%, 85%, 90% or 95% homologous to SEQ ID NO. 15 and has the same or similar functions.

[0017] Preferably, the light chain amino acid sequence of the monoclonal antibody comprises SEQ ID NO. 17, or an amino acid sequence that is at least 80%, 85%, 90% or 95% homologous to SEQ ID NO. 17 and has the same or similar functions.

[0018] Preferably, the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.15; the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.17.

[0019] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-BP230 high-affinity monoclonal antibody according to the first aspect.

[0020] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0021] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains at least one copy of the expression vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into the genome of the host cell.

[0022] In a fifth aspect, the present invention provides a composition for detecting BP230 protein, wherein the composition comprises the anti-BP230 high-affinity monoclonal antibody according to the first aspect.

[0023] In a sixth aspect, the present invention provides a kit for detecting BP230 protein in a sample, the kit comprising the anti-BP230 high-affinity monoclonal antibody described in the first aspect and / or the composition for detecting BP230 protein described in the fifth aspect.

[0024] In a specific embodiment, the kit for detecting BP230 protein in a sample is an immunofluorescence detection kit, and the immunofluorescence detection kit comprises the anti-BP230 high-affinity monoclonal antibody described in the first aspect.

[0025] In a specific embodiment, the kit for detecting BP230 protein in a sample is an immunohistochemical detection kit, and the immunohistochemical detection kit comprises the anti-BP230 high-affinity monoclonal antibody described in the first aspect.

[0026] In a specific embodiment, the kit for detecting BP230 protein in a sample is a flow cytometry detection kit, and the flow cytometry detection kit comprises the anti-BP230 high-affinity monoclonal antibody described in the first aspect.

[0027] In a specific embodiment, the kit for detecting BP230 protein in a sample is a protein immunoblotting detection kit, and the protein immunoblotting detection kit comprises the anti-BP230 high-affinity monoclonal antibody described in the first aspect.

[0028] In a specific embodiment, the kit for detecting BP230 protein in a sample is an ELISA detection kit, and the ELISA detection kit comprises the anti-BP230 high-affinity monoclonal antibody described in the first aspect.

[0029] In a seventh aspect, the present invention provides the use of any one or a combination of at least two of the high-affinity anti-BP230 monoclonal antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, the composition for detecting BP230 protein described in the fifth aspect, or the kit for detecting BP230 protein in a sample described in the sixth aspect in the preparation of a product for diagnosing bullous pemphigoid.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The recombinant antibodies screened in this study were functionally validated using ELISA to verify their specific binding to the BP230 antigen. These recombinant antibodies exhibited high activity, stability, and specificity, making them suitable for use as reference standards for qualitative detection of BP230 positivity and for quantitative detection of anti-BP230 autoantibody levels in BP patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the SDS-PAGE electrophoresis diagram of the recombinant protein BP230.

[0033] Figure 2 It is the result of the specific binding effect between recombinant antibodies and antigens. DETAILED DESCRIPTION

[0034] The screening and preparation process of the anti-BP230 high-affinity monoclonal antibody of the present invention is as follows:

[0035] (1) The artificially synthesized BP230 gene was recombined into the expression vector plasmid pET28b to obtain the BP230-pET28b expression vector. The BP230-pET28b expression vector was transfected into BL21 (DE3) competent cells for culture and purified to obtain the BP230 antigen protein.

[0036] (2) Rabbits were immunized with BP230 antigen protein multiple times, and serum titers were tested after immunization. Spleen tissue and peripheral blood of immunized rabbits with high serum titers were sterilely separated and tested, and PBMC cells were isolated from both materials using lymphocyte separation fluid. 960 single B cell clones were isolated and cultured (10×96-well plates, after antigen enrichment) by B cell staining and flow cytometry, and positive clones that specifically recognized the antigen were screened by ELISA.

[0037] (3) Using SuperScript TM The IV Single Cell / Low-Input cDNA PreAmp Kit (Thermo, Cat. No. 11752048) reverse transcribes RNA from single B cell positive clones into cDNA, amplifying all antibody VH and VL gene segments. The in vitro amplified VH and VL gene segments are cloned into an expression vector containing the human IgG constant region for recombinant expression.

