Antibodies and kits for detecting human dsc2 protein

Abstract

The application 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, wherein the light chain variable region comprises CDR-H1, CDR-H2 and CDR-H3 with the amino acid sequences shown in SEQ ID NO: 1, 2 and 3 respectively, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3 with the amino acid sequences shown in SEQ ID NO: 6, 7 and 8 respectively. The antibody has high affinity, and the kit adopting the antibody can realize high-sensitivity quantitative detection of DSC2, which is not only helpful to basic research, but also can provide valuable reference information for clinical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to antibodies and kits for detecting human DSC2 protein. Background Technology

[0002] Desmocollin-2 (DSC2) is an intercellular adhesion protein belonging to the glycoprotein family. It plays a crucial role in cell adhesion by connecting cells on the cell membrane surface. 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 primary function is to maintain the connections between cardiomyocytes and ensure stable cardiac function. DSC2 deletion or mutation can lead to disruption of intercellular connections, affecting cardiomyocyte stability and even causing heart disease. Therefore, immunizing animals with DSC2 protein to obtain antibodies targeting human DSC2 protein, and subsequently developing kits (such as ELISA kits) for detecting DSC2 protein in clinical patients, is of great significance. Summary of the Invention

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

[0004] Specifically, in this invention, DSC2 protein is expressed and purified using eukaryotic cells and used as an antigen to immunize rabbits. Blood is collected from rabbits periodically, and spleens of rabbits with the highest titer are taken. RNA is extracted, a phage immunoglobulin library is constructed, and antibodies are screened. The antibody titer and immunization effect are determined using an indirect ELISA method. From the spleens of rabbits with the highest titer, total RNA is extracted from spleen cells and reverse transcribed into cDNA. This cDNA is used as a template to amplify the VH / VL variable region gene fragment, which is then ligated into Scfv using a linker (G4S). After ligation of the Scfv gene fragment and the phage plasmid, an anti-Scfv phage antibody library is constructed by electroporation, and panning is performed to enrich phages with high affinity. Finally, after recombination, expression, and purification, anti-DSC2 antibodies are obtained.

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

[0006] A first aspect of the present invention provides 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 CDR-H1 is as shown in SEQ ID NO:1, the amino acid sequence of CDR-H2 is as shown in SEQ ID NO:2, the amino acid sequence of CDR-H3 is as shown in SEQ ID NO:3, the amino acid sequence of CDR-L1 is as shown in SEQ ID NO:6, the amino acid sequence of CDR-L2 is as shown in SEQ ID NO:7, and the amino acid sequence of CDR-L3 is as 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-derived, mouse-derived, or rabbit-derived framework region.

[0008] In some embodiments of the present 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 with 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, wherein the Fv is preferably scFv.

[0012] In some embodiments of the present 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 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.

[0014] A second aspect of the present invention provides an isolated nucleic acid that encodes an antibody as described in 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 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.

[0016] A third aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids 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, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0018] A fourth aspect of the present invention provides a transformant comprising 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, wherein 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 HEK293 cells or CHO cells.

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

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

[0022] The antibody is obtained from the culture by culturing the transformant as described in the fourth aspect of the present invention.

[0023] A sixth aspect of the present invention provides the use of antibodies as described in the first aspect of the present invention, isolated nucleic acids as described in the second aspect of the present invention, recombinant expression vectors as described in the third aspect of the present invention, or transformants as described in the fourth aspect of the present invention in the preparation of a kit for detecting human DSC2 protein.

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

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

[0026] In some embodiments of the present invention, the kit is based on a double-antibody sandwich ELISA for detecting human DSC2 protein, comprising a capture antibody and a detection antibody; the capture antibody is an 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, wherein the heavy chain variable region of the detection antibody comprises amino acid sequences CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO:21, 22 and 23, respectively, and the light chain variable region of the detection antibody comprises amino acid sequences CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: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 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.

[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 a horseradish peroxidase-labeled antibody.

