CN119192366B

CN119192366B - Antibodies and kits for detecting human DSC2 protein

Info

Publication number: CN119192366B
Application number: CN202411586806.3A
Authority: CN
Inventors: 杜兰兰; 梁青; 方水琴; 李胜; 张旺萍; 赵海利
Original assignee: Shanghai Lanque Biomedical Co ltd
Current assignee: Shanghai Lanque Biomedical Co ltd
Priority date: 2024-11-07
Filing date: 2024-11-07
Publication date: 2025-11-04
Anticipated expiration: 2044-11-07
Also published as: CN119192366A

Abstract

Antibodies and kits for detecting human DSC2 protein are disclosed. 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 amino acid sequences shown as SEQ ID NO. 1,2 and 3 respectively, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3 with amino acid sequences shown as 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 on DSC2, thereby not only being beneficial to basic research, but also providing valuable reference information for clinical diagnosis.

Description

Antibodies and kits for detection of human DSC2 protein

Technical Field

The invention belongs to the field of biological detection, and in particular relates to an antibody and a kit for detecting human DSC2 protein.

Background

Desmoglein 2 (DSC 2, desmocollin-2) is an intercellular adhesion protein belonging to the glycoprotein family. It acts to attach cells to the surface of cell membranes 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. The protein is expressed in myocardial cells, and the main function is to maintain the connection between myocardial cells and maintain the stable function of heart. Loss or mutation of DSC2 may lead to a disturbance of intercellular junctions, affecting the stable function of cardiomyocytes and even to heart lesions. Therefore, immunization of animals with DSC2 protein to obtain antibodies targeting human DSC2 protein, and development of a kit (e.g., ELISA kit) for detecting DSC2 are of great significance for detecting DSC2 protein in clinical patients.

Disclosure of Invention

The present invention has been made to solve the above problems, and provides an antibody and a kit for detecting human DSC2 protein.

Specifically, the DSC2 protein is expressed and purified by utilizing eukaryotic cells, and is used as an antigen for immunizing rabbits. And (3) regularly taking blood of the rabbits, taking spleens of the rabbits with optimal titers, extracting RNA, constructing a phage immune library, and carrying out antibody screening. The method comprises the steps of determining antibody titer and immune effect by an indirect ELISA method, taking the spleen of a rabbit with optimal titer, extracting total RNA of spleen cells, carrying out reverse transcription to obtain cDNA, using the cDNA as a template for amplification to obtain a VH VL gene fragment of a heavy and light chain variable region, and connecting the VH gene fragment to Scfv through a linker (G ₄ S). After the Scfv gene fragment is connected with phage plasmid, an anti-Scfv phage antibody library is constructed by electric shock, panning is carried out, phage with high affinity is obtained through enrichment, and finally, the anti-DSC 2 antibody is obtained through expression and purification after recombination.

The technical problems are solved by the following technical scheme.

In a first aspect, the 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, and is characterized in that the amino acid sequence of the CDR-H1 is shown as SEQ ID NO. 1, the amino acid sequence of the CDR-H2 is shown as SEQ ID NO. 2, the amino acid sequence of the CDR-H3 is shown as SEQ ID NO. 3, the amino acid sequence of the CDR-L1 is shown as SEQ ID NO. 6, the amino acid sequence of the CDR-L2 is shown as SEQ ID NO. 7, and the amino acid sequence of the CDR-L3 is shown as SEQ ID NO. 8.

In some embodiments of the invention, the framework regions of the light chain variable region and/or heavy chain variable region are human, murine, or rabbit framework regions.

In some embodiments of the invention, the light chain variable region has an amino acid sequence 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;

And/or the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 4 or 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.

In some embodiments of the 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.

In some embodiments of the invention, the antibody is a full length antibody, fab ', F (ab') ₂, or Fv, with scFv being preferred.

In some embodiments of the invention, the antibody is a full length antibody.

In some embodiments of the 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.

In a second aspect the invention provides an isolated nucleic acid encoding an antibody according to the first aspect of the invention.

In some embodiments of the invention, the nucleotide sequence of the light chain of the antibody is shown as SEQ ID NO. 20 and/or the nucleotide sequence of the heavy chain of the antibody is shown as SEQ ID NO. 15.

In a third aspect the invention provides a recombinant expression vector comprising an isolated nucleic acid according to the second aspect of the invention.

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

In a fourth aspect the invention provides a transformant comprising an isolated nucleic acid according to the second aspect of the invention or a recombinant expression vector according to the third aspect of the invention, the host cell of the transformant being a prokaryotic cell or a eukaryotic cell.

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

In some embodiments of the invention, the host cell is a 293F cell.

