Antibody and kit for targeting human desmomere glycoprotein 2

Abstract

The invention discloses an antibody and a kit for targeting human desmogranuloprotein 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 with deodontoprotein-2, and a kit developed by using the antibody can realize high-sensitivity quantitative detection of DSC2, thereby not only being beneficial to basic research, but also being capable of providing valuable reference information for clinical diagnosis.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular 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 sites, providing structural integrity for maintaining tissues that experience 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 staging, tumor recurrence rate, and survival of a variety of other primary malignant tumors such as esophageal squamous cell carcinoma, colorectal cancer, urothelial carcinoma, and lung cancer, and participate 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 help basic research, but also provide powerful tools for clinical diagnosis, which has broad application prospects and important social significance.

[0003] The most commonly used method for antigen detection is enzyme-linked immunosorbent assay, and the performance of the kit is highly correlated with the affinity and specificity of the antibody. Most ELISA kit antibodies on the market are mainly prepared and produced using mouse hybridoma technology. Compared with mice and other rodents, rabbits have a better immune system and produce antibodies with stronger specificity and higher affinity. However, there are currently no effective rabbit-derived antibodies targeting human DSC2 and related detection kits. Summary of the invention

[0004] The present invention is made to solve the above problems, and the purpose is to provide an antibody and a kit targeting human desmosome glycoprotein 2. Specifically, the present invention uses eukaryotic cells to express and purify DSC2 protein, and uses it as an antigen to immunize mice and rabbits. Blood is collected from mice regularly, and the spleen of the mouse with the best titer is taken and fused with myeloma cells for hybridoma screening and affinity determination. The best rabbit spleen is taken, and the total RNA of the spleen cells is extracted for reverse transcription into cDNA, which is used as a template for amplification to obtain the Fab gene fragment, and is connected to the phage plasmid, and an anti-DSC2 phage antibody library is constructed by electric shock, and panning and sequencing are performed. Mouse antibodies and rabbit antibodies are subjected to affinity screening and epitope competition tests, paired antibodies are screened, and an ELISA kit is constructed to achieve high-sensitivity detection of DSC2.

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

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

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

[0008] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO:41, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:42, the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:43, the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:46, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:47, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:48.

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

[0010] In some embodiments of the invention, the amino acid sequence of the light chain variable region is as shown 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 to SEQ ID NO: 16, 24 or 48;

[0011] And / or, the amino acid sequence of the heavy chain variable region is as shown 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 present invention, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:12.

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

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

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

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

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

[0018] In some specific embodiments of the present invention, the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO:2, and the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO:1.

[0019] In some specific embodiments of the present invention, the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:3.

[0020] In some specific embodiments of the present invention, the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:50, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:45.

[0021] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody according to the first aspect of the present invention.

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

[0023] In some specific embodiments of the present invention, the nucleotide sequence of the light chain of the antibody is shown as SEQ ID NO:6, and the nucleotide sequence of the heavy chain of the antibody is shown as SEQ ID NO:5.

[0024] In some specific embodiments of the present invention, the nucleotide sequence of the light chain of the antibody is shown as SEQ ID NO:8, and the nucleotide sequence of the heavy chain of the antibody is shown as SEQ ID NO:7.

[0025] In some specific embodiments of the present invention, the nucleotide sequence of the light chain of the antibody is shown as SEQ ID NO:60, and the nucleotide sequence of the heavy chain of the antibody is shown as SEQ ID NO:55.

[0026] The third aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid as described in the second aspect of the present invention.

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

[0028] The fourth aspect of the present invention provides a transformant, which comprises the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0029] In some embodiments of the present invention, the host cell is a yeast cell or a mammalian cell, such as a HEK293 cell or a CHO cell.

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

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

[0032] The transformant according to the fourth aspect of the present invention is cultured, and the antibody is obtained from the culture.

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

[0034] The seventh aspect of the present invention provides a kit for detecting human desmoglein 2, the kit comprising the antibody as described in the first aspect of the present invention.

