An antibody and kit targeting human desmoglein 2

Abstract

The application discloses an antibody and a kit for targeting human desmocollin 2. The antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3. The antibody has high affinity to desmocollin 2, and the kit developed by using the antibody can realize high-sensitivity quantitative detection of DSC2, which is not only helpful to basic research, but also can provide valuable reference information for clinical diagnosis.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and particularly relates to an antibody and a kit for targeting human desmocollin 2 (DSC2 / Desmocollin-2). Background Art

[0002] Desmosomes (DSM) are large macromolecular protein aggregates that mechanically connect the intermediate filament cytoskeleton with cadherin-mediated cell adhesion points, providing structural integrity for maintaining tissues undergoing mechanical stress (such as skin, myocardium, and gastrointestinal mucosa). Desmocollin-2 (DSC2) is an important cell adhesion molecule that belongs to the cadherin family. DSC2 is mainly present in cardiomyocytes and epithelial cells, and plays an important role in cell adhesion and signal transduction. Its abnormal expression is closely related to a variety of diseases. Literature has reported that reduced DSC2 protein expression levels are closely related to clinical indicators such as pathological type, degree of differentiation, lymph node metastasis, TNM stage, tumor recurrence rate, and survival of many other primary malignant tumors such as esophageal squamous cell carcinoma, colorectal cancer, urothelial carcinoma, and lung cancer, and are involved in tumor progression. Quantitative detection of DSC2 provides very valuable reference information for clinical evaluation of tumor staging and prognosis. Therefore, the development of antibodies and kits targeting DSC2 will not only contribute to basic research, but also provide powerful tools for clinical diagnosis, and has broad application prospects and important social significance.

[0003] Currently, the most commonly used method for antigen detection is the enzyme-linked immunosorbent assay (ELISA). The performance of the kit is highly correlated with the affinity and specificity of the antibody. Most ELISA kit antibodies on the market are primarily prepared and produced using mouse hybridoma technology. Compared to mice and other rodents, rabbits have a superior immune system, producing antibodies with greater specificity and higher affinity. However, there are currently no effective rabbit-derived antibodies targeting human DSC2, nor are there any related detection kits. Summary of the Invention

[0004] The present application is to solve the above problems, and aims to provide an antibody and a kit targeting human desmocollin 2. Specifically, in the present application, DSC2 protein is expressed and purified by using eukaryotic cells, and is used as an antigen for immunizing mice and rabbits. The mice are regularly bled, and the spleen of the mouse with the optimal titer is fused with myeloma cells, hybridoma screening and affinity determination are performed. The spleen of the optimal rabbit is taken, total RNA of the spleen cells is extracted for reverse transcription into cDNA, the cDNA is used as a template to amplify a Fab gene fragment, and the Fab gene fragment is connected with a phage plasmid to construct an anti-DSC2 phage antibody library by electroporation, and panning and sequencing are performed. The mouse and rabbit antibodies are screened by affinity screening and epitope competition test, paired antibodies are screened, an ELISA kit is established, and high-sensitivity detection of DSC2 is realized.

[0005] The present application provides the following technical solutions to solve the above technical problems.

[0006] The first aspect of the present application provides an antibody targeting human desmocollin 2, which comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-H1 being as shown in SEQ ID NO: 9, the amino acid sequence of CDR-H2 being as shown in SEQ ID NO: 10, the amino acid sequence of CDR-H3 being as shown in SEQ ID NO: 11, the amino acid sequence of CDR-L1 being as shown in SEQ ID NO: 13, the amino acid sequence of CDR-L2 being as shown in SEQ ID NO: 14, and the amino acid sequence of CDR-L3 being as shown in SEQ ID NO: 15; or

[0007] the amino acid sequence of CDR-H1 being as shown in SEQ ID NO: 17, the amino acid sequence of CDR-H2 being as shown in SEQ ID NO: 18, the amino acid sequence of CDR-H3 being as shown in SEQ ID NO: 19, the amino acid sequence of CDR-L1 being as shown in SEQ ID NO: 21, the amino acid sequence of CDR-L2 being as shown in SEQ ID NO: 22, and the amino acid sequence of CDR-L3 being as shown in SEQ ID NO: 23; or

[0008] the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 42, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 43, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 46, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 47, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 48.

[0009] In some embodiments of the application, the framework region of the light chain variable region and / or the heavy chain variable region is a human, murine or rabbit framework region.

[0010] In some embodiments of the application, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16, 24 or 48, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 16, 24 or 48;

[0011] and / or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 12, 20 or 44, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 12, 20 or 44.

[0012] In some specific embodiments of the application, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 12.

[0013] In some specific embodiments of the application, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 24, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20.

[0014] In some specific embodiments of the application, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 48, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 44.

[0015] In some embodiments of the application, the antibody is a full-length antibody, a Fab, a Fab', a F(ab')2, or a Fv, preferably a scFv.

[0016] In some embodiments of the application, the antibody is a full-length antibody.

[0017] In some embodiments of the application, the amino acid sequence of the light chain of the antibody is as set forth in SEQ ID NO: 2, 4 or 50; and / or, the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 1, 3 or 45.

[0018] In some specific embodiments of the application, the amino acid sequence of the light chain of the antibody is as set forth in SEQ ID NO: 2, and the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 1.

[0019] In some specific embodiments of the application, the amino acid sequence of the light chain of the antibody is as set forth in SEQ ID NO: 4, and the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 3.

[0020] In some specific embodiments of the application, the amino acid sequence of the light chain of the antibody is as set forth in SEQ ID NO: 50, and the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 45.

[0021] A second aspect of the application provides an isolated nucleic acid encoding an antibody as described in the first aspect of the application.

[0022] In some embodiments of the application, the nucleotide sequence of the light chain of the antibody is as set forth in SEQ ID NO: 6, 8 or 60; and / or, the nucleotide sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 5, 7 or 55.

[0023] In some specific embodiments of the application, the nucleotide sequence of the light chain of the antibody is as set forth in SEQ ID NO: 6, and the nucleotide sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 5.

[0024] In some specific embodiments of the application, the nucleotide sequence of the light chain of the antibody is as set forth in SEQ ID NO: 8, and the nucleotide sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 7.

