Antibody and kit for detecting human DSC2 protein

Abstract

The invention discloses an antibody and a kit for detecting human DSC2 protein. The antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 of which the amino acid sequences are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3 of which the amino acid sequences are respectively shown as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The antibody is high in affinity, and a kit adopting the antibody can realize high-sensitivity quantitative detection on DSC2, so that basic research is facilitated, and valuable reference information can be provided 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 reagent kit for detecting human DSC2 protein. Background Art

[0002] Desmocollin-2 (DSC2) is an intercellular adhesion protein that belongs to the glycoprotein family. It plays a role in connecting cells on the cell membrane surface and is a key factor in cell adhesion. The main structure of DSC2 includes a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain. This protein is expressed in cardiomyocytes, and its main function is to maintain the connection between cardiomyocytes and maintain the stable function of the heart. DSC2 deletion or mutation may lead to disorder of intercellular connections, affect the stable function of cardiomyocytes, and even cause heart disease. Therefore, it is of great significance to immunize animals with DSC2 protein to obtain antibodies targeting human DSC2 protein, and then develop a kit for detecting DSC2 (such as an ELISA kit) for the detection of DSC2 protein in clinical patients. Summary of the invention

[0003] The present invention has been made to solve the above-mentioned problems and provides an antibody and a kit for detecting human DSC2 protein.

[0004] Specifically, the present invention uses eukaryotic cells to express and purify DSC2 protein, and uses it as an antigen to immunize rabbits. Rabbits are regularly blooded, the spleen of the rabbit with the best titer is taken, RNA is extracted, a phage immune library is constructed, and antibody screening is performed. The antibody titer and immune effect are determined by indirect ELISA; the spleen of the rabbit with the best titer is taken, the total RNA of the spleen cells is extracted and reverse transcribed into cDNA, and the heavy and light chain variable region VH VL gene fragments are amplified using this as a template, and connected to Scfv through a linker (G4S). After the Scfv gene fragment and the phage plasmid are connected, an anti-Scfv phage antibody library is constructed by electric shock, and panning is performed to enrich the phage with high affinity, and finally the antibody against DSC2 is expressed and purified after recombinant.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The first aspect of the present invention provides an antibody for detecting human DSC2 protein, which 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, characterized in that the amino acid sequence of the CDR-H1 is shown in SEQ ID NO:1, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:2, the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:3, the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:6, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:7, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:8.

[0007] 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.

[0008] In some embodiments of the invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 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:9;

[0009] And / or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 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:4.

[0010] In some specific embodiments of the present invention, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4.

[0011] 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.

[0012] In some embodiments of the invention, the antibody is a full-length antibody.

[0013] 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:10; and / or, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:5.

[0014] The second aspect of the present invention provides an isolated nucleic acid encoding the antibody according to the first aspect of the present invention.

[0015] In some embodiments of the present invention, the nucleotide sequence of the light chain of the antibody is shown in SEQ ID NO:20; and / or, the nucleotide sequence of the heavy chain of the antibody is shown in SEQ ID NO:15.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In some specific embodiments of the present invention, the host cell is a 293F cell.

[0021] The fifth aspect of the present invention provides a method for detecting antibodies against human DSC2 protein, the method comprising the following steps:

[0022] The transformant according to the fourth aspect of the present invention is cultured, and the antibody is obtained from the culture.

[0023] 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 or the transformant described in the fourth aspect of the present invention in the preparation of a kit for detecting human DSC2 protein.

[0024] The seventh aspect of the present invention provides a kit for detecting human DSC2 protein, the kit comprising the antibody as described in the first aspect of the present invention;

[0025] Preferably, the kit further comprises other reagents for detecting human DSC2 protein based on enzyme-linked immunosorbent assay (ELISA), Western Blot, immunohistochemistry (IHC), flow cytometry or protein chip technology.