[0038] (4) The expressed recombinant antibodies were functionally verified using the ELISA method to verify their specific binding ability to the BP230 antigen. Finally, humanized monoclonal antibodies with good specificity and strong affinity were screened.

[0039] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] Example 1 Method for constructing BP230 protein

[0042] The BP230 gene sequence was artificially synthesized and recombined into the expression vector plasmid pET28b to obtain the BP230-pET28b expression vector; the cloning site was NcoI / HindIII, and the amino acid sequence of BP230 was SEQ ID NO.1:

[0043] MGSSHHHHHHSSGLEVLFQGPGSKSTAKDCTFKPDFEMTVKECQHSGELSSRNTGHLHPTPRSPLLRWTQEPQPLEEKWQHRVVEQIPKEVQFQPPGAPLEKEKSQQCYSEYFSQTSTELQITFDETNPITRLSEIEKIRDQALNNSRPPVRYQDNACEMELVKVLTPLEIAKNKQYDMHTEVTTLKQEKNPVPSAEEWMLEGCRASGGLKKGDFLKKGLEPETFQNFDGDHACSVRDDEFKFQGLRHTVTARQLVEAKLLDMRTIEQLRLGLKTVEEVQKTLNKFLTKATSIAGLYLESTKEKISFASAAERIIIDKMVALAFLEAQAATGFIIDPISGQTYSVEDAVLKGVVDPEFRIRLLEAEKAAVGYSYSSKTLSVFQAMENRMLDRQKGKHILEAQIASGGVIDPVRGIRVPPEIALQQGLLNNAILQFLHEPSSNTRVFPNPNNKQALYYSELLRMCVFDVESQCFLFPFGERNISNLNVKKTHRISVVDTKTGSELTVYEAFQRNLIEKSIYLELSGQQYQWKEAMFFESYGHSSHMLTDTKTGLHFNINEAIEQGTIDKALVKKYQEGLITLTELADSLLSRLVPKKDLHSPVAGYWLTASGERISVLKASRRNLVDRITALRCLEAQVSTGGIIDPLTGKKYRVAEALHRGLVDEGFAQQLRQCELVITGIGHPITNKMMSVVEAVNANIINKEMGIRCLEFQYLTGGLIEPQVHSRLSIEEALQVGIIDVLIATKLKDQKSYVRNIICPQTKRKLTYKEALEKADFDFHTGLKLLEVSEPLMTGISSLYYSS。

[0044] The gene sequence is SEQ ID NO.2: [[ID=&&]] [[ID=&&]]

[0045]

[0046] The BP230-pET28b expression vector was transfected into BL21 (DE3) competent cells and cultured. The precipitate was collected and subjected to His tag affinity chromatography to obtain the BP230 protein. The purified BP230 also needed to be subjected to SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis) to verify its purity. The SDS-PAGE electrophoresis of the purified BP230 is shown in the figure below. Figure 1 As shown, the protein size is 90.54 KDa and the purity is greater than 85%.

[0047] Example 2 Preparation of BP230 Rabbit Monoclonal Antibody

[0048] The main instruments used in this example are shown in Table 1, and the main reagents used in this example are shown in Table 2.

[0049] Table 1

[0050] Instrument name Model / Manufacturer High-speed refrigerated centrifuge Neofuge 15R Biological safety cabinets Heal Force Electric constant temperature water bath HHW21.600AⅡ Constant temperature incubator Heal force Automated cell counter Count Star flow cytometry Beckman Inverted phase contrast microscope Olympus IX71 Shaking incubator ZCZY-AS8

[0051] Table 2

[0052] Reagent name Item No. factory Lymphatic fluid 7111011 Dakota RPMI-1640 350-000-CL WISEENT Fetal bovine serum 10099-141 Gibco L-glutamine solution 200mM SH30034.01 Hyclone

[0053] 1. Animal immunization

[0054] New Zealand white rabbits (Hubei Provincial Experimental Animal Research Center) were used for immunization. For the first immunization, 250 μg of BP230 protein was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites on the rabbit's back. Fifteen days later, a second immunization was performed, with 250 μg of BP230 protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple sites on the rabbit's back. Ten days later, a third immunization was performed, with 250 μg of BP230 protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple sites on the rabbit's back. Five days later, a fourth immunization was performed, with 250 μg of BP230 protein emulsified with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple sites on the rabbit's back. Serum titers were determined by ELISA after four immunizations.