[0025] In some specific embodiments of the application, the nucleotide sequence of the light chain of the antibody is as set forth in SEQ ID NO: 60, and the nucleotide sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 55.

[0026] A third aspect of the application provides a recombinant expression vector comprising an isolated nucleic acid as described in the second aspect of the application.

[0027] In some embodiments of the present application, the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector, preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0028] The fourth aspect of the present application provides a transformant comprising the isolated nucleic acid according to the second aspect of the present application or the recombinant expression vector according to the third aspect of the present application, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0029] In some embodiments of the present application, the host cell is a yeast cell or a mammalian cell, for example, a HEK293 cell or a CHO cell.

[0030] In some specific embodiments of the present application, the host cell is a 293F cell.

[0031] The fifth aspect of the present application provides a method for preparing an antibody targeting human desmocollin 2, comprising the steps of:

[0032] culturing the transformant according to the fourth aspect of the present application to obtain the antibody from the culture.

[0033] The sixth aspect of the present application provides use of the antibody according to the first aspect of the present application, the isolated nucleic acid according to the second aspect of the present application, the recombinant expression vector according to the third aspect of the present application and / or the transformant according to the fourth aspect of the present application in the preparation of a kit for detecting human desmocollin 2.

[0034] The seventh aspect of the present application provides a kit for detecting human desmocollin 2, comprising the antibody according to the first aspect of the present application.

[0035] In some embodiments of the present application, the kit further comprises other reagents for detecting human desmocollin 2 based on enzyme-linked immunosorbent assay (ELISA), Western Blot, immunohistochemistry (IHC), flow cytometry or protein chip technology.

[0036] In some embodiments of the present application, the kit detects human desmocollin 2 based on double antibody sandwich ELISA, which comprises a capture antibody and a detection antibody; the capture antibody and / or the detection antibody are the antibody according to the first aspect of the present application, and the amino acid sequences of the two are different.

[0037] In some embodiments of the present application, the amino acid sequence of the light chain of the capture antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain of the capture antibody is shown in SEQ ID NO: 1; and / or,

[0038] The amino acid sequence of the light chain of the detection antibody is shown as SEQ ID NO: 4, and the amino acid sequence of the heavy chain of the detection antibody is shown as SEQ ID NO: 3; or the amino acid sequence of the light chain of the detection antibody is shown as SEQ ID NO: 50, and the amino acid sequence of the heavy chain of the detection antibody is shown as SEQ ID NO: 45; optionally, the detection antibody is a biotinylated antibody.

[0039] In some embodiments of the present application, the kit further comprises one or more of an enzyme-labeled secondary antibody, a substrate corresponding to the enzyme, and a complex of the enzyme and streptavidin; the enzyme is preferably horseradish peroxidase, and the substrate is preferably TMB.

[0040] The eighth aspect of the present application provides a method for detecting human desmocollin 2, which comprises contacting a sample to be tested with the antibody of the first aspect of the present application or the kit of the seventh aspect of the present application, and detecting the expression level of human desmocollin 2 in the sample to be tested.

[0041] In some embodiments of the present application, the method is for non-diagnostic purposes.

[0042] In some embodiments of the present application, the mass ratio of the capture antibody to the detection antibody is 5: (1-5), for example, for antibody H3 / R3, the ratio of the two is 5:2, and for antibody H3 / H11-biotin, the ratio of the two is 5:3.

[0043] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred embodiments of the present application.

[0044] The reagents and raw materials used in the present application are commercially available.

[0045] The positive progress effect of the present application is that:

[0046] The detection antibody for human desmocollin 2 (DSC2 / Desmocollin-2) and the kit according to the present application can realize high-sensitivity quantitative detection of DSC2, which not only helps basic research, but also provides valuable reference information for clinical diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is a determination of the titer of mouse serum of Example 2 of the present application.

[0048] Figure 2 is a non-reduced SDS-PAGE electropherogram of DSC2 mouse-derived antibodies H3 and H11.

[0049] Figure 3Figure 2 is a reduced SDS-PAGE electrophoretogram of DSC2 mouse antibodies H3 and H11.

[0050] Figure 4 Figure 3 is a binding curve of DSC2 mouse antibodies H3 and H11.

[0051] Figure 5 Figure 4 is a non-reduced and reduced SDS-PAGE electrophoretogram of DSC2 rabbit antibodies R3.

[0052] Figure 6 Figure 5 is a binding curve of DSC2 rabbit antibodies R3.

[0053] Figure 7 Figure 6 is a calibration curve of a human DSC2 protein detection kit provided by the H3-R3 pairing of the embodiment 4 of the present application.

[0054] Figure 8 Figure 7 is a calibration curve of a human DSC2 protein detection kit provided by the H3-H11 pairing of the embodiment 4 of the present application. DETAILED DESCRIPTION

[0055] The present application will be further described by way of examples below, but the present application is not limited to the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0056] The main reagents and instruments used in the embodiments of the present application are as follows:

[0057] (1) Main reagents

[0058] Goat anti-mouse secondary antibody-HRP, Luoyang Baoatong Experimental Material Center; Donkey anti-rabbit IgG (H+L), Jackson; Fetal bovine serum, Biosun; 50*HAT, Sigma; 50*HT, Sigma; Hybridoma supplement, Boaolong; DMEM, Shanghai Yuanpei; Trizol, Ambion; Chloroform, SCR; DEPC water, Switzerland; PrimeScript RTase 6110A-1, TaKaRa.

[0059] (2) Main instruments

[0060] Plate washer, Agilent; Microplate reader, Thermo; Incubator, Shanghai Zhichu; BTX ECM 2001+ electrofusion instrument, BTX; Protein molecular interaction platform, Cytiva; Countess 3 automatic cell counter, Thermo; CO2 incubator, HealForce.

[0061] Example 1

[0062] The present embodiment provides a combination of monoclonal antibodies for detecting human desmocollin 2 (DSC2 / Desmocollin-2), comprising monoclonal antibody #H3 and monoclonal antibody #R3.

[0063] The amino acid sequence of monoclonal antibody #H3 is shown in Table 1; the DNA sequence of monoclonal antibody #H3 is shown in Table 2; the amino acid sequence of monoclonal antibody #R3 is shown in Table 3; and the DNA sequence of monoclonal antibody #R3 is shown in Table 4. The complementarity determining region (CDR) amino acid sequences in the tables are shown according to the definition of IMGT numbering rules.