[0026] In some embodiments of the present invention, the kit detects human DSC2 protein based on double antibody sandwich ELISA, which includes a capture antibody and a detection antibody; the capture antibody is the antibody as described in the first aspect of the present invention.

[0027] In some embodiments of the present invention, the detection antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region of the detection antibody comprises CDR-H1, CDR-H2 and CDR-H3 whose amino acid sequences are shown in SEQ ID NOs: 21, 22 and 23, respectively, and the light chain variable region of the detection antibody comprises CDR-L1, CDR-L2 and CDR-L3 whose amino acid sequences are shown in SEQ ID NOs: 26, 27 and 28, respectively.

[0028] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of the detection antibody is shown as SEQ ID NO:29, and the amino acid sequence of the heavy chain variable region of the detection antibody is shown as SEQ ID NO:24.

[0029] In some embodiments of the present invention, the amino acid sequence of the light chain of the detection antibody is shown in SEQ ID NO:30, and the amino acid sequence of the heavy chain of the detection antibody is shown in SEQ ID NO:25; the detection antibody is preferably an antibody labeled with horseradish peroxidase.

[0030] In some embodiments of the present invention, the capture antibody and the horseradish peroxidase are mixed at a mass ratio of (1-4):1 to prepare the horseradish peroxidase-labeled antibody.

[0031] The eighth aspect of the present invention provides a method for detecting human DSC2 protein, 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 DSC2 protein in the sample to be tested.

[0032] In some embodiments of the invention, the method is for non-diagnostic purposes.

[0033] In some embodiments of the present invention, the added mass ratio of the capture antibody to the detection antibody is (1-10):1, preferably (3-9):1, for example 8:1.

[0034] 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.

[0035] The reagents and raw materials used in the present invention are commercially available.

[0036] The positive and progressive effects of the present invention are:

[0037] The antibody and kit for detecting human DSC2 protein according to the present invention can realize 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

[0038] Figure 1 This is a graph showing the rabbit serum titer determination in Example 2 of the present invention.

[0039] Figure 2 It is the reducing and non-reducing SDS-PAGE electrophoresis diagram of the antibody of Example 2 of the present invention.

[0040] Figure 3 This is the antibody binding curve provided in Example 2 of the present invention.

[0041] Figure 4 It is a calibration curve diagram of a human DSC2 protein detection kit of the R2-R3 pairing provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0042] 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.

[0043] Reagents and instruments used in the following examples:

[0044] (1) Main reagents

[0045] PrimeScript 1st Strand cDNA Synthesis kit and LA Taq (5U / μl) were purchased from TaKaRa; TG1Electrocompetent Cells4 were purchased from Dakoway; M-280Streptavidin was purchased from Invitrogen; SfiI restriction endonuclease and T4 DNA ligase were purchased from NEB; M13KO7 helper phage was purchased from Chengdu Renyu Biotechnology; BSA was purchased from Shanghai Jieyi Biotechnology Co., Ltd.; goat anti-rabbit was purchased from Luoyang Bio-Tone Experimental Materials Center.

[0046] (2) Main instruments

[0047] The high-pressure steam sterilizer was purchased from TOMY, Japan; the varioskan lux microplate reader was purchased from Thermo; the Nanodrop one was purchased from Thermo Scientific; the electroporator BLO-RAD was purchased from BIO-RAD; the shaking incubator ZQZY-88AH was purchased from Shanghai Zhichu; the high-speed centrifuge LYNX6000 was purchased from Thermo Scientific; and the biological clean bench BCM-1300A-Ⅱ was purchased from Suzhou Antai Air Technology Co., Ltd.

[0048] Example 1

[0049] This embodiment provides a combination of human DSC2 protein antibodies, including antibody #R2 and antibody #R3.

[0050] The amino acid sequence of antibody #R2 is shown in Table 1; the DNA sequence of antibody #R2 is shown in Table 2. The following complementarity determining region (CDR) amino acid sequences are all shown in accordance with the definition of IMGT numbering rules.