[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, the method comprising contacting a sample to be tested with an antibody as described in the first aspect of the present invention or a kit as 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 present invention, the method is for non-diagnostic purposes.

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

[0034] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0035] The reagents and raw materials used in this invention are all commercially available.

[0036] The positive and progressive effects of this invention are as follows:

[0037] The antibody and kit for detecting human DSC2 protein according to the present invention can achieve highly sensitive quantitative detection of DSC2, which not only helps basic research, but also provides valuable reference information for clinical diagnosis. Attached Figure Description

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

[0039] Figure 2 These are reduced and non-reduced SDS-PAGE electrophoresis images of the antibody in Example 2 of this invention.

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

[0041] Figure 4 This is a calibration curve of a human DSC2 protein detection kit with R2-R3 pairing provided in Example 3 of the present invention. Detailed Implementation

[0042] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0043] The reagents and instruments used in the following examples:

[0044] (1) Main reagents

[0045] PrimeScript 1st Strand cDNA Synthesis Kit and LA Taq (5 U / μl) were purchased from TaKaRa; TG1 Electrocompetent Cells 4 were purchased from Dakota. M-280 Streptavidin 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.; and sheep anti-rabbit was purchased from Luoyang Baitong Experimental Materials Center.

[0046] (2) Main instruments

[0047] The autoclave was purchased from TOMY, Japan; the varioskan lux microplate reader was purchased from Thermo; the Nanodrop One was purchased from Thermo Scientific; the BLO-RAD electroporator was purchased from BIO-RAD; the ZQZY-88AH shaking incubator was purchased from Shanghai Zhichu; the LYNX6000 high-speed centrifuge was purchased from Thermo Scientific; and the BCM-1300A-Ⅱ biological clean bench 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 amino acid sequences of the complementarity determining region (CDR) are shown in accordance with the definition of the IMGT numbering rules.

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

[0052]

[0053] Table 2. DNA sequence listing of antibody #R2

[0054]

[0055]

[0056]

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

[0058]

[0059] Table 4. DNA sequence listing of antibody #R3

[0060]

[0061]

[0062]

[0063] Example 2: Preparation of DSC2 rabbit antibody

[0064] I. 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] II. Rabbit Immunization and Antibody Screening

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

[0069] 1. Protein immunity:

[0070] Wipe the emulsifier needle twice with 75% alcohol (use a new cotton ball for the second wipe). The volume of liquid emulsified at one time should be approximately 250 μl to 20 ml. Dilute the DSC2 protein to 2 mg / mL, take 1.2 mg of protein and 600 μL of complete or incomplete adjuvant, and emulsify using an emulsifier, moving from top to bottom and occasionally oscillating up and down in specific areas. Generally, 1-10 ml of emulsion can be emulsified in 3-5 minutes, and should not exceed 10 minutes.

[0071] Administration method: Subcutaneous multi-site immunization, pulse immunization is administered intravenously.

[0072] Breed: New Zealand White Rabbit, 2-3 months old, 2-2.5kg, female.

[0073] Quantity: 2 control mice, 2 sample mice for phage screening, and 1 backup mouse. Immunization dose: 500 μg / mouse / dose, at two-week intervals.

[0074] 2. Determination of antibody titer and cell supernatant in rabbit serum

[0075] One week after the fourth DSC2 immunization, blood was collected from the rabbit serum to determine the 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 of 1×PBST each time. Shake the plate as dry as possible on absorbent paper after the last wash.

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

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

[0081] (6) Wash the plate 4 times, adding 300 μL of 1×PBST each time. Shake the plate as dry as possible on the last wash and blot dry on absorbent paper.

[0082] (7) Dilute serum or cell supernatant with NAYO 96-well pipette and transfer 100 μL to each well of a sealed 96-well plate. For positive control screening of cell supernatant, add 1000-fold diluted positive serum (without control antibody).

[0083] (8) Incubate at 37℃ in the dark for 1 hour;

[0084] (9) Wash the plate 4 times, adding 300 μL of 1×PBST each time. Shake the plate as dry as possible on the last wash and blot dry on absorbent paper.