[0035] In some embodiments of the present invention, the kit further comprises other reagents for detecting human desmoglein 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 invention, the kit detects human desmoglein 2 based on a double antibody sandwich ELISA, which includes a capture antibody and a detection antibody; the capture antibody and / or the detection antibody is the antibody as described in the first aspect of the present invention, and the amino acid sequences of the two are different.

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

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

[0039] In some embodiments of the present invention, 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; the enzyme is preferably horseradish peroxidase, and the substrate is preferably TMB.

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

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

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

[0043] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0045] The positive and progressive effects of the present invention are:

[0046] The antibody and kit for detecting human desmocollin-2 (DSC2 / Desmocollin-2) according to the present invention can achieve highly sensitive quantitative detection of DSC2, which is not only helpful for basic research, but also can provide valuable reference information for clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0048] Figure 2 This is the non-reduced SDS-PAGE electrophoresis of DSC2 mouse antibodies H3 and H11.

[0049] Figure 3This is the reduced SDS-PAGE electrophoresis of DSC2 mouse antibody H3 and H11.

[0050] Figure 4 This is the binding curve of DSC2 mouse antibody H3 and H11.

[0051] Figure 5 This is the SDS-PAGE electrophoresis diagram of the non-reduced and reduced forms of the DSC2 rabbit antibody R3.

[0052] Figure 6 This is the binding curve of DSC2 rabbit antibody R3.

[0053] Figure 7 It is a calibration curve diagram of a human DSC2 protein detection kit for H3-R3 pairing provided in Example 4 of the present invention.

[0054] Figure 8 A calibration curve diagram of a human DSC2 protein detection kit for H3-H11 pairing provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0055] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

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

[0057] (1) Main reagents

[0058] Sheep anti-mouse secondary antibody-HRP, Luoyang Bio-Tong Experimental Material Center; donkey anti-rabbit IgG (H+L), Jackson; fetal bovine serum, Biosun; 50*HAT, Sigma; 50*HT, Sigma; Hybridoma supplementary factor, Bio-Long; DMEM, Shanghai Yuanpei; Trizol, Ambion; chloroform, SCR; DEPC water, Switzerland; PrimeScript RTase 6110A-1, TaKaRa.

[0059] (2) Main instruments

[0060] Microplate 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] This embodiment provides a monoclonal antibody combination for detecting human desmocollin-2 (DSC2 / Desmocollin-2), including 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 amino acid sequence of monoclonal antibody #R3 is shown in Table 4. The complementarity determining region (CDR) amino acid sequences in the table are all shown in accordance with the definition of the IMGT numbering rules.

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

[0065]

[0066]

[0067] Table 2 DNA sequence of antibody #H3

[0068]

[0069]

[0070]

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

[0072]

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

[0074]

[0075]

[0076]

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

[0078]

[0079]

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

[0081]

[0082]

[0083]

[0084] Example 2 Preparation of mouse monoclonal antibodies for detecting DSC2

[0085] 1. Selection of immunogens

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

[0087] >DSC2(Human):RWAPIPCSMLENSLGPFPLFLQQVQSDTAQNYTI YYSIRGPGVDQEPRNLFYVERDTGNLYCTRPVDREQYESFEIIAFATTPDGYTPELPLPLIIKIEDENDNYPIFTEETYTFTIFENCRVGTTVGQVCATDKDEPDTMHTRLKYSIIGQVPPSPTLFS MHPTTGVITTTSSQLDRELIDKYQLKIKVQDMDGQYFGLQTTSTCIINIDDVNDHLPTFTRTSYVTSVEENTVDVEILRVTVEDKDLVNTANWRANYTILKGNENGNFKIVTDAKTNEGVLCVVKPLN YEEKQQMILQIGVVNEAPFSREASPRSAMSTATVTVNVEDQDEGPECNPPIQTVRMKENAEVGTTSNGYKAYDPETRSSSGIRYKKLTDPTGWVTIDENTGSIKVFRSLDREAETIKNGIYNITVLA SDQGGRTCTGTLGIILQDVNDNSPFIPKKTVIICKPTMSSAEIVAVDPDEPIHGPPFDFSLESSTSEVQRMWRLKAINDTAARLSYQNDPPFGSYVVPITVRDRLGMSSVTSLDVTLCDCITENDCTH RVDPR(SEQ ID NO:61)