[0064] Table 1 Amino acid sequence table of antibody #H3

[0065]

[0066]

[0067] Table 2 DNA sequence table of antibody #H3

[0068]

[0069]

[0070]

[0071] Table 3 Amino acid sequence table of antibody #R3

[0072]

[0073] Table 4 DNA sequence table of antibody #R3

[0074]

[0075]

[0076]

[0077] Table 5 Amino acid sequence table of antibody #H11

[0078]

[0079]

[0080] Table 6 DNA sequence table of antibody #H11

[0081]

[0082]

[0083]

[0084] Preparation of murine monoclonal antibodies detecting DSC2

[0085] I. Selection of immunogen

[0086] The amino acid sequence of the immunogen is as follows:

[0087] >DSC2 (Human): RWAPIPCSMLENSLGPFPLFLQQVQSDTAQNYTI YYSIRGPGVDQEPRNLFYVERDTGNLYCTRPVDREQYESFEIIAFATTPDGYTPELPLPLIIKIEDENDNYPIFTEETYTFTIFENCRVGTTVGQVCATDKDEPDTMHTRLKYSIIGQVPPSPTLFSMHPTTGVITTTSSQLDRELIDKYQLKIKVQDMDGQYFGLQTTSTCIINIDDVNDHLPTFTRTSYVTSVEENTVDVEILRVTVEDKDLVNTANWRANYTILKGNENGNFKIVTDAKTNEGVLCVVKPLNYEEKQQMILQIGVVNEAPFSREASPRSAMSTATVTVNVEDQDEGPECNPPIQTVRMKENAEVGTTSNGYKAYDPETRSSSGIRYKKLTDPTGWVTIDENTGSIKVFRSLDREAETIKNGIYNITVLASDQGGRTCTGTLGIILQDVNDNSPFIPKKTVIICKPTMSSAEIVAVDPDEPIHGPPFDFSLESSTSEVQRMWRLKAINDTAARLSYQNDPPFGSYVVPITVRDRLGMSSVTSLDVTLCDCITENDCTH RVDPR (SEQ ID NO: 61)

[0088] II. Immunization of mice and antibody screening

[0089] Human DSC2 was used as a specific antigen to prepare mouse monoclonal antibodies.

[0090] 1. Protein immunization:

[0091] The emulsifier needle is wiped twice with 75% alcohol (the second time with a new cotton ball), and the single emulsion liquid volume ranges from about 250 μl to 20 ml. The DSC2 protein is diluted to 2 mg / mL, 400 μg of protein and 400 μL of complete adjuvant or incomplete adjuvant are taken, and emulsification is performed using an emulsifier. The whole is moved from top to bottom, and the local is moved up and down. Generally, 1-10 ml of emulsion can be emulsified for 3-5 minutes, and the maximum time should not exceed 10 minutes.

[0092] Dosing method: subcutaneous multi-point immunization, impact immunization is muscle immunization.

[0093] Strain: Balb / C, 6-8 weeks, female.

[0094] Number: 2 controls, 2 samples for hybridoma screening, and 3 alternatives. Immunization dose: 100 μg per mouse per time, with a 2-week interval.

[0095] 2. Mouse serum antibody titer and cell supernatant determination

[0096] After the third immunization, blood was taken one week later to determine the mouse serum antibody titer, and the specific method is as follows:

[0097] (1) 100 μl of antigen (100 ng) was added to a 96-well plate for coating, and incubated at 4°C in the dark overnight.

[0098] (2) Wash the plate 4 times, each time add 300 μl of 1×PBST, and finally try to spin dry and absorb on absorbent paper. Add 200 μl of 5% skim milk or 3% BSA or 1% casein solution to each well for blocking. Incubate at 37°C in the dark for 2 hours.

[0099] (3) Wash the plate 4 times, each time add 300 μl of 1×PBST, and finally try to spin dry and absorb on absorbent paper.

[0100] (4) Take the cell supernatant or diluted serum to the blocked 96-well plate using the NAYO 96-well pipetting station, 100 μL per well, and add positive and negative controls at the same time. Incubate at 37°C in the dark for 1 hour.

[0101] (5) Wash the plate 4 times, each time add 300 μl of 1×PBST, and finally try to spin dry and absorb on absorbent paper. Add 100 μl of 1×secondary antibody (10,000-20,000-fold dilution) to each well. Incubate at 37°C in the dark for 1 hour.

[0102] (6) Wash the plate 4 times, each time add 300 μl of 1×PBST, and finally try to spin dry and absorb on absorbent paper. Add 50 μl of TMB developing solution (the developing solution itself is colorless, and cannot be used if a color change occurs). Incubate at 37°C in the dark for 10-15 minutes, add 100 μl of stop solution to stop the reaction, and read the OD value at 450 nm.

[0103] As shown in Figure 1 Figure 1, the serum titers of mice No. 1-3 all reached 1 million, and mice No. 1-2 were selected for hybridoma fusion.

[0104] 3. Cell fusion

[0105] The fusion buffer was taken out in advance to restore to room temperature. The mixed cells were washed twice with DMEM medium, and the last washing was performed with the electrofusion buffer. After washing, the B cells and myeloma cells were fused, and the total amount of cells required was calculated according to the experimental requirements. The fusion buffer placed at room temperature was added to dilute the cell concentration to 1 x 10 7 cells / mL. The mixed cells were added to 2 ml or 9 ml fusion pools, and the lid was covered for electrofusion. After completing the electrofusion, the cells were gently added to a conical tube containing 40 ml of culture medium containing 20% FBS, and incubated at 37°C for 60 minutes. After incubation, the cells in the conical tube were diluted and plated.

[0106] 4. Positive hybridoma cell screening

[0107] After 10-14 days of fusion, positive hybridoma cells were screened by indirect ELISA method, and the specific method was the same as the method for determining the antibody titer of mouse serum and cell supernatant described above.