[0051] Table 1 Amino acid sequence of antibody #R2

[0052]

[0053] Table 2 DNA sequence list of antibody #R2

[0054]

[0055]

[0056]

[0057] Table 3 Amino acid sequence of antibody #R3

[0058]

[0059] Table 4 DNA sequence list of antibody #R3

[0060]

[0061]

[0062]

[0063] Example 2 Preparation of DSC2 Rabbit Antibody

[0064] 1. Selection of immunogens

[0065] The amino acid sequence of the immunogen is as follows:

[0066] >DSC2(Human):RWAPIPCSMLENSLGPFPLFLQQVQSDTAQNYTI YYSIRGPGVDQEPRNLFYVERDTGNLYCTRPVDREQYESFEIIAFATTPDGYTPELPLPLIIKIEDENDNYPIFTEETYTFTIFENCRVGTTVGQVCATDKDEPDTMHTRLKYSIIGQVPPSPTLFSMH PTTGVITTTSSQLDRELIDKYQLKIKVQDMDGQYFGLQTTSTCIINIDDVNDHLPTFTRTSYVTSVEENTVDVEILRVTVEDKDLVNTANWRANYTILKGNENGNFKIVTDAKTNEGVLCVVKPLNYEEK QQMILQIGVVNEAPFSREASPRSAMSTATVTVNVEDQDEGPECNPPIQTVRMKENAEVGTTSNGYKAYDPETRSSSGIRYKKLTDPTGWVTIDENTGSIKVFRSLDREAETIKNGIYNITVLASDQGGRT CTGTLGIILQDVNDNSPFIPKKTVIICKPTMSSAEIVAVDPDEPIHGPPFDFSLESSTSEVQRMWRLKAINDTAARLSYQNDPPFGSYVVPITVRDRLGMSSVTSLDVTLCDCITENDCTHRVDPR(SEQ ID NO:41)

[0067] 2. Rabbit Immunization and Antibody Screening

[0068] Human DSC2 is used as a specific antigen to prepare rabbit antibodies, and the preparation method is as follows.

[0069] 1. Protein Immunization:

[0070] Wipe the needle of the emulsifier twice with 75% alcohol (use a new cotton ball for the second time). The range of emulsified liquid volume for a single time is about 250μl to 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.

[0071] Administration: Subcutaneous multiple-point immunization, and impact immunization is intravenous injection.

[0072] Strain: New Zealand white rabbit, 2-3 months old, 2-2.5 kg, female.

[0073] Quantity: 2 for control, 2 for sample for phage screening, 1 for backup. Immunization dose: 500 μg / mouse / time, every two weeks.

[0074] 2. Rabbit serum antibody titer and cell supernatant determination

[0075] After the fourth immunization of DSC2, blood was collected one week apart to determine the rabbit serum antibody titer. The specific method is as follows:

[0076] (1) Add 100 μL of DSC2 antigen (100 ng) to a 96-well plate for coating;

[0077] (2) Incubate overnight at 4°C in the dark;

[0078] (3) Wash the plate 4 times, adding 300 μL 1× PBST each time. After the last wash, shake off as much as possible and blot on absorbent paper.

[0079] (4) Add 200 μL of 5% BSA blocking solution to each well for blocking;

[0080] (5) Incubate at 37°C in the dark for 2 hours;

[0081] (6) 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.

[0082] (7) Use the NAYO 96-well pipetting station to dilute the serum or cell supernatant into the blocked 96-well plate, 100 μL per well. For positive quality control screening, add 1000-fold diluted positive serum (without control antibody) to the cell supernatant.

[0083] (8) Incubate at 37°C in the dark for 1 h;

[0084] (9) 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.

[0085] (10) Add 20,000-fold diluted goat anti-rabbit IgG H+L secondary antibody to each well;

[0086] (11) Incubate at 37°C in the dark for 1 h;

[0087] (12) 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.