In a fifth aspect the present invention provides a method for detecting antibodies to human DSC2 protein, the method comprising the steps of:

Culturing the transformant according to the fourth aspect of the present invention, and obtaining the antibody from the culture.

In a sixth aspect, the invention provides the use of an antibody according to the first aspect of the invention, an isolated nucleic acid according to the second aspect of the invention, a recombinant expression vector according to the third aspect of the invention or a transformant according to the fourth aspect of the invention in the preparation of a kit for detecting human DSC2 protein.

In a seventh aspect the invention provides a kit for detecting human DSC2 protein, the kit comprising an antibody according to the first aspect of the invention;

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

In some embodiments of the invention, the kit is based on a double antibody sandwich ELISA for detecting human DSC2 protein, which comprises a capture antibody and a detection antibody, wherein the capture antibody is an antibody according to the first aspect of the invention.

In some embodiments of the invention, the detection antibody comprises a light chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 having amino acid sequences as shown in SEQ ID NOS: 21, 22, 23, respectively, and a heavy chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 having amino acid sequences as shown in SEQ ID NOS: 26, 27, 28, respectively.

In some embodiments of the invention, the amino acid sequence of the light chain variable region of the test antibody is shown in SEQ ID NO. 29 and the amino acid sequence of the heavy chain variable region of the test antibody is shown in SEQ ID NO. 24.

In some embodiments of the invention, the amino acid sequence of the light chain of the detection antibody is shown as SEQ ID NO. 30, and the amino acid sequence of the heavy chain of the detection antibody is shown as SEQ ID NO. 25, and the detection antibody is preferably a horseradish peroxidase-labeled antibody.

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

In an eighth aspect, the invention provides a method of detecting human DSC2 protein, the method comprising contacting a test sample with an antibody according to the first aspect of the invention or a kit according to the seventh aspect of the invention, and detecting the expression level of human DSC2 protein in the test sample.

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

In some embodiments of the invention, the capture antibody and the detection antibody are added in a mass ratio of (1-10): 1, preferably (3-9): 1, for example 8:1.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

according to the human DSC2 protein antibody and the kit, high-sensitivity quantitative detection of DSC2 can be realized, so that basic research is facilitated, and valuable reference information can be provided for clinical diagnosis.

Drawings

FIG. 1 is a chart of rabbit serum titer assay of example 2 of the present invention.

FIG. 2 is a SDS-PAGE electrophoresis of the reduced and non-reduced antibodies of example 2 of the present invention.

FIG. 3 is a graph showing the binding curves of the antibodies provided in example 2 of the present invention.

FIG. 4 is a calibration graph of a human DSC2 protein detection kit paired R2-R3 provided in example 3 of the present invention.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Reagents and instrumentation used in the following examples:

(1) Main reagent

PRIMESCRIPT 1st Strand cDNA Synthesis kit, LA Taq (5U/. Mu.l) were purchased from TaKaRa, and TG1 Electrocompetent Cells was purchased from Daidae; M-280 strepitavidins from Invitrogen, sfiI restriction enzyme, T4 DNA ligase from NEB, M13KO7 helper phage from Du-Ke-Domain organisms, BSA from Shanghai Jie Biotechnology Co., ltd, sheep anti-rabbit from Luoyang Baiao laboratory materials center.

(2) Main instrument

The autoclave was purchased from TOMY, japan, the microplate reader varioskan lux from Thermo, the Nanodrop one from Thermo Scientific, the electrotransducer BLO-RAD from BIO-RAD, the shaking incubator ZQZY-88AH from Shanghai know Chu, the high-speed centrifuge LYNX6000 from Thermo Scientific, and the biological clean bench BCM-1300A-II from Soncha air technologies Co.

Example 1

The present example provides a combination of human DSC2 protein antibodies, including antibody #r2 and antibody #r3.

The amino acid sequence of antibody #r2 is shown in table 1, the DNA sequence of antibody #r2 is shown in table 2, and the amino acid sequences of the complementarity determining regions (complementarity determining region, CDRs) are all shown according to the definition of IMGT numbering convention.