[0108] 5. Hybridoma cell subcloning

[0109] The positive wells initially identified as positive were first expanded and cryopreserved, and then when the remaining cells grew to near 80%, they were gently resuspended and counted for viable cells, and 5000 viable cells were transferred to a centrifuge tube containing 5 mL of complete medium (DMEM containing 20% FBS with 1 x HAT (H0262, Sigma)) with 1 x HAT (H0262, Sigma). At this time, the cell concentration was 1000 cells / mL. 200 μL of diluted cell suspension was added to the first row, and 2-fold gradient dilution was performed in turn, and finally 200 μL of cell culture medium was added to each cell well.

[0110] 6. Antibody sequencing

[0111] The mRNA of hybridoma cells secreting monoclonal antibodies was extracted, and cDNA was synthesized by reverse transcription. Ten heavy chain variable region primers and 12 light chain variable region primers were selected for PCR experiments, and correct size bands were obtained. The correct bands were sent to the company for sequencing, and the sequencing results were analyzed and compared.

[0112] 7. Antibody preparation and affinity determination

[0113] (1) Preparation of ascites antibody

[0114] Abdominal injection of Balb / c mice 0.3-0.5ml liquid paraffin sensitized 7-15d; the need for injection of hybridoma cells blow, 1000rpm centrifugation for 5min, discard the supernatant; add 1ml DMEM resuspended, cell counting; the cells with DMEM diluted to 2x10 6 / ml; with 1ml syringe 1ml cells, each mouse injection of 0.5ml about 10 6 cells. The specific injection method: pinch the mouse tail rotation several weeks to reduce the activity of the mouse, the left hand pinch mouse ear and the back of the neck, ring finger and little finger hold the mouse tail, let the mouse in a natural state of expansion, with alcohol cotton ball wipe the mouse abdomen, between the two legs of the mouse slightly to the right of the insertion of the syringe, let the mouse 45 degrees downward tilt injection of cells, spin half a week pull out the needle, observe the mouse state into the new bedding cage, make a good mark. 7-10 days to observe the growth of mice and whether the abdomen is larger. The abdomen of the mouse is larger, and the dislocation is killed, and the ascites is collected. 12000rpm centrifugation for 10min, take the supernatant, and precipitate with equal volume of saturated ammonium sulfate. Resuspend with equal volume of 1xPBS, dialysis 3 times, and measure the concentration with nanodrop.

[0115] Resuspend the magnetic beads by shaking or vortexing; take the appropriate amount of magnetic beads (magnetic bead load 25mg / 4ml) into a 50ml centrifuge tube, place it on the magnetic base of the magnetic stand, discard the supernatant; add 10 times the volume of 0.1% PBST, invert and mix thoroughly, place it on the magnetic base of the magnetic stand, discard the supernatant; repeat the washing 1 time, add equal volume of PBST resuspend for standby; add the ascites antibody to the resuspended magnetic beads, rotate overnight at 4°C; place it on the magnetic base of the magnetic stand, discard the supernatant or save for standby; wash with PBS three times, discard the supernatant, and finally use 1ml gun head to clean; add equal volume of elution buffer, incubate at room temperature for 10min, resuspend 2-3 times during the period; place it on the magnetic base of the magnetic stand, collect the liquid into a clean 50ml centrifuge tube, repeat the elution once; add neutralizing solution to the centrifuge tube according to 100μl / ml, mix gently; place the centrifuge tube on the magnetic base to adsorb the magnetic beads, transfer the supernatant to the prepared EP tube with 1ml gun, measure the concentration; dialysis three times with 1xPBS, change the liquid every 4h, or ultrafiltration concentration change liquid, measure the concentration; wash the magnetic beads with PBS 3 times, resuspend the magnetic beads with 20% ethanol, and store at 4°C.

[0116] (2) Antibody SDS-PAGE

[0117] Take 5 μg of the sample to be tested into a 1.5 ml centrifuge tube, add water to 9 μl for non-reduced and reduced samples, add 3 μl of 4xLDS Buffer (NP0008, Thermo) for non-reduced samples, and add 3 μl of 4xLDS Buffer containing 10 mM DTT for reduced samples, mix well, and then cook the reduced sample at 95°C for 10 min. Take a piece of protein electrophoresis gel, tear off the protective film, insert the side with the switch, and insert a baffle on the other side, then close the two switches, and place it in the electrophoresis tank. Add freshly prepared 1xMOPS buffer to the electrophoresis tank, and add 1xMOPS recovery liquid outside. Pull out the comb, add 5 μl of protein marker to the first column, and then add the prepared sample to be tested, making sure to add it to the center of the hole. Cover the cover, pay attention to the positive and negative electrodes, and run the strip at 170V for 55 min until it stops below the red color. Pour the MOPS into the MOPS recovery liquid bottle, open the switch and take out the protein gel, and use scissors to pry it open, making sure not to dry the gel. Use a comb to cut off the loading part, and transfer the protein gel to clean water, boil, and repeat three times. Transfer the treated gel to the Coomassie brilliant blue solution, boil, shake on the shaker for 5-30 min, and then change the water to remove the color until clear bands can be seen.

[0118] Figure 2 、 Figure 3 is the non-reduced and reduced SDS-PAGE electrophoresis diagram of the antibody DSC2 antibody (H3 and H11) after purification in this example, the antibody has high purity, and is further used for performance determination and analysis of the antibody.

[0119] (3) Antibody binding curve determination

[0120] Prepare an antigen dilution solution with a final concentration of 1 μg / ml, mix well, and then use a multichannel pipette to add 100 μl per well to a 96-well plate, 100 ng / well, and incubate overnight at 4°C. The next day, wash the plate three times with 0.05% PBST, then add 5% skim milk and incubate at 37°C for 1-3 hours. After the blocking is completed, wash the plate three times with 0.05% PBST, add gradient-diluted antibody samples, and incubate at 37°C for 1 hour. After the primary antibody incubation is completed, wash the plate three times with 0.05% PBST, add 20000-fold diluted goat anti-mouse IgG (H+L)-HRP, and incubate at 37°C for 1 hour. After the secondary antibody incubation is completed, wash the plate three times with 0.05% PBST, add 50 μL TMB, incubate at room temperature for 5-10 minutes in the dark, add 100 μl of 1M concentrated sulfuric acid to terminate the reaction, and then read the absorbance at 450 nm.