[0088] (13) Add 100 μL of TMB colorimetric solution (the colorimetric solution itself is colorless and cannot be used if it changes color);

[0089] (14) Incubate at 37°C in the dark for 10-15 minutes, add 50 μL of stop solution to terminate the reaction, and read the OD value at 450 nm. The specific results are as follows: Figure 1 shown.

[0090] 3. Immune library construction

[0091] 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.

[0092] 4. Panning of Phage Library

[0093] 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.

[0094] 5. Screening of single positive phage

[0095] Monoclonal antibodies were selected one by one from the above panned phage enrichment library 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 sequence was obtained by sending the bacterial solution to the second-generation sequencing.

[0096] 6. Antibody cloning construction and plasmid preparation

[0097] 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).

[0098] 7. Antibody Preparation and Affinity Determination

[0099] (1) Preparation of recombinant rabbit antibodies and determination of binding curves

[0100] 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.

[0101] 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℃.

[0102] (2) Antibody SDS-PAGE

[0103] 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. The results are as follows Figure 2 shown.

[0104] (3) Antibody binding curve determination

[0105] 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 results are as follows Figure 3 As shown, sino-Ab (Sino Biological, 10809-MM04) is a positive control.

[0106] (4) Antibody affinity determination

[0107] 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 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 performs data fitting, and the output results are shown in Table 5.

[0108] Table 5 DSC2 antibody affinity determination

[0109] Antibody Substances analyzed ka(1 / Ms) kd(1 / s) KD(M) DSC2-R2 DSC2 3.35E+05 3.51E-03 1.02E-08 DSC2-R3 DSC2 1.79E+05 6.99E-04 3.90E-09

[0110] (5) Antibody epitope competition and pairing

[0111] According to the affinity determination results, anti-huamn IgG Fc was fixed on the CM5 chip, and then an antibody 1 was captured, and then the excess Fc was blocked. After that, antibody 1 was used as a negative, and antibody 2 and DSC2 premix were added for competition analysis. This method has a signal value for the presence of different epitope binding and no competition. The results are shown in Table 6. The epitopes of the two antibodies R2 and R3 (the specific sequences of these two antibodies are shown in Tables 1-4) are different.

[0112] Table 6 Epitope analysis of mouse and rabbit antibodies

[0113] Antibody Name Positive Negative R3 Antibody Name Positive Negative R2 R2 29 -5 12 R3 131 -3 113

[0114] Example 3

[0115] 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.

[0116] 1. Human Desmocollin-2 (DSC2 / Desmocollin-2) quantitative detection kit (double antibody sandwich quantitative method).

[0117] 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 human desmocollin 2 (DSC2 / Desmocollin-2). The capture antibody is pre-coated on the microporous surface of the microplate and selectively binds to human desmocollin 2 (DSC2 / Desmocollin-2). After washing, the horseradish peroxidase-labeled second antibody is added to bind to the second site of human desmocollin 2 (DSC2 / Desmocollin-2) to form a sandwich complex. Finally, TMB substrate is used directly for color development. The depth of color in the microplate is positively correlated with the concentration of human desmocollin 2 (DSC2 / Desmocollin-2). The concentration of human desmocollin 2 (DSC2 / Desmocollin-2) can be quantitatively detected by reading at 450nm with an enzyme reader.

[0118] 2. R2-R3 kit

[0119] It includes capture antibody R2, detection antibody horseradish peroxidase labeled R3, NEST detachable Elisa plate, dilution buffer, etc.

[0120] Linear detection: The concentrations of human desmocollin 2 (DSC2 / Desmocollin-2) calibrators are 0ng / ml, 0.078ng / ml, 0.156ng / ml, 0.312ng / ml, 0.625ng / ml, 1.25ng / ml, 2.5ng / ml, and 5ng / ml. The calibrators are added to a 96-well plate and each concentration is measured in parallel 3 times. After 30 minutes of reaction, the reaction signal intensity is detected by an ELISA instrument to obtain examples of the corrected absorbance signal values ​​corresponding to each calibrator, as shown in Table 7.