[0088] 2. Mouse Immunization and Antibody Screening

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

[0090] 1. Protein Immunization:

[0091] Wipe the needle of the emulsifier twice with 75% alcohol (use a new cotton ball for the second time). The range of the single emulsified liquid is about 250μl to 20ml. Dilute the DSC2 protein to 2mg / mL, take 400μg protein and 400μL complete adjuvant or incomplete adjuvant, and use an emulsifier to emulsify, from top to bottom as a whole, and swing up and down locally. Generally, 1-10ml of emulsification volume can be emulsified in 3-5 minutes, and no more than 10 minutes at most.

[0092] Administration method: subcutaneous multiple-point immunization, and impact immunization is muscle immunization.

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

[0094] Quantity: 2 for control, 2 for sample for hybridoma screening, 3 for alternative. Immunization dose: 100 μg / mouse / time, every 2 weeks.

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

[0096] After the third immunization, blood was collected at intervals of one week to determine the mouse serum antibody titer. The specific method is as follows:

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

[0098] (2) Wash the plate 4 times, adding 300 μl 1×PBST each time. Shake off as much as possible and blot on absorbent paper for the last wash. Add 200 μl 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 four times, adding 300 μl 1× PBST each time. After the last wash, shake off as much as possible and blot on absorbent paper.

[0100] (4) Use the NAYO 96-well pipette station to transfer cell supernatant or diluted serum to a blocked 96-well plate, 100 μL per well, and add positive and negative controls. Incubate at 37°C in the dark for 1 h.

[0101] (5) Wash the plate 4 times, adding 300 μl 1× PBST each time. Shake off as much as possible and blot on absorbent paper for the last wash. Add 100 μl 1× secondary antibody (10,000-20,000 times dilution) to each well. Incubate at 37°C in the dark for 1 h.

[0102] (6) Wash the plate 4 times, adding 300 μl 1× PBST each time. Drain and blot on absorbent paper for the last wash. Add 50 μl TMB colorimetric solution (the colorimetric solution itself is colorless and cannot be used if it changes color). Incubate at 37°C in the dark for 10-15 minutes, add 100 μl stop solution to terminate the reaction, and read the OD value at 450 nm.

[0103] like Figure 1 As shown, 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] Take out the fusion buffer in advance and let it return to room temperature. Wash the mixed cells twice with DMEM medium by centrifugation, and wash them with electrofusion buffer for the last time. After washing, fuse B cells and myeloma cells, and calculate the total amount of cells required according to experimental needs. Add fusion buffer that has been placed at room temperature and dilute the cell concentration to 1×10 7 cells / mL. Add the mixed cells to a 2ml or 9ml fusion pool, cover it, and perform electrofusion. After electrofusion, gently add the cells to a conical tube containing 40ml of culture medium containing 20% ​​FBS and incubate at 37℃ for 60 minutes. After the incubation, gently dilute the cells in the conical tube and plate them.

[0106] 4. Positive hybridoma cell screening

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

[0108] 5. Hybridoma Cell Subcloning

[0109] For the positive wells initially identified as positive, expand the cells and freeze them. When the remaining cells grow to nearly 80%, gently resuspend them and count the live cells. Take 5,000 live cells and transfer them into a centrifuge tube containing 5 mL of DMEM complete medium containing 1×HAT (H0262, Sigma) and 20% FBS (do not change the medium composition during the subcloning process). At this time, the cell concentration is 1,000 cells / ml. Add 200 μL of the diluted cell suspension to the first row, perform a 2-fold gradient dilution in sequence, and finally add 200 μL of cell culture medium to each cell well.

[0110] 6. Antibody Sequencing

[0111] Extract the mRNA of hybridoma cells that can secrete monoclonal antibodies, reverse transcribe and synthesize cDNA, select 10 heavy chain variable region primers and 12 light chain variable region primers for PCR experiment, obtain the band of correct size, send the correct band to the company for sequencing, and analyze and compare the sequencing results.