[0121] Figure 4 is the H3 and H11 antibody binding curve determination in this example, and the positive control antibody (10809-MM04, Yiqiao Shenzhou) is used at a dilution of one thousand times. As shown in the figure, H3 and H11 have strong binding to DSC2 protein.

[0122] (4) Antibody affinity assay

[0123] According to the ELISA results, select the appropriate chip, and use the manual mode to explore the antigen-antibody binding Ru value at a certain concentration. Use Protein G chip or CM5 chip coupled with anti-mouse antibody chip, select Multi-cycle kinetics affinity using GST Capture kit program, set injection and dissociation time, flow rate, regeneration solution, Startup cycle number, analyte concentration gradient and sample position. In Biacore Insight Evaluation Software, click "Predefined" to find the corresponding analysis method (Kinetics / affinity - Antibody / general - Multi-cycle kinetics affinity using GST Capture kit) (corresponding to the experimental method), double-click or click the lower right corner "Open" button for analysis. The analysis software automatically performs data fitting and outputs the results. The results are shown in Table 7, and the antibody affinity range is 3.0-5.2 x 10 -8 .

[0124] Table 7 DSC2 antibody affinity and force measurement

[0125]

[0126] (5) Antibody epitope competition and pairing

[0127] According to the affinity assay results, select high-affinity antibodies to be immobilized on the CM5 chip, then pre-mix the competing antibodies with the antigen, and then perform competition analysis. H10+: DSC2 mouse anti-H10 antibody is pre-mixed with the antigen, Antigen: only the antigen is combined for testing, and the results are shown in Table 8, H3 and H11 do not compete.

[0128] Table 8 Antibody competition Ru value

[0129]

[0130] Example 3 Preparation of rabbit monoclonal antibody for detecting DSC2

[0131] Human DSC2 (SEQ ID NO: 41) was used as a specific antigen to prepare a rabbit monoclonal antibody.

[0132] 1. Protein immunization

[0133] The emulsifier needle is wiped with 75% alcohol twice (the second time with a new cotton ball), and the single emulsion liquid volume ranges from about 250 μl to 20 ml. The DSC2 protein is diluted to 2 mg / mL, 1.2 mg of protein and 600 μL of complete adjuvant or incomplete adjuvant are taken, and the emulsifier is used for emulsification. The whole is moved from top to bottom, and the local is moved up and down. Generally, 3-5 minutes are needed for 1-10 ml of emulsion, and the time should not exceed 10 minutes at most.

[0134] Dosage: subcutaneous multi-point immunization, impact immunization is intravenous injection.

[0135] Strain: New Zealand white rabbits, 2-3 months old, 2-2.5 kg, female.

[0136] Quantity: 2 controls, immunization dose: 500 μg per rabbit per time, interval of two weeks.

[0137] 2. Rabbit serum antibody titer and cell supernatant determination

[0138] After the fourth immunization of DSC2, blood was taken one week later to determine the antibody titer of rabbit serum, and the specific method was the same as that of mouse serum titer determination in Example 2.

[0139] 3. Construction of immune library

[0140] The total RNA of rabbit spleen cells was extracted and reverse transcribed into cDNA. The heavy and light chain variable regions were amplified using the cDNA as a template, connected by a linker, and then connected to a phagemid vector. An immune library of 9 times was constructed by electroporation, and was packaged into scfv-phage for screening.

[0141] 4. Screening of phage library

[0142] After incubating the scfv-phage with biotin-labeled Human DSC2 antigen at room temperature for 1 hour, the blocked SA-coated magnetic beads were added and incubated at room temperature for 15 minutes. After the magnetic beads were washed with PBST for 10 times, the specifically bound phage was eluted with trypsin at room temperature and used to infect logarithmic phase E. coli TG1. After 3 rounds of screening, a highly enriched phage library was obtained.

[0143] 5. Screening of single positive phage

[0144] The above screened phage-enriched library was picked one by one to form scfv-phage in a 96-well round-bottom deep-well plate. Then, ELISA was performed using the phage, and the positive wells were specific antibodies against DSC2 antigen. The corresponding antibody R3 variable region sequence was obtained by sending the bacterial solution for second-generation sequencing.

[0145] 6. Antibody clone construction and plasmid preparation

[0146] Design primers to amplify the heavy and light chain variable regions and clone into vectors with the constant regions of the heavy and light chains of the mouse antibody to construct the whole antibody molecule and prepare plasmids using a kit (MN740410.100).

[0147] 7. Antibody preparation and affinity determination

[0148] (1) Recombinant rabbit antibody preparation and binding curve determination

[0149] The recombinant plasmid was transfected into 293F cells at a ratio of 1:1, and the supernatant was purified to purify the antibody when the cell viability decreased to 80% on the fifth day.

[0150] Resuspend the magnetic beads by shaking or vortexing; take an appropriate amount of magnetic beads (25 mg of magnetic beads per 4 ml) and place them in a 50 ml centrifuge tube, place them on the magnetic base of the magnetic stand, and discard the supernatant; add 10 times the volume of 0.1% PBST, invert to mix thoroughly, place on the magnetic base of the magnetic stand, and discard the supernatant; repeat the washing once, add an equal volume of PBST to resuspend, and store for later use; add the expression supernatant to the resuspended magnetic beads and rotate at 4°C overnight; place on the magnetic base of the magnetic stand, discard the supernatant or store for later use; wash three times with PBS, discard the supernatant, and finally use 1 ml of a gun to dry completely; add an equal volume of elution buffer, incubate at room temperature for 15 minutes, resuspend 2-3 times during the incubation; place on the magnetic base of the magnetic stand, collect the liquid into a clean 50 ml centrifuge tube, and repeat the elution once; add neutralizing solution to the centrifuge tube at a ratio of 100 μl / ml, mix gently; place the centrifuge tube on the magnetic base to adsorb the magnetic beads, use a 1 ml gun to transfer the supernatant to a prepared EP tube, and measure the concentration; concentrate the antibody using an ultrafiltration tube, replace 3 times with 1 x PBS, measure the concentration, and store at -20°C.