[0121] Table 7 Absorbance signal values ​​corresponding to the calibrators

[0122]

[0123] The calibration curve is drawn by linear fitting based on the concentration of the calibration substance and its corresponding absorbance signal value. Figure 4 As shown, R 2 =0.99, indicating that in the range of 0.078-5ng / ml, the absorbance signal value is highly linearly correlated with the detection concentration.

[0124] 3. Preparation of horseradish peroxidase labeled R3

[0125] Use HRP labeling kit Proteintech-PK20001.

[0126] Follow these steps to mark:

[0127] 1) Take out the HRP labeling kit from -20℃ and equilibrate it at room temperature for 30 minutes to fully thaw the reaction start solution and the reaction stop solution and mix them evenly.

[0128] 2) Add 1 μl of reaction start solution to every 10 μl of antibody or other protein molecule solution to be labeled, and mix thoroughly by pipetting several times to avoid bubbles. For example: add 170 μl of 1×PBS to label 400 μg of R3 (30 μl), mix well, then add 20 μl of start solution and pipette to mix well.

[0129] 3) Open the cap of the horseradish peroxidase tube (bottle), add the activated antibody or other protein solution directly into the tube (bottle), pipette repeatedly to mix well to avoid bubbles, and place at room temperature for 3 hours.

[0130] 4) Add the reaction stop solution to the horseradish peroxidase reaction tube (bottle) at a ratio of 1 μl of the reaction stop solution for every 10 μl of the antibody or other protein solution, mix thoroughly, and leave at room temperature for 1 hour.

[0131] 5) After termination, add an equal volume of product protection solution, mix thoroughly, and store at -20°C. (This step is not required when the antibody or other protein solution to be labeled already contains 50% glycerol) Mark the concentration date and store in a -20°C refrigerator.

[0132] Notice:

[0133] a) The titer of the antibody to be labeled should be above 1:20.

[0134] b) Antibody buffer: PBS with a concentration of 0.01M and a pH of 7.4 is preferred, preferably free of glycerol, sodium azide, and amino substances (including glycine, Tris, etc.). However, a small amount of amino substances (<0.05M) and a small amount of sodium azide (<0.02%) will not have a serious impact on the labeling results. If the antibody to be labeled contains substances higher than the above concentration, it needs to be fully dialyzed with the above PBS buffer solution.

[0135] c) Antibody dosage: The concentration should be between 0.5-5 mg / ml. For specific dosage, refer to Table 8 below:

[0136] Table 8 Relationship between labeling amount and volume of corresponding antibodies or other protein molecules

[0137]

[0138] d) The amount of antibody can be adjusted according to the experimental needs. Within the corresponding HRP dosage range, the less the amount of antibody, the more efficient the HRP labeling.

[0139] e) HRP is ready to use after it is dissolved. It is not recommended to dissolve HRP and then use it in aliquots.

[0140] 4. Instructions for use of the R2-R3 kit:

[0141] 4.1 Reagent preparation

[0142] (1) All reagents were equilibrated to room temperature (18-25°C) before use.

[0143] (2) Capture Antibody R2 Solution The R2 protein was diluted to 2 μg / ml using 1× PBS.

[0144] (3)DSC2 standard protein should be diluted with 1% casein PBS solution and used freshly prepared.

[0145] First, the DSC2 standard was diluted to a 10 ng / ml standard stock solution using a 1% casein PBS solution (or the kit standard stock solution was directly used), and then two-fold serial gradient dilutions were performed, see Table 9 below.

[0146] Table 9 Dilution concentration settings

[0147]

[0148] (4) The detection antibody R3-HRP should be diluted to 0.25 μg / ml with 1% casein PBS solution before use.