[0112] 7. Antibody Preparation and Affinity Determination

[0113] (1) Ascites antibody preparation

[0114] Balb / c mice were sensitized by intraperitoneal injection of 0.3-0.5 ml liquid paraffin for 7-15 days; the hybridoma cells to be injected were blown up, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; 1 ml DMEM was added to resuspend and the cells were counted; the cells were diluted to 2×10 6 / ml; use a 1ml syringe to draw 1ml of cells, and inject 0.5ml per mouse for about 10 6 cells. Specific injection method: pinch the mouse's tail and rotate it for a few weeks to reduce the mouse's activity, pinch the mouse's ears and back of the neck with the left hand, clamp the mouse's tail with the ring finger and the little finger, let the mouse unfold naturally, wipe the mouse's abdomen with an alcohol cotton ball, insert the syringe slightly to the right between the mouse's legs, let the mouse tilt downward at 45 degrees to inject the cells, rotate half a circle to pull out the needle, observe the mouse's condition, put it in a cage with new bedding, and mark it. Observe the growth status of the mouse and whether the abdomen becomes larger for 7-10 days. Mice with enlarged abdomen are dislocated and killed, ascites are collected, centrifuged at 12000rpm for 10 minutes, the supernatant is taken, and precipitated with an equal volume of saturated ammonium sulfate. Redissolve with an equal volume of 1×PBS, dialyze 3 times, and measure the concentration with nanodrop.

[0115] Resuspend the magnetic beads by shaking or vortexing; take an appropriate amount of magnetic beads (magnetic bead load 25mg / 4ml) and put them into a 50ml centrifuge tube, place it on the magnetic base of the magnetic stand, and 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, and discard the supernatant; repeat washing once, add an equal volume of PBST to resuspend for use; add ascites antibody to the resuspended magnetic beads, rotate at 4℃ overnight; place it on the magnetic base of the magnetic stand, discard the supernatant or save it for use; wash three times with PBS, discard the supernatant, and use a 1ml pipette tip to suck it clean for the last time; add an equal volume of Add elution buffer and incubate at room temperature for 10 minutes, resuspending 2-3 times during the process; place on the magnetic base of the magnetic stand, collect the liquid into a clean 50ml centrifuge tube, and repeat the elution once; add neutralizing solution to the centrifuge tube at 100μl / ml and mix gently; place the centrifuge tube on the magnetic base to adsorb the magnetic beads, transfer the supernatant to the prepared EP tube with a 1ml gun, and measure the concentration; dialyze three times with 1×PBS, change the liquid every 4h, aspirate the dialyzed liquid, or change the liquid by ultrafiltration and concentration, and measure the concentration; wash the magnetic beads 3 times with PBS, resuspend the magnetic beads with 20% ethanol, and store at 4℃.

[0116] (2) Antibody SDS-PAGE

[0117] Take 5μg of the sample to be tested into a 1.5ml centrifuge tube, add water to the non-reduced and reduced samples to 9μl, add 3μl 4×LDS Buffer (NP0008, Thermo) to the non-reduced sample, and add 3μl 4×LDS Buffer (containing 10mM DTT) to the reduced sample. After mixing, boil the reduced sample at 95℃ for 10min. Take a piece of protein electrophoresis gel, tear off the protective film, insert the side with the switch, insert a baffle on the other side, turn off both switches at the same time, put it into the electrophoresis tank, add the newly prepared 1×MOPS buffer to the electrophoresis tank, and add 1×MOPS recovery solution to the outside. Unplug the comb, add 5μl protein marker to the first column, and then add the prepared sample to be tested. Note that the sample is added to the center of the well. Cover the lid, pay attention to the positive and negative poles, and stop the electrophoresis band at 170V for 55min until it runs below the red. Pour MOPS into the MOPS recovery liquid bottle, turn on the switch and take out the protein gel, pry it open with scissors, be careful to avoid the gel from drying, cut off the sample part with a comb, transfer the protein gel to clean water, boil, and repeat three times. Transfer the treated gel to Coomassie brilliant blue solution, boil, shake on a shaker for 5-30 minutes, change to clean water to de-brighten, until clear bands are visible.