[0151] (2) SDS-PAGE of the antibody

[0152] Take the sample to be tested 5 μg into a 1.5 ml centrifuge tube, add water to 9 μl for non-reduced and reduced samples, add 3 μl of 4xLDS Buffer (NP0008, Thermo) for non-reduced samples, and add 3 μl of 4xLDS Buffer containing 10 mM DTT for reduced samples. Mix well and then cook the reduced sample at 95°C for 10 min. Take a piece of protein electrophoresis gel, tear off the protective film, insert the side with switches, and insert a baffle on the other side. Close the two switches and place them in the electrophoresis tank. Add freshly prepared 1xMOPS buffer to the electrophoresis tank and add 1xMOPS recovery liquid outside. Pull out the comb, add 5 μl of protein marker to the first column, and then add the prepared sample to be tested. Make sure the sample is added to the center of the hole. Cover the cover, pay attention to the positive and negative electrodes, and electrophorese the strip at 170V for 55 min until it stops below the red color. Pour the MOPS into the MOPS recovery liquid bottle, open the switch and take out the protein gel. Use scissors to pry it open, be careful not to dry the gel. Use a comb to cut off the loading part and transfer the protein gel to clean water. Boil and repeat three times. Transfer the treated gel to the Coomassie brilliant blue solution, boil, shake on the shaker for 5-30 min, and then change the water to remove the light. Until clear bands can be seen, the R3 antibody SDS-PAGE results are shown in Figure 5 .

[0153] (3) Antibody binding curve determination

[0154] Prepare an antigen dilution solution with a final concentration of 1 μg / ml. After mixing well, use a multichannel pipette to add 100 μl per well to a 96-well plate, 100 ng / well, and incubate overnight at 4°C. The next day, wash the plate three times with 0.05% PBST, then add 5% skim milk and incubate at 37°C for 1-3 hours. After blocking, wash the plate three times with 0.05% PBST, add gradient-diluted antibody samples, and incubate at 37°C for 1 hour. After incubation of the primary antibody, wash the plate three times with 0.05% PBST, add 20000-fold diluted goat anti-rabbit IgG (H+L)-HRP, and incubate at 37°C for 1 hour. After incubation of the secondary antibody, wash the plate three times with 0.05% PBST, add 50 μL TMB, incubate at room temperature for 5-10 minutes in the dark, add 100 uL of 1M concentrated sulfuric acid to stop the reaction, and read the absorbance at 450 nm. The R3 antibody binding curve results are shown in Figure 6 .

[0155] The affinity determination method is the same as that in Example 2. The DSC2 rabbit antibody affinity is shown in Table 9.

[0156] Table 9 DSC2 antibody affinity determination

[0157] Antibody ka (1 / Ms) kd (1 / s) KD (M) DSC2-R3 3.07E+05 1.07E-03 3.47E-09

[0158] 7. Mouse anti compete with rabbit anti epitope test

[0159] According to the affinity determination results, high affinity antibodies were selected and immobilized on the CM5 chip, and then the competing antibodies were pre-mixed with the antigen, and then the competition analysis was carried out. R3+: DSC2 rabbit anti R3 antibody was pre-mixed with the antigen, Antigen: only the antigen was subjected to binding test, and the results are shown in Table 10, H3 and R3 did not compete.

[0160] Table 10 Mouse anti compete with rabbit anti epitope test

[0161] Channel Immobilized antibody Antigen R3+ H3+ H11+ 4 H3 137.9 93.1 3.1 5 H11 35 5.7 -2.1

[0162] Example 4

[0163] This embodiment discloses the application of a human desmocollin-2 (DSC2 / Desmocollin-2) monoclonal antibody in an immunological tool: an immunological detection tool includes a detection kit.

[0164] 1. Human desmocollin-2 quantitative detection kit (double antibody sandwich quantitative method)

[0165] Detection principle: The detection principle of the human desmocollin-2 (DSC2 / Desmocollin-2) quantitative detection kit (double antibody sandwich quantitative method) is to use a pair of antibodies that can bind to at least two different epitopes on DSC2. The capture antibody is pre-coated on the surface of the microwells of the microplate and selectively binds to the DSC2 protein. After washing, the detection antibody is added to bind to the second site of DSC2 to form a sandwich complex. After adding the enzyme-labeled second antibody to bind to the detection antibody, finally the TMB substrate is colored, and the color depth in the microwells is positively correlated with the concentration of DSC2 protein. Reading at 450 nm by an enzyme-labeled instrument can quantitatively detect the concentration of DSC2.

[0166] 2. H3-R3 kit

[0167] It includes H3, R3, NEST detachable Elisa plate, dilution buffer, donkey anti-rabbit HRP (H+L), etc.

[0168] Linearity test: The concentration of human Desmocollin-2 (DSC2) calibrator was 0 ng / ml, 0.078 ng / ml, 0.156 ng / ml, 0.312 ng / ml, 0.625 ng / ml, 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml. The calibrator was added to the 96-well plate, and each concentration was determined in triplicate. After 30 min of reaction, the reaction signal intensity was detected by a microplate reader, and the absorbance signal value corresponding to each calibrator was obtained, as shown in Table 11.

[0169] Table 11 Absorbance signal value corresponding to each calibrator

[0170]

[0171] The linear fitting of the calibrator concentration and its corresponding absorbance signal value was performed to draw the calibration curve, as shown in Figure 7 R 2 = 1, indicating that the absorbance signal value was highly linearly correlated with the detection concentration in the range of 0.078-5 ng / ml.

[0172] 3. H3-R3 kit usage method:

[0173] (1) Add 100 μL of capture antibody H3 solution (5 μg / mL) to the appropriate well. Cover the well with a well cover and incubate at 4°C for 16 hours.

[0174] (2) Discard the solution in the well and wash 4 times with 1x PBST. Invert the plate and dry with a clean paper towel.

[0175] (3) Add 200 μL of 1% casein solution to each well. Cover the well with a well cover and block at 37°C for 2 hours.

[0176] (4) Wash according to step (2).

[0177] (5) Add the standard and test sample to the appropriate well. Cover the well with a well cover and incubate at 25°C for 2.5 hours with gentle shaking.

[0178] (6) Wash according to step (2).

[0179] (7) Add 100 μL of detection antibody R3 (2 μg / mL) solution to each well. Cover the well with a well cover and incubate at 25°C for 1 hour with gentle shaking.

[0180] (8) Wash according to step (2).

[0181] (9) Add 100 μl of prepared Donkey anti-rabbit HRP (H+L) solution to each well. Cover the well with a well cover and incubate at 25°C for 45 minutes with gentle shaking.