[0149] 4.2 Operating procedures

[0150] (1) Add 100 μl / well of capture antibody R2 solution (2 μg / ml) to appropriate wells, cover the wells, and incubate at 4°C for 16 hours.

[0151] (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.

[0152] (3) Add 200 μl of 1% casein PBS solution to each well, cover the wells, and incubate at 37°C for 2 hours.

[0153] (4) Wash according to step (2).

[0154] (5) Add 100 μl / well of the standard and test samples to the appropriate wells, cover the wells, and incubate at 25°C with gentle shaking for 2.0 hours.

[0155] (6) Wash according to step (2).

[0156] (7) Add 100 μl / well of the detection antibody horseradish peroxidase-labeled R3 solution to each well, cover the wells, and incubate at 25°C with gentle shaking for 1 hour.

[0157] (8) Wash twice according to step (2).

[0158] (9) Add TMB substrate solution to each well at a volume of 100 μl / well and incubate at 25°C for 30 minutes in a dark environment with gentle shaking.

[0159] (10) 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.

[0160] Example 4 Performance Evaluation of Human Desmocollin-2 (DSC2 / Desmocollin-2) H3-R3 Kit

[0161] 1. Sensitivity

[0162] The minimum detection value of the R2-R3 kit of Example 3 is 0.078 ng / ml, indicating that the kit has a high sensitivity.

[0163] 2. Accuracy

[0164] The recovery rate of the R2-R3 kit of Example 3 is 97-111%, indicating that the kit has high accuracy.

[0165] 3. Linear range

[0166] The R2-R3 test kit of Example 3 has a correlation coefficient of ≥0.99 within the range of 0.078 ng / ml to 5 ng / ml.

[0167] In summary, it can be determined that the performance of the kit of the present invention is good, and it has the advantages of high sensitivity, strong specificity and high accuracy.

Claims

1. An antibody for detecting human DSC2 protein, 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:1, the amino acid sequence of the CDR-H2 is shown in SEQ ID NO:2, the amino acid sequence of the CDR-H3 is shown in SEQ ID NO:3, the amino acid sequence of the CDR-L1 is shown in SEQ ID NO:6, the amino acid sequence of the CDR-L2 is shown in SEQ ID NO:7, and the amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

8.

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:9 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:9; and / or, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 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:4; More preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

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; More preferably, the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:10; and / or, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:

5.

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:20; and / or, the nucleotide sequence of the heavy chain of the antibody is as shown in SEQ ID NO:

15.

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 detecting antibodies against human DSC2 protein, 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 or the transformant according to claim 6 in the preparation of a kit for detecting human DSC2 protein.

9. A kit for detecting human DSC2 protein, 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 DSC2 protein based on enzyme-linked immunosorbent assay, protein 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 DSC2 protein, and comprises a capture antibody and a detection antibody; the capture antibody is the antibody according to any one of claims 1 to 3; Preferably, the detection antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region of the detection antibody comprises CDR-H1, CDR-H2 and CDR-H3 whose amino acid sequences are shown in SEQ ID NOs: 21, 22 and 23, respectively, and the light chain variable region of the detection antibody comprises CDR-L1, CDR-L2 and CDR-L3 whose amino acid sequences are shown in SEQ ID NOs: 26, 27 and 28, respectively; Further preferably, the amino acid sequence of the light chain variable region of the detection antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the heavy chain variable region of the detection antibody is shown in SEQ ID NO: 24; More preferably, the amino acid sequence of the light chain of the detection antibody is as shown in SEQ ID NO:30, and the amino acid sequence of the heavy chain of the detection antibody is as shown in SEQ ID NO:25; the detection antibody is preferably an antibody labeled with horseradish peroxidase.

11. A method for detecting human DSC2 protein, 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 DSC2 protein 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 (1-10):1, preferably (3-9):1, for example 8:1.

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