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

[0086] (11) Incubate at 37℃ in the dark for 1 hour;

[0087] (12) Wash the plate 4 times, adding 300 μL of 1×PBST each time. Shake the plate as dry as possible on absorbent paper after the last wash.

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

[0089] (14) Incubate at 37℃ 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. Specific results are as follows: Figure 1 As shown.

[0090] 3. Construction of the immune repositories

[0091] Total RNA was extracted from rabbit spleen cells and reverse transcribed into cDNA. The variable regions of the heavy and light chains were amplified using cDNA as a template, ligated into scfv using a linker, and then ligated into the phagemid vector. A 9th power immune library was constructed by electroporation and packaged into scfv-phage for screening.

[0092] 4. Selection of phage libraries

[0093] After incubating scfv-phage with biotin-labeled Human DSC2 antigen at room temperature for 1 hour, it was added to blocked SA-coated magnetic beads and incubated at room temperature for 15 minutes. The magnetic beads were washed 10 times with PBST, and then the specifically bound phages were eluted with trypsin at room temperature. The phages were then used to infect E. coli TG1 in the logarithmic growth phase. After three rounds of panning, a highly enriched phage library was obtained.

[0094] 5. Screening for single positive phages

[0095] The selected phage enrichment library was used to extract monoclonal antibodies one by one, which were then packaged into scfv-phage in 96-well round-bottom deep-well plates. ELISA was then performed using the phage; positive wells contained specific antibodies against the DSC2 antigen. The corresponding antibody sequences were obtained by next-generation sequencing of the bacterial culture.

[0096] 6. Antibody Cloning and Plasmid Preparation

[0097] Primers were designed to amplify the variable region of the heavy and light chains and insert it into a vector containing the constant region of the heavy and light chains of mouse antibodies to construct a total antibody molecule. The plasmid was then prepared using a kit (MN740410.100).

[0098] 7. Antibody preparation and affinity determination

[0099] (1) Preparation of recombinant rabbit antibody and determination of binding curve

[0100] The recombinant plasmid was transfected into 293F cells at a 1:1 ratio. The supernatant was collected and the antibody purified when the cell viability dropped to 80% at about the fifth day.

[0101] Resuspend the magnetic beads by shaking or vortexing; place an appropriate amount of magnetic beads (magnetic bead loading 25mg / 4ml) into a 50ml centrifuge tube, place it on the magnetic base of a magnetic rack, and discard the supernatant; add 10 times the volume of 0.1% PBST, invert and mix thoroughly, place it on the magnetic base of a magnetic rack, and discard the supernatant; repeat the washing once, add an equal volume of PBST to resuspend for later use; add the expression supernatant to the resuspended magnetic beads, and incubate overnight at 4°C; place it on the magnetic base of a magnetic rack, discard the supernatant or store for later use; wash three times with PBS, discard the supernatant, and finally... Aspirate the solution thoroughly with a 1ml pipette tip; add an equal volume of elution buffer and incubate at room temperature for 15 minutes, resuspending 2-3 times during this period; place the centrifuge tube on a magnetic rack and collect the liquid into a clean 50ml centrifuge tube, repeating the elution once; add neutralization buffer to the centrifuge tube at a rate of 100μl / ml and mix gently; place the centrifuge tube on a magnetic rack to attract magnetic beads, and transfer the supernatant to a prepared EP tube using a 1ml pipette to measure the concentration; concentrate the antibody using an ultrafiltration tube, replace with 1×PBS 3 times, aspirate to measure the concentration, and store in aliquots at -20℃.