[0055] 2. B lymphocyte acquisition

[0056] Take one immunized rabbit with a high serum titer, aseptically dissect the spleen, and collect peripheral blood. Isolate PBMCs using Ficoll density gradient centrifugation. Wash the cells twice with sterile PBS, then count the suspended cells. PBMCs can be stored long-term in liquid nitrogen.

[0057] 3. B lymphocyte sorting and culture

[0058] 3.1 Resuspend PBMC in PBS and adjust the cell concentration to 1×10 7 cells / mL, add flow cytometry antibodies to stain B cells according to the volume of resuspended cells. P (Proteintech, Cat No. SA00003-2) and BSA-specific-APC were added at a ratio of 1:100, respectively. Be sure to set up blank controls and single-staining controls. Incubate at room temperature in the dark for 30 minutes; wash twice with PBS, and centrifuge at 300g, 4°C for 5 minutes.

[0059] 3.2 Resuspend cells in PBS and filter through a 100-mesh strainer before loading. 5 minutes before loading, add 2% PI to stain to remove dead cells. Use controls to adjust voltage and fluorescence compensation settings. Use FSC-A / SSC-A to select lymphocytes and FSC-W / FSC-A to remove adherent cells. Select IgM-negative, antigen-positive, and PI-negative B cells.

[0060] 3.3 By flow cytometry, one B cell was added to each well of a 96-well plate containing 100 μL of complete 1640 medium (containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, and 2 μg / mL human IL-2). The 96-well plate was centrifuged at 300 g for 5 minutes and cultured at 37°C, 5% CO2 for 7 days.

[0061] 4. Identification of B lymphocytes

[0062] ELISA was performed on the supernatant of cells cultured on the seventh day. The ELISA plate was coated with 100 μL (5 μg / mL) of BP230 antigen per well and incubated at 37°C for 2 hours.

[0063] Discard the plate and wash each well three times with 300 μL of 0.05% PBST. Add 300 μL of 3% BSA (dissolved in PBS) blocking buffer to each well and block at 37°C for 1.5 hours. Discard the plate and wash each well three times with 300 μL of 0.05% PBST. Add 100 μL of the two-fold diluted supernatant to be tested to each well and incubate at 37°C for 1 hour. Discard the plate and wash each well three times with 300 μL of 0.05% PBST. Add 100 μL of secondary antibody (Goat Anti-Rabbit IgG H&L Polyclonal antibody, HRP (PTB96431) brand: Antibodysystem) diluted in blocking buffer to each well and incubate at 37°C for 45 minutes. Discard the plate and wash each well three times with 300 μL of 0.05% PBST. Add 100 μL of TMB to each well and incubate at 37°C for 5-10 minutes. Then, add 50 μL of 2M HCl to each well to terminate the reaction. Read the values ​​at 450-630 nm using a microplate reader. The results are shown in Table 3.

[0064] Table 3

[0065] Clone number OD450 4-F6 3.73 6-D2 3.4 10-A5 3.09 9-B9 3.79 6-E9 2.92

[0066] 5. Amplification and sequencing analysis of variable regions of positive cloned antibodies

[0067] 5.1 The positive B cells were collected and RNA was extracted using conventional methods and then reverse transcribed into cDNA. PCR amplification was performed using amplification primers including:

[0068] Antibody heavy chain gene primers:

[0069] Upstream primer (SEQ ID NO. 3) 5′-cagtcggtggaggagtccrgg-3′;

[0070] Downstream primer (SEQ ID NO. 4) 5′-actcgagayggtgaccagggtgcc-3′.

[0071] Antibody light chain kappa chain gene primers:

[0072] Upstream primer (SEQ ID NO. 5) 5′-gagctcgtgmtgacccagactcca-3′;

[0073] Downstream primer (SEQ ID NO. 6) 5′-tttgatttccacattggtgcc-3′.