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

[0119] (3) Antibody binding curve determination

[0120] Prepare an antigen dilution with a final concentration of 1 μg / ml, mix well, and 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 block 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 the primary antibody incubation is completed, wash the plate three times with 0.05% PBST, add 20,000-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 in the dark for 5-10 minutes, add 100 μl 1M concentrated sulfuric acid to stop, and read the absorbance at 450nm.

[0121] Figure 4 This is the H3 and H11 antibody binding curve determination in this example. The positive control antibody (10809-MM04, Sino Biological) was diluted 1,000 times and used. As shown in the figure, H3 and H11 bind strongly to the DSC2 protein.

[0122] (4) Antibody affinity determination

[0123] According to the ELISA results, select the appropriate chip, and use the manual mode to explore the binding Ru value of the antigen-antibody at a certain concentration. Use the anti-mouse antibody chip coupled with the Protein G chip or the CM5 chip, select the Multi-cycle kinetics affinity using GST Capture kit program, set the injection and dissociation time, flow rate, regeneration solution, Startup cycle number, analyte concentration gradient and sample position. In Biacore Insight EvaluationSoftware, 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 "Open" button in the lower right corner for analysis. The analysis software automatically fits the data and outputs the results. The results are shown in Table 7. The antibody affinity range is 3.0 to 5.2×10 -8 .

[0124] Table 7 DSC2 antibody affinity test

[0125]

[0126] (5) Antibody epitope competition and pairing

[0127] According to the affinity determination results, high affinity antibodies were selected and immobilized on the CM5 chip, and then the competing antibodies were premixed with the antigens, and then the competition analysis was performed. H10+: DSC2 mouse anti-H10 antibody was premixed with the antigen, Antigen: only the antigen was tested for binding, and the results are shown in Table 8. H3 and H11 did not compete.

[0128] Table 8 Antibody Competition Ru Values

[0129]

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

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

[0132] 1. Protein Immunization

[0133] Wipe the needle of the emulsifier twice with 75% alcohol (use a new cotton ball for the second time). The range of the single emulsified liquid volume is about 250μl~20ml. Dilute the DSC2 protein to 2mg / mL, take 1.2mg protein and 600μL complete adjuvant or incomplete adjuvant, and use an emulsifier to emulsify, moving from top to bottom as a whole and swinging up and down locally. Generally, 1-10ml of emulsification volume can be emulsified in 3-5 minutes, and should not exceed 10 minutes at most.

[0134] Administration method: subcutaneous multiple-point immunization, and impact immunization is intravenous injection.

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

[0136] Quantity: 2 controls, immunization dose: 500 μg / mouse / time, two weeks apart.

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

[0138] After the fourth immunization of DSC2, blood was collected at intervals of one week to determine the rabbit serum antibody titer. The specific method was the same as the mouse serum titer determination method in Example 2.

[0139] 3. Immune library construction

[0140] Total RNA from rabbit spleen cells was extracted and reverse transcribed into cDNA. The variable regions of heavy and light chains were amplified using cDNA as template, connected into scfv with linkers and then connected to the phagemid vector. A 9-fold immune library was constructed by electroporation and packaged into scfv-phage for screening.

[0141] 4. Panning of phage library

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

[0143] 5. Screening of single positive phage

[0144] The selected phage enrichment library was used to pick single clones one by one and packaged into scFv-phage in a 96-well round-bottom deep-well plate. Then, ELISA was performed with phage, and the positive wells were specific antibodies against the DSC2 antigen. The corresponding antibody R3 variable region sequence was obtained by sending the bacterial solution to the second-generation sequencing.

[0145] 6. Antibody cloning construction and plasmid preparation

[0146] Primers were designed to amplify the heavy and light chain variable regions and loaded into a vector carrying the heavy and light chain constant regions of mouse antibodies to construct a full antibody molecule, and a plasmid was prepared using a kit (MN740410.100).