[0182] (10) Wash twice according to step (2).

[0183] (11) Add TMB substrate solution to each well. Incubate at 25°C for 30 minutes in the dark with gentle shaking.

[0184] (12) Add 50 μl 0.2M sulfuric acid to each well to stop the color development. Immediately read the optical density at 450nm using a microplate reader.

[0185] 4. H3-H11 kit

[0186] H3, H11-Biotin, NEST 96-well Elisa plate, dilution buffer, SA-HRP, etc.

[0187] Linear detection: The concentration of human desmocollin-2 (DSC2) calibration samples was 0 ng / ml, 0.312 ng / ml, 0.625 ng / ml, 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml, and 10 ng / ml. The calibration samples were added to the 96-well plate, and each concentration was determined in triplicate. After 30 minutes of reaction, the reaction signal intensity was detected by a microplate reader to obtain the absorbance signal value corresponding to each calibration sample, as shown in Table 12.

[0188] Table 12 Absorbance signal value corresponding to calibration samples

[0189]

[0190] The calibration curve was drawn by linear fitting of the calibration sample concentration and its corresponding absorbance signal value, as shown in Figure 8 R 2 = 0.9999, indicating that the absorbance signal value was highly linearly correlated with the detection concentration in the range of 0.312-10 ng / ml.

[0191] 5. Preparation method of H11-Biotin (LK03, Dojindo)

[0192] (1) Add 100 μl of WS Buffer and a sample solution containing 100 μg of antibody H11 to a filter tube.

[0193] (2) Centrifuge at 8000-10000 g for 10 minutes.

[0194] (3) Add 10 μl of DMSO to NH2-Reactive Biotin and dissolve it by pipetting.

[0195] (4) Add 100 μl Reaction Buffer and 8 μl NH2-Reactive Biotin solution into the filter tube, mix by flicking.

[0196] (5) Put the filter tube into the incubator, incubate at 37°C for 10 min.

[0197] (6) Add 100 μl WS Buffer into the filter tube, centrifuge at 8000-10000 g for 10 min, remove the filtrate.

[0198] (7) Add 200 μl WS Buffer into the filter tube, centrifuge at 8000-10000 g for 10 min, repeat this step once.

[0199] (8) Add 200 μl WS Buffer into the filter tube, mix by flicking 10-15 times to recover the labeled product. Transfer the solution into a 1.5 mL tube, store at 0-5°C.

[0200] 6. H3-H11 Kit Usage Method:

[0201] (1) Add 100 μL of capture antibody H3 solution (5 μg / mL) into the appropriate well. Cover the well, incubate at 4°C for 16 hours.

[0202] (2) Discard the solution in the well, wash 4 times with 1x PBST. Invert the plate and blot dry with a clean paper towel.

[0203] (3) Add 200 μL of 1% casein solution to each well. Cover the well, block at 37°C for 2 hours.

[0204] (4) Wash according to step (2).

[0205] (5) Add the standard and test sample into the appropriate well. Cover the well, incubate at 25°C for 2.5 hours with gentle shaking.

[0206] (6) Wash according to step (2).

[0207] (7) Add 100 μL of detection antibody H11-Biotin (3 μg / mL) solution to each well. Cover the well, incubate at 25°C for 1 hour with gentle shaking.

[0208] (8) Wash according to step (2).

[0209] (9) Add 100 μl of prepared SA-HRP (M00091, KPL) solution to each well. Cover the well, incubate at 25°C for 45 minutes with gentle shaking.

[0210] (10) Wash twice according to step (2).

[0211] (11) Add TMB substrate solution to each well. Incubate at 25°C for 30 minutes in the dark with gentle shaking.

[0212] (12) Add 50 μl 0.2M sulfuric acid to each well to stop the color development. Then immediately read at 450nm with a microplate reader.

[0213] 7. Sensitivity comparison between H3-R3 kit and H3-H11 kit

[0214] Kit Linear range (pg / ml) Sensitivity (pg / ml) H3-R3 78-5000 78 H3-H11 312-10000 312

[0215] Example 5

[0216] Performance evaluation of human desmoglein 2 (DSC2 / Desmocollin-2) H3-R3 kit

[0217] 1. Sensitivity

[0218] The minimum detection value of H3-R3 kit of Example 4 is 0.078 ng / ml, which indicates that the kit has high sensitivity.

[0219] 2. Accuracy

[0220] The recovery rate of H3-R3 kit of Example 4 is 102-114%, which indicates that the kit has high accuracy.

[0221] 3. Linear range

[0222] The correlation coefficient of H3-R3 kit of Example 4 is ≥0.99 in the range of 0.078 ng / ml-5 ng / ml.

[0223] In summary, it can be determined that the performance of the kit is good, and it has the advantages of high sensitivity, strong specificity, and high accuracy.

Claims

1. An antibody targeting human desmoglein 2, comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising a CDR-H1, a CDR-H2 and a CDR-H3, the light chain variable region comprising a CDR-L1, a CDR-L2 and a CDR-L3, characterized in that, the amino acid sequence of the CDR-H1 is set forth in SEQ ID NO: 9, the amino acid sequence of the CDR-H2 is set forth in SEQ ID NO: 10, the amino acid sequence of the CDR-H3 is set forth in SEQ ID NO: 11, the amino acid sequence of the CDR-L1 is set forth in SEQ ID NO: 13, the amino acid sequence of the CDR-L2 is set forth in SEQ ID NO: 14, and the amino acid sequence of the CDR-L3 is set forth in SEQ ID NO:

15. The framework region of the light chain variable region and / or the heavy chain variable region is a human, murine or rabbit framework region.

2. The antibody of claim 1, wherein The amino acid sequence of the light chain variable region has at least 85% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 85% sequence identity to SEQ ID NO:

12.

3. The antibody of claim 2, wherein The amino acid sequence of the light chain variable region has at least 90% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 90% sequence identity to SEQ ID NO:

12.

4. The antibody of claim 3, wherein The amino acid sequence of the light chain variable region has at least 95% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 95% sequence identity to SEQ ID NO:

12.

5. The antibody of claim 4, wherein The amino acid sequence of the light chain variable region has at least 96% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 96% sequence identity to SEQ ID NO:

12.