[0102] (2) Antibody SDS-PAGE

[0103] Add 5 μg of the sample to a 1.5 ml centrifuge tube. For both non-reduced and reduced samples, add water to a final volume of 9 μl. Add 3 μl of 4×LDS Buffer (NP0008, Thermo) to the non-reduced sample and 3 μl of 4×LDS Buffer (containing 10 mM DTT) to the reduced sample. Mix well and boil the reduced sample at 95°C for 10 min. Take a protein electrophoresis gel, remove the protective film, insert the side with the switch, and insert a baffle into the other side. Turn off both switches simultaneously and place the gel in the electrophoresis tank. Add freshly prepared 1×MOPS buffer to the tank and 1×MOPS recovery buffer to the outside. Remove the comb. Add 5 μl of protein marker to the first column, followed by the prepared sample, ensuring the sample is centered in the well. Cover the tank, paying attention to the positive and negative electrodes. Incubate at 170V for 55 min until the bands turn below the red line. Pour MOPS into a MOPS recovery bottle, turn on the valve, remove the protein gel, pry it open with scissors (avoid drying), and cut off the sample portion with a comb. Transfer the protein gel to distilled water, boil, and repeat three times. Transfer the treated gel to Coomassie Brilliant Blue solution, boil, and shake on a shaker for 5-30 minutes. Change the water to remove the whitening agent until clear bands are visible. The results are as follows: Figure 2 As shown.

[0104] (3) Antibody binding curve determination

[0105] Prepare an antigen dilution buffer to a final concentration of 1 μg / ml. After thorough mixing, add 100 μl per well to a 96-well plate using a multichannel pipette (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 serially diluted antibody samples, and incubate at 37°C for 1 hour. After primary antibody incubation, 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 secondary antibody incubation, wash the plate three times with 0.05% PBST, add 50 μL TMB, incubate at room temperature in the dark for 5-10 minutes, and stop the incubation with 100 μL of 1M concentrated sulfuric acid. Read the absorbance at 450 nm. The results are as follows: Figure 3 As shown, sino-Ab (Sino-Ab, 10809-MM04) is a positive control.

[0106] (4) Antibody affinity assay

[0107] Based on the ELISA results, a suitable chip was selected, and the binding Ru value of the antigen and antibody at a certain concentration was determined using manual mode. An anti-mouse antibody chip coupled with a Protein G or CM5 chip was used, and the "Multi-cycle kinetics affinity using GST Capture kit" program was selected. The injection and dissociation times, flow rates, regeneration solutions, startup cycles, analyte concentration gradient, and sample location were set. In Biacore Insight Evaluation Software, the corresponding analytical method (Kinetics / affinity–Antibody / general–Multi-cycle kinetics affinity using GST Capture kit) was located by clicking "Predefined" (corresponding to the experimental method), and analysis was initiated by double-clicking or clicking the "Open" button in the lower right corner. The analysis software automatically fitted the data, and the output results are shown in Table 5.

[0108] Table 5. DSC2 Antibody Affinity Assay

[0109] Antibody The substance being 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] Based on affinity assay results, anti-huamn IgG Fc was immobilized on a CM5 chip. Then, antibody 1 was captured, and excess Fc was blocked. Antibody 1 was then used as a negative result, followed by competitive analysis of antibody 2 and the DSC2 premix. This method showed a signal indicating the presence of different epitopes binding, but no competition. The results are shown in Table 6. Antibodies R2 and R3 (the specific sequences of which are shown in Tables 1-4) have different epitopes.

[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 application of a human desmocollin-2 (DSC2 / Desmocollin-2) monoclonal antibody in an immunoassay tool: the immunoassay tool includes a test kit.

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

[0117] Detection Principle: The Human Desmocollin-2 (DSC2 / Desmocollin-2) Quantitative Detection Kit (Double Antibody Sandwich Quantitative Method) utilizes a pair of antibodies that can bind to at least two different epitopes on human desmocollin-2 (DSC2 / Desmocollin-2). Capture antibodies are pre-coated onto the surface of the wells of a microplate and selectively bind to human desmocollin-2 (DSC2 / Desmocollin-2). After washing, a second antibody labeled with horseradish peroxidase is added, which binds to the second epitope of human desmocollin-2 (DSC2 / Desmocollin-2), forming a sandwich complex. Finally, TMB substrate is used for color development. The color intensity 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 450 nm using a microplate reader.