TABLE 1 amino acid sequence listing of antibody #R2

TABLE 2 DNA sequence listing of antibody #R2

TABLE 3 amino acid sequence listing of antibody #R3

TABLE 4 DNA sequence listing of antibody #R3

EXAMPLE 2DSC2 preparation of rabbit antibody

1. Selection of immunogens

The amino acid sequence of the immunogen is as follows:

>DSC2(Human)：RWAPIPCSMLENSLGPFPLFLQQVQSDTAQNYTI YYSIRGPGVDQEPRNLFYVERDTGNLYCTRPVDREQYESFEIIAFATTPDGYTPELPLPLIIKIEDENDNYPIFTEETYTFTIFENCRVGTTVGQVCATDKDEPDTMHTRLKYSIIGQVPPSPTLFSMHPTTGVITTTSSQLDRELIDKYQLKIKVQDMDGQYFGLQTTSTCIINIDDVNDHLPTFTRTSYVTSVEENTVDVEILRVTVEDKDLVNTANWRANYTILKGNENGNFKIVTDAKTNEGVLCVVKPLNYEEKQQMILQIGVVNEAPFSREASPRSAMSTATVTVNVEDQDEGPECNPPIQTVRMKENAEVGTTSNGYKAYDPETRSSSGIRYKKLTDPTGWVTIDENTGSIKVFRSLDREAETIKNGIYNITVLASDQGGRTCTGTLGIILQDVNDNSPFIPKKTVIICKPTMSSAEIVAVDPDEPIHGPPFDFSLESSTSEVQRMWRLKAINDTAARLSYQNDPPFGSYVVPITVRDRLGMSSVTSLDVTLCDCITENDCTHRVDPR(SEQ ID NO:41)

2. rabbit immunization and antibody screening

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

1. Protein immunization:

The emulsifying needle head is wiped by 75% alcohol twice (new cotton balls are used for the second time), and the single emulsified liquid amount is about 250 mu l-20 ml. DSC2 protein is diluted to 2mg/mL, 1.2mg protein and 600 mu L of complete adjuvant or incomplete adjuvant are taken, emulsification is carried out by adopting an emulsifier, and the whole is moved up and down in a swinging way. The emulsifying amount is generally 1-10ml, and is 3-5 min, and no more than 10 min.

The administration mode is subcutaneous multipoint immunity, and the impact immunity is intravenous injection.

The strain is New Zealand white rabbits, 2-3 months old, 2-2.5 kg, female.

Number control 2, sample 2 was used for phage selection, 1 alternative. The immunization dose was 500. Mu.g/dose, two weeks apart.

2. Rabbit serum antibody titer and cell supernatant assay

After the fourth immunization of DSC2, blood is taken at intervals of one week, and the antibody titer of rabbit serum is measured by the following specific method:

(1) 100. Mu.L of DSC2 antigen (100 ng) was added to the well plate for coating;

(2) Incubation overnight at 4 ℃ in the dark;

(3) Washing the plate for 4 times, adding 300 mu L of 1 XPBST each time, spin-drying for the last time as much as possible, and sucking on water-absorbing paper;

(4) Blocking by adding 200. Mu.L of 5% BSA blocking solution to each well;

(5) Incubation at 37 ℃ for 2 hours in the dark;

(6) Washing the plate for 4 times, adding 300 mu L of 1 XPBST each time, spin-drying for the last time as much as possible, and sucking on water-absorbing paper;

(7) Serum or cell supernatants diluted with NAYO96 well pipetting stations were added to the 96 well plates with 100 μl per well. Cell supernatant selection positive quality control positive serum (no control antibody) was added at 1000-fold dilution.

(8) Incubation for 1h at 37 ℃ in the dark;

(9) Washing the plate for 4 times, adding 300 mu L of 1 XPBST each time, spin-drying for the last time as much as possible, and sucking on water-absorbing paper;

(10) Adding goat anti-rabbit IgG H+L secondary antibody diluted by 2 ten thousand times into each hole;

(11) Incubation for 1h at 37 ℃ in the dark;

(12) Washing the plate for 4 times, adding 300 mu L of 1 XPBST each time, spin-drying for the last time as much as possible, and sucking on water-absorbing paper;

(13) 100. Mu.L of TMB developing solution (the developing solution itself is colorless, and the color change occurs and cannot be used) was added;

(14) Incubation at 37℃for 10-15 min in the dark was stopped by adding 50. Mu.L of stop solution and reading the OD at 450nm, the specific results are shown in FIG. 1.

3. Immune repertoire construction

Extracting total RNA of rabbit spleen cells, performing reverse transcription to obtain cDNA, amplifying heavy and light chain variable regions by taking the cDNA as a template, connecting the cDNA to a phagemid carrier after connecting the cDNA to scfv, constructing an immune library to the power of 9 through electric rotation, and packaging the immune library into scfv-phase for screening.

4. Panning of phage libraries

After scfv-phage is incubated with the biological marked Human DSC2 antigen for 1 hour at room temperature, the mixture is added into the magnetic beads coated by the SA and incubated for 15 minutes at room temperature, the magnetic beads are washed by PBST for 10 times, then the specifically bound phage is eluted by trypsin at room temperature, and the phage is used for infecting E.coli TG1 in the log phase, and a highly enriched phage library is obtained through 3 rounds of panning.