[0074] Gene primers for the lambda chain of the antibody light chain:

[0075] Upstream primer (SEQ ID NO. 7) 5′-gagctcgtgctgactcagtcgccctc-3′;

[0076] Downstream primer (SEQ ID NO. 8) 5′-gcctgtgacggtcagctgggtccc-3′.

[0077] The PCR amplification procedure was as follows: a) initial denaturation at 96°C for 5 minutes; b) denaturation at 96°C for 15 seconds; c) annealing at 58°C for 15 seconds; d) extension at 72°C for 15 seconds; e) final extension at 72°C for 10 minutes; f) storage at 10°C. Steps b to d were repeated 25 times.

[0078] 5.2 The amplified products were subcloned into an expression vector containing the human IgG constant region for sequencing analysis. One of the recombinant antibodies was named ATGM929 (as shown in Table 4).

[0079] Table 4

[0080]

[0081] The heavy chain and light chain sequences of the antibody are shown in Table 5-7:

[0082] Table 5

[0083]

[0084] Table 6

[0085] serial number Heavy chain variable region heavy chain Light chain variable region light chain ATGM929 SEQ ID NO.14 SEQ ID NO.15 SEQ ID NO.16 SEQ ID NO.17

[0086] Table 7

[0087]

[0088] Example 3 Functional verification of purified antibodies

[0089] Coat the ELISA plate with 100 μL (5 μg / mL) of BP230 antigen per well and incubate at 37°C for 2 hours. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST. Add 300 μL of 3% BSA (dissolved in PBS) blocking solution to each well and block at 37°C for 1.5 hours. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST. Add 100 μL of serially diluted recombinant antibody to each well and incubate at 37°C for 1 hour. Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST. Add 100 μL of secondary antibody (Goat Anti-Human IgG Polyclonal Antibody, HRP (PHB96431), brand: Antibodysystem) diluted in blocking solution to each well and incubate at 37°C for 45 minutes. Discard the liquid in the microplate and wash each well three times with 300 μL of 0.05% PBST. Add 100 μL of TMB to each well and incubate at 37°C for 5-10 minutes. Then add 50 μL of 2M HCl to each well to terminate the reaction. Use a microplate reader to read the values ​​at 450nm-630nm. The test results are shown in Figure 2 .from Figure 2 It can be seen that the screened human anti-BP230 monoclonal antibody has a strong specific binding ability to BP230.

[0090] In summary, the monoclonal antibodies screened in the present invention have strong specific binding ability to the BP230 antigen. The obtained recombinant antibodies are highly active, stable, and have strong specificity. They can be used as a reference standard for qualitative detection of BP230 positivity and can also be used to quantitatively detect the level of anti-BP230 autoantibodies in BP patients.

[0091] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A high-affinity monoclonal antibody against BP230, characterized in that: The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is shown in SEQ ID NO.10, and the amino acid sequence of CDR3 is shown in SEQ ID NO.11; the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID NO.12, the amino acid sequence of CDR2 is RAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

13.

2. The anti-BP230 high-affinity monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.14; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

16.

3. The anti-BP230 high-affinity monoclonal antibody according to claim 1, characterized in that The heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.15; the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.

17.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-BP230 high-affinity monoclonal antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 5, or the nucleic acid molecule according to claim 4 is integrated into the genome of the host cell.

7. A composition for detecting BP230 protein, characterized in that: The composition comprises the anti-BP230 high-affinity monoclonal antibody according to any one of claims 1 to 3.

8. A kit for detecting BP230 protein in a sample, characterized in that: The kit comprises the anti-BP230 high-affinity monoclonal antibody according to any one of claims 1 to 3 and / or the composition for detecting BP230 protein according to claim 7.

9. Use of any one or a combination of at least two of the anti-BP230 high-affinity monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, the composition for detecting BP230 protein according to claim 7, or the kit for detecting BP230 protein in a sample according to claim 8 in the preparation of a product for diagnosing bullous pemphigoid.

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