[0147] 7. Antibody Preparation and Affinity Determination

[0148] (1) Preparation of recombinant rabbit antibodies and determination of binding curves

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

[0150] Resuspend the magnetic beads by shaking or vortexing; take an appropriate amount of magnetic beads (magnetic bead load 25mg / 4ml) and put them into a 50ml centrifuge tube, place it on the magnetic base of the magnetic stand, and 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, and discard the supernatant; repeat washing once, add an equal volume of PBST to resuspend for use; add the expression supernatant to the resuspended magnetic beads, rotate at 4℃ overnight; place it on the magnetic base of the magnetic stand, discard the supernatant or save it for use; wash three times with PBS, discard the supernatant, and finally Aspirate clean with a 1ml pipette tip once; add an equal volume of elution buffer, incubate at room temperature for 15 minutes, and resuspend 2-3 times during this period; place on the magnetic base of the magnetic stand, collect the liquid into a clean 50ml centrifuge tube, and repeat the elution once; add neutralizing solution to the centrifuge tube at 100μl / ml and mix gently; place the centrifuge tube on the magnetic base to adsorb the magnetic beads, transfer the supernatant to the prepared EP tube with a 1ml gun, and measure the concentration; concentrate the antibody with an ultrafiltration tube, replace 1×PBS 3 times, aspirate to measure the concentration, and store in aliquots at -20℃.

[0151] (2) Antibody SDS-PAGE

[0152] Take 5μg of the sample to be tested into a 1.5ml centrifuge tube, add water to the non-reduced and reduced samples to 9μl, add 3μl 4×LDS Buffer (NP0008, Thermo) to the non-reduced sample, and add 3μl 4×LDS Buffer (containing 10mM DTT) to the reduced sample. After mixing, boil the reduced sample at 95℃ for 10min. Take a piece of protein electrophoresis gel, tear off the protective film, insert the side with the switch, insert a baffle on the other side, turn off both switches at the same time, put it into the electrophoresis tank, add the newly prepared 1×MOPS buffer to the electrophoresis tank, and add 1×MOPS recovery solution to the outside. Unplug the comb, add 5μl protein marker to the first column, and then add the prepared sample to be tested. Note that the sample is added to the center of the well. Cover the lid, pay attention to the positive and negative poles, and stop the electrophoresis band at 170V for 55min until it runs below the red. Pour MOPS into the MOPS recovery liquid bottle, turn on the switch and take out the protein gel, pry it open with scissors, be careful to avoid the gel from drying, cut off the sample part with a comb, transfer the protein gel to clean water, boil, and repeat three times. Transfer the treated gel to Coomassie brilliant blue solution, boil, shake on a shaker for 5-30 minutes, change to clean water to deblur, until clear bands are visible. The R3 antibody SDS-PAGE results are as follows Figure 5 shown.

[0153] (3) Antibody binding curve determination

[0154] Prepare an antigen dilution with a final concentration of 1μg / ml, mix well, and use a multichannel pipette to add 100μl to each well of a 96-well plate, 100ng / 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 block 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 the primary antibody incubation is completed, wash the plate three times with 0.05% PBST, add 20,000-fold diluted goat anti-rabbit 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 50μL TMB, incubate at room temperature in the dark for 5-10 minutes, add 100uL 1M concentrated sulfuric acid to terminate, and read the absorbance at 450nm. The R3 antibody binding curve results are as follows Figure 6 As shown; wherein, Sino-Ab is a positive control (Sino Biological, 10809-MM04).

[0155] The affinity determination method is the same as the antibody affinity determination method in Example 2. The affinity of DSC2 rabbit antibody 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 and rabbit anti-epitope competition 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 premixed with the antigens, and then the competition analysis was performed. R3+: DSC2 rabbit anti-R3 antibody was premixed with the antigen, Antigen: only the antigen was tested for binding, and the results are shown in Table 10. There was no competition between H3 and R3.

[0160] Table 10 Mouse and rabbit anti-epitope competition test

[0161] aisle Immobilized Antibodies 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 use of a human desmocollin-2 (DSC2 / Desmocollin-2) monoclonal antibody in an immunological tool: the immunological detection tool includes a detection kit.