6. The antibody of claim 5, wherein The amino acid sequence of the light chain variable region has at least 97% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 97% sequence identity to SEQ ID NO:

12.

7. The antibody of claim 6, wherein The amino acid sequence of the light chain variable region has at least 98% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 98% sequence identity to SEQ ID NO:

12.

8. The antibody of claim 7, wherein The amino acid sequence of the light chain variable region has at least 99% sequence identity to SEQ ID NO: 16; and / or, the amino acid sequence of the heavy chain variable region has at least 99% sequence identity to SEQ ID NO:

12.

9. The antibody of claim 8, wherein The amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:

12.

10. The antibody of claim 9, wherein The antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv.

11. The antibody of any one of claims 1-10, wherein ​ 12. The antibody of any one of claims 1-10, wherein The antibody is a scFv.

13. The antibody of claim 11, wherein The antibody is a full-length antibody.

14. The antibody of claim 13, wherein The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 2; and / or, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:

1.

15. An isolated nucleic acid, comprising: The isolated nucleic acid encodes the antibody of any one of claims 1-14.

16. The isolated nucleic acid of claim 15, wherein, The nucleotide sequence of the light chain of the antibody is shown in SEQ ID NO: 6; and / or, the nucleotide sequence of the heavy chain of the antibody is shown in SEQ ID NO:

5.

17. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 15 or 16.

18. The recombinant expression vector of claim 17, wherein, The recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector.

19. The recombinant expression vector of claim 18, wherein, The viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

20. A transformant characterized in that, The transformant comprises the isolated nucleic acid of claim 15 or 16 or the recombinant expression vector of any one of claims 17-19, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

21. The transformant of claim 20, wherein, The host cell is a yeast cell or a mammalian cell.

22. The transformant of claim 21, wherein, The host cell is a HEK293 cell or a CHO cell.

23. The transformant of any one of claims 20 to 22, wherein the nucleic acid molecule is integrated into the genome of the host cell. The host cell is a 293F cell.

24. A method of making an antibody that targets human desmoglein 2, comprising, The method comprises the following steps: The antibody is obtained from the culture of the transformant of any one of claims 20-23.

25. Use of the antibody of any one of claims 1-14, the isolated nucleic acid of claim 15 or 16, the recombinant expression vector of any one of claims 17-19 and / or the transformant of any one of claims 20-23 in the preparation of a kit for detecting human desmocollin 2.

26. A kit for detecting human desmoglein 2, characterized by, The kit comprises the antibody of any one of claims 1-14.

27. The kit of claim 26, wherein The kit further comprises reagents for detecting human desmocollin 2 based on enzyme-linked immunosorbent assay, Western blot, immunohistochemistry, flow cytometry or protein chip technology.

28. The kit of claim 27, wherein The kit detects human desmocollin 2 based on double antibody sandwich ELISA, which comprises a capture antibody and a detection antibody targeting human desmocollin 2; the capture antibody is the antibody of any one of claims 1-14.

29. The kit of claim 28, wherein The amino acid sequence of CDR-H1 of the detection antibody is shown in SEQ ID NO: 17, the amino acid sequence of CDR-H2 of the detection antibody is shown in SEQ ID NO: 18, the amino acid sequence of CDR-H3 of the detection antibody is shown in SEQ ID NO: 19, the amino acid sequence of CDR-L1 of the detection antibody is shown in SEQ ID NO: 21, the amino acid sequence of CDR-L2 of the detection antibody is shown in SEQ ID NO: 22, and the amino acid sequence of CDR-L3 of the detection antibody is shown in SEQ ID NO: 23; or the amino acid sequence of CDR-H1 of the detection antibody is shown as SEQ ID NO: 41, the amino acid sequence of CDR-H2 of the detection antibody is shown as SEQ ID NO: 42, the amino acid sequence of CDR-H3 of the detection antibody is shown as SEQ ID NO: 43, the amino acid sequence of CDR-L1 of the detection antibody is shown as SEQ ID NO: 46, the amino acid sequence of CDR-L2 of the detection antibody is shown as SEQ ID NO: 47, and the amino acid sequence of CDR-L3 of the detection antibody is shown as SEQ ID NO:

48.

30. The kit of claim 29, wherein the amino acid sequence of the light chain variable region of the detection antibody is shown as SEQ ID NO: 24, and the amino acid sequence of the heavy chain variable region of the detection antibody is shown as SEQ ID NO: 20; or the amino acid sequence of the light chain variable region of the detection antibody is shown as SEQ ID NO: 48, and the amino acid sequence of the heavy chain variable region of the detection antibody is shown as SEQ ID NO:

44.

31. The kit of claim 28, wherein the amino acid sequence of the light chain of the capture antibody is shown as SEQ ID NO: 2, and the amino acid sequence of the heavy chain of the capture antibody is shown as SEQ ID NO: 1; and / or, the amino acid sequence of the light chain of the detection antibody is shown as SEQ ID NO: 4, and the amino acid sequence of the heavy chain of the detection antibody is shown as SEQ ID NO: 3; or, the amino acid sequence of the light chain of the detection antibody is shown as SEQ ID NO: 50, and the amino acid sequence of the heavy chain of the detection antibody is shown as SEQ ID NO:

45.

32. The kit of any one of claims 28-31, wherein, the detection antibody is a biotinylated antibody.

33. The kit of any one of claims 28-31, wherein, the kit further comprises one or more of an enzyme-labeled secondary antibody, a substrate corresponding to the enzyme, and a complex formed by the enzyme and streptavidin.

34. The kit of claim 33, wherein the enzyme is horseradish peroxidase.

35. The kit according to claim 33, wherein the substrate is TMB.

36. A method of detecting human desmoglein 2 for a non-diagnostic purpose, comprising contacting a sample with an antibody of claim 1. 5 the method comprises contacting a sample to be tested with the antibody of any one of claims 1-14 or the kit of any one of claims 26-35, and detecting the expression level of human desmoglein 2 in the sample to be tested.

37. The method of claim 36, wherein, the mass ratio of the capture antibody to the detection antibody is 5: (1-5).

38. The method of claim 37, wherein, the mass ratio of the capture antibody to the detection antibody is 5:2 or 5:3.

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