[0118] 2. R2-R3 reagent kit

[0119] This includes capture antibody R2, detection antibody horseradish peroxidase labeled R3, NEST removable ELISA plate, dilution buffer, etc.

[0120] 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 96-well plates, and each concentration was measured in triplicate. After 30 min of reaction, the reaction signal intensity was detected using a microplate reader. Examples of corrected absorbance signal values ​​for each calibrator are shown in Table 7.

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

[0122]

[0123] A calibration curve is plotted by linearly fitting the calibrator concentration to its corresponding absorbance signal value, as shown below. Figure 4 As shown, R 2 =0.99, indicating that within the range of 0.078-5 ng / ml, the absorbance signal value is highly linearly correlated with the detection concentration.

[0124] 3. Preparation method of horseradish peroxidase labeled R3

[0125] The HRP labeling kit Proteintech-PK20001 was used.

[0126] Follow these steps to label:

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

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

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

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

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

[0132] Notice:

[0133] a) The antibody to be labeled should have a titer of 1:20 or higher.

[0134] b) Antibody buffer: 0.01M PBS at pH 7.4 is preferred, and it should ideally be free of glycerol, sodium azide, and amino substances (including glycine, Tris, etc.). However, small amounts of amino substances (<0.05M) and sodium azide (<0.02%) will not significantly affect the labeling results. If the antibody to be labeled contains substances at concentrations higher than these, thorough dialysis with the above-mentioned PBS buffer solution is necessary.

[0135] c) Antibody dosage: The concentration should be between 0.5–5 mg / ml. Refer to Table 8 below for specific dosages:

[0136] Table 8. Relationship between the amount and volume of corresponding antibody or other protein molecular markers

[0137]

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

[0139] e) HRP should be dissolved and used immediately. It is not recommended to dissolve HRP and then repackage it for use.

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

[0141] 4.1 Reagent Preparation

[0142] (1) Equilibrate all reagents to room temperature (18-25℃) before use.

[0143] (2) Capture antibody R2 solution: Dilute R2 protein to 2 μg / ml with 1×PBS.

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

[0145] First, dilute the DSC2 standard to a 10 ng / ml standard stock solution using 1% casein PBS solution (or use the kit standard stock solution directly), and then perform a two-fold serial dilution, as shown in Table 9 below.

[0146] Table 9 Dilution Concentration Settings

[0147]

[0148] (4) The 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 of the capture antibody R2 solution (2 μg / ml) to the appropriate wells. Cap 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 seal at 37°C for 2 hours.

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

[0154] (5) Add the standard and test sample at a rate of 100 μl / well to the appropriate well. Cover the well and incubate gently with shaking at 25°C for 2.0 hours.

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

[0156] (7) Add 100 μl of horseradish peroxidase-labeled R3 solution to each well. Cover the wells and incubate gently at 25°C for 1 hour.

[0157] (8) Wash twice as per step (2).

[0158] (9) Add 100 μl of TMB substrate solution to each well. Incubate gently with shaking for 30 minutes at 25°C in the dark.

[0159] (10) Add 50 μl of 0.2 M sulfuric acid to each well to stop the color development. Then immediately take a reading at 450 nm using an ELISA reader.

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

[0161] 1. Sensitivity

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

[0163] 2. Accuracy

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

[0165] 3. Linear range

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

[0167] In summary, the reagent kit of the present invention can be determined to have good performance and the advantages of high sensitivity, strong specificity and high accuracy.

Claims

1. An antibody against 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 CDR-H1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 8; the antibody binds to human DSC2 protein.

2. The antibody as described in claim 1, characterized in that, The framework regions of the light chain variable region and / or heavy chain variable region are human-derived, mouse-derived, or rabbit-derived framework regions.

3. The antibody as described in claim 2, characterized in that, The amino acid sequence of the light chain variable region has at least 85% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 85% sequence identity with SEQ ID NO:

4.

4. The antibody as described in claim 3, characterized in that, The amino acid sequence of the light chain variable region has at least 90% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO:

4.