[0164] 1. Human Desmoglein 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 microporous surface 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. Then, the enzyme-labeled second antibody is added and combined with the detection antibody, and finally the TMB substrate is used for color development. The depth of the color in the microplate is positively correlated with the concentration of the DSC2 protein. The concentration of DSC2 can be quantitatively detected by reading at 450nm with an enzyme reader.

[0166] 2.H3-R3 kit

[0167] Including H3, R3, NEST detachable Elisa plates, dilution buffer, Donkey anti-rabbit HRP (H+L), etc.

[0168] Linear detection: The concentrations of human desmocollin 2 (DSC2 / Desmocollin-2) calibrators were 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, and 5 ng / ml. The calibrators were added to a 96-well plate and each concentration was measured in parallel 3 times. After 30 minutes of reaction, the reaction signal intensity was detected by an ELISA instrument to obtain the absorbance signal value corresponding to each calibrator, as shown in Table 11.

[0169] Table 11 Absorbance signal values ​​corresponding to the calibration products

[0170]

[0171] The calibration curve is drawn by linear fitting based on the concentration of the calibration substance and its corresponding absorbance signal value. Figure 7 As shown, R 2 =1, indicating that in the range of 0.078-5ng / ml, the absorbance signal value is highly linearly correlated with the detection concentration.

[0172] 3. Instructions for use of the H3-R3 kit:

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

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

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

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

[0177] (5) Add the standard and test samples to the appropriate wells, cover the wells, and incubate with gentle shaking at 25°C for 2.5 hours.

[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 wells, and incubate at 25°C with gentle shaking for 1 hour.

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

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

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

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

[0184] (12) Add 50 μl of 0.2 M sulfuric acid to each well to stop color development, and then immediately read the sample at 450 nm using an ELISA reader.

[0185] 4.H3-H11 kit

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

[0187] Linearity detection: The concentrations of human desmocollin 2 (DSC2 / Desmocollin-2) calibrators were 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 calibrators were added to a 96-well plate, and each concentration was measured in parallel 3 times. After 30 minutes of reaction, the reaction signal intensity was detected by an ELISA instrument to obtain the absorbance signal value corresponding to each calibrator, as shown in Table 12.

[0188] Table 12 Absorbance signal values ​​corresponding to the calibration products

[0189]

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

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

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

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

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

[0195] (4) Add 100 μl of Reaction Buffer and 8 μl of NH2-Reactive Biotin solution to the filter tube and pipette to mix.

[0196] (5) Place the filter tube in an incubator and incubate at 37°C for 10 min.

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

[0198] (7) Add 200 μl of WS Buffer to the filter tube and centrifuge at 8000-10000 g for 10 min. Repeat this step once.

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

[0200] 6. Instructions for use of H3-H11 kit:

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

[0202] (2) Discard the solution in the wells and wash 4 times with 1×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 wells, and incubate at 37°C for 2 hours.

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

[0205] (5) Add the standard and test samples to the appropriate wells, cover the wells, and incubate with gentle shaking at 25°C for 2.5 hours.

[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 wells, and incubate at 25°C with gentle shaking for 1 hour.

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

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

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

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

[0212] (12) Add 50 μl of 0.2 M sulfuric acid to each well to stop color development, and then immediately read the sample at 450 nm using an ELISA reader.

[0213] 7. Comparison of sensitivity between H3-R3 kit and H3-H11 kit

[0214] Reagent test 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 Desmocollin-2 (DSC2 / Desmocollin-2) H3-R3 Kit

[0217] 1. Sensitivity

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

[0219] 2. Accuracy

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

[0221] 3. Linear range

[0222] The H3-R3 test kit of Example 4 has a correlation coefficient of ≥0.99 within the range of 0.078 ng / ml to 5 ng / ml.

[0223] In summary, it can be determined that the performance of this 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, wherein 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, characterized in that: The amino acid sequence of the CDR-H1 is shown in SEQ ID NO:9, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:10, the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:11, the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:13, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:14, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:15; or The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 17, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 18, the amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 19, the amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 21, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 22, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 23; or The amino acid sequence of the CDR-H1 is shown in SEQ ID NO:41, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:42, the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:43, the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:46, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:47, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

48.

2. The antibody according to claim 1, characterized in that The framework region of the light chain variable region and / or the heavy chain variable region is a human, mouse or rabbit framework region; Preferably, the amino acid sequence of the light chain variable region is as shown 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; and / or, the amino acid sequence of the heavy chain variable region is as shown 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 to SEQ ID NO: 12, 20 or 44; More preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 12; or The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20; or The amino acid sequence of the light chain variable region is shown in SEQ ID NO:48, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

44.

3. The antibody according to claim 1 or 2, characterized in that The antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv, and the Fv is preferably scFv; Preferably, the antibody is a full-length antibody; Further preferably, the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 2, 4 or 50; and / or, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 1, 3 or 45; More preferably, the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 1; or The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:3; or The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:50, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:

45.

4. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the antibody according to any one of claims 1 to 3; Preferably, the nucleotide sequence of the light chain of the antibody is as shown in SEQ ID NO: 6, 8 or 60; and / or, the nucleotide sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 5, 7 or 55; More preferably, the nucleotide sequence of the light chain of the antibody is as shown in SEQ ID NO:6, and the nucleotide sequence of the heavy chain of the antibody is as shown in SEQ ID NO:5; or The nucleotide sequence of the light chain of the antibody is shown in SEQ ID NO:8, and the nucleotide sequence of the heavy chain of the antibody is shown in SEQ ID NO:7; or The nucleotide sequence of the light chain of the antibody is shown in SEQ ID NO:60, and the nucleotide sequence of the heavy chain of the antibody is shown in SEQ ID NO:

55.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 4; Preferably, the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

6. A transformant, characterized in that The transformant comprises the isolated nucleic acid according to claim 4 or the recombinant expression vector according to claim 5, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is a yeast cell or a mammalian cell, such as a HEK293 cell or a CHO cell; More preferably, the host cell is a 293F cell.

7. A method for preparing an antibody targeting human desmoglein 2, characterized in that: The method comprises the following steps: The transformant according to claim 6 is cultured, and the antibody is obtained from the culture.

8. Use of the antibody according to any one of claims 1 to 3, the isolated nucleic acid according to claim 4, the recombinant expression vector according to claim 5 and / or the transformant according to claim 6 in the preparation of a kit for detecting human desmoglein 2.

9. A kit for detecting human desmoglein 2, characterized in that: The kit comprises the antibody according to any one of claims 1 to 3; Preferably, the kit further comprises other reagents for detecting human desmoglein 2 based on enzyme-linked immunosorbent assay, western blotting, immunohistochemistry, flow cytometry or protein chip technology.

10. The kit according to claim 9, characterized in that The kit is based on double antibody sandwich ELISA to detect human desmosome glycoprotein 2, which includes a capture antibody and a detection antibody; the capture antibody and / or the detection antibody is the antibody according to any one of claims 1 to 3, and the amino acid sequences of the two are different; Preferably, the amino acid sequence of the light chain of the capture antibody is as shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain of the capture antibody is as shown in SEQ ID NO: 1; and / or, The amino acid sequence of the light chain of the detection antibody is shown in SEQ ID NO:4, and the amino acid sequence of the heavy chain of the detection antibody is shown in SEQ ID NO:3; or, the amino acid sequence of the light chain of the detection antibody is shown in SEQ ID NO:50, and the amino acid sequence of the heavy chain of the detection antibody is shown in SEQ ID NO:45; optionally, the detection antibody is a biotinylated antibody; More preferably, 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; the enzyme is preferably horseradish peroxidase, and the substrate is preferably TMB.

11. A method for detecting human desmoglein 2, characterized in that: The method comprises contacting the antibody according to any one of claims 1 to 3 or the kit according to claim 9 or 10 with a sample to be tested, and detecting the expression level of human desmoglein 2 in the sample to be tested; Preferably, the method is for non-diagnostic purposes; and / or, The added mass ratio of the capture antibody to the detection antibody is 5:(1-5), such as 5:2 or 5:3.

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