5. The antibody as described in claim 4, characterized in that, The amino acid sequence of the light chain variable region has at least 95% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 95% sequence identity with SEQ ID NO:

4.

6. The antibody as described in claim 5, characterized in that, The amino acid sequence of the light chain variable region has at least 96% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 96% sequence identity with SEQ ID NO:

4.

7. The antibody as described in claim 6, characterized in that, The amino acid sequence of the light chain variable region has at least 97% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 97% sequence identity with SEQ ID NO:

4.

8. The antibody as described in claim 7, characterized in that, The amino acid sequence of the light chain variable region has at least 98% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 98% sequence identity with SEQ ID NO:

4.

9. The antibody as described in claim 8, characterized in that, The amino acid sequence of the light chain variable region has at least 99% sequence identity with SEQ ID NO:9; and / or, the amino acid sequence of the heavy chain variable region has at least 99% sequence identity with SEQ ID NO:

4.

10. The antibody as described in claim 9, characterized in that, 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.

11. The antibody according to any one of claims 1-10, characterized in that, The antibody is a full-length antibody, Fab, Fab', F(ab')2, or Fv.

12. The antibody according to any one of claims 1-10, characterized in that, The antibody is scFv.

13. The antibody as claimed in claim 11, characterized in that, The antibody is a full-length antibody.

14. The antibody as described in claim 13, characterized in that, 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.

15. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the antibody as described in any one of claims 1-14.

16. The isolated nucleic acid as described in claim 15, characterized in that, 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.

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

18. The recombinant expression vector as described in claim 17, characterized in that, The recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

19. The recombinant expression vector as described in claim 18, characterized in that, The viral vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

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

21. The transformant as described in claim 20, characterized in that, The host cell is a yeast cell or a mammalian cell.

22. The transformant as described in claim 21, characterized in that, The host cells are HEK293 cells or CHO cells.

23. The transformant according to any one of claims 20-22, characterized in that, The host cell was a 293F cell.

24. A method for preparing an antibody for detecting human DSC2 protein, characterized in that, The method includes the following steps: The antibody is obtained from the culture by culturing the transformant as described in any one of claims 20-23.

25. The use of the antibody as described in any one of claims 1-14, the isolated nucleic acid as described in claim 15 or 16, the recombinant expression vector as described in any one of claims 17-19, or the transformant as described in any one of claims 20-23 in the preparation of a kit for detecting human DSC2 protein.

26. A kit for detecting human DSC2 protein, characterized in that, The kit includes the antibody as described in any one of claims 1-14.

27. The kit as claimed in claim 26, characterized in that, The kit also includes reagents for detecting human DSC2 protein based on enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, flow cytometry, or protein chip technology.

28. The kit according to claim 27, characterized in that, The kit is based on a double-antibody sandwich ELISA for detecting human DSC2 protein, comprising a capture antibody and a detection antibody that binds to human DSC2 protein; the capture antibody is the antibody as described in any one of claims 1-14.

29. The kit according to claim 28, characterized in that, 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 amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 21, 22, and 23, respectively. The light chain variable region of the detection antibody comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 26, 27, and 28, respectively.

30. The kit according to claim 29, characterized in that, 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.

31. The kit according to claim 30, characterized in that, 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.

32. The kit according to any one of claims 28-31, characterized in that, The detection antibody is a horseradish peroxidase-labeled antibody.

33. A method for detecting human DSC2 protein for non-diagnostic purposes, characterized in that, The method includes contacting a test sample with an antibody as described in any one of claims 1-14 or a kit as described in any one of claims 26-32 to detect the expression level of human DSC2 protein in the test sample.

34. The method as described in claim 33, characterized in that, The mass ratio of the capture antibody to the detection antibody is (1-10):

1.

35. The method as described in claim 34, characterized in that, The mass ratio of the capture antibody to the detection antibody is (3-9):

1.

36. The method as described in claim 35, characterized in that, The mass ratio of the capture antibody to the detection antibody is 8:1.

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