5. Screening of Single Positive phages

And (3) picking the monoclonal antibodies one by one from the phage enrichment library, and packaging the monoclonal antibodies into scfv-phage in a 96-well round-bottomed deep well plate. And then ELISA is carried out by using phage, and the positive hole is the specific antibody aiming at DSC2 antigen. And (5) carrying out second generation sequencing on the bacterial liquid to obtain the corresponding antibody sequence.

6. Antibody clone construction and plasmid preparation

Primers were designed to amplify the heavy and light chain variable region and loaded into a vector with the mouse antibody heavy and light chain constant region to construct a whole antibody molecule, and plasmid preparation was performed using the kit (MN 740410.100).

7. Antibody preparation and affinity assay

(1) Recombinant rabbit antibody preparation and binding curve determination

The recombinant plasmid was transfected into 293F cells at 1:1, and supernatant purified antibodies were harvested approximately the fifth day the cell viability was reduced to 80%.

The method comprises the steps of shaking or vortexing, resuspension of magnetic beads, placing a proper amount of magnetic beads (25 mg/4ml of magnetic bead load) into a 50ml centrifuge tube, placing the 50ml centrifuge tube on a magnetic frame magnetic base, discarding supernatant, adding 10 times of 0.1% PBST, inversely and fully mixing, placing the supernatant on the magnetic frame magnetic base, discarding the supernatant, repeatedly washing for 1 time, adding an equal volume of PBST for resuspension, adding the expression supernatant into the resuspended magnetic beads, rotating the magnetic beads at 4 ℃ overnight, placing the magnetic beads on the magnetic frame magnetic base, discarding the supernatant or preserving the supernatant for later use, washing the supernatant with PBS for three times, discarding the supernatant, finally sucking the supernatant with 1ml gun head, adding an equal volume of elution buffer solution, incubating for 15min at room temperature, resuspension for 2-3 times, placing the supernatant on the magnetic frame magnetic base, collecting the liquid into a clean 50ml centrifuge tube, repeatedly eluting once, adding the neutralization solution into the centrifuge tube according to 100 mu l/ml, lightly mixing, adsorbing the magnetic beads on the magnetic frame, transferring the supernatant into a prepared EP tube by using a 1ml gun, measuring the concentration, replacing the antibody by using the 1X 3 ml gun, and preserving the antibody by preserving the centrifugation tube at 20 ℃.

(2) Antibody SDS-PAGE

Taking 5 mug of a sample to be detected into a 1.5ml centrifuge tube, adding water into the non-reduced sample and the reduced sample to supplement 9 mug, adding 3 mug of 4 XLDS Buffer (NP 0008, thermo) into the non-reduced sample, adding 3 mug of 4 XLDS Buffer (containing 10mM DTT) into the reduced sample, uniformly mixing, and boiling the reduced sample at 95 ℃ for 10min. Taking a piece of protein electrophoresis gel, tearing off the protective film, inserting the protective film into the side with the switch, inserting the baffle into the other side, closing two switches at the same time, placing the two switches into an electrophoresis tank, adding a newly prepared 1 xMOPS buffer solution into the electrophoresis tank, and externally adding a 1 xMOPS recovery solution. The comb was pulled off and 5. Mu.l of protein Marker was added to the first column followed by the formulated sample to be tested, taking care that the sample was added to the center of the well. The cover was closed, note positive and negative electrodes, and 170v 55min electrophoresis strips were run to below red and stopped. Pouring MOPS into MOPS recovery liquid bottle, opening switch to take out the albumin glue, prying open with scissors, taking care to avoid drying the glue, cutting off the sample part with comb, transferring the albumin glue into clear water, boiling, repeating for three times. The treated gum was transferred to coomassie blue solution, boiled, shaken for 5-30min, and the clear bands were removed by changing the water until clear bands were visible, the results are shown in fig. 2.

(3) Antibody binding curve assay

Antigen dilutions were prepared at a final concentration of 1 μg/ml, mixed well and added to 96-well plates at 100 μl per well using a multichannel pipette, and incubated at 100 ng/well overnight at 4 ℃. The next day, plates were washed three times with 0.05% PBST, followed by 5% skim milk, and blocked at 37 ℃ for 1-3 hours. After blocking was completed, plates were washed three times with 0.05% pbst, gradient diluted antibody samples were added, and incubated at 37 ℃ for 1 hour. After the end of the primary antibody incubation, the plates were washed three times with 0.05% PBST, and 20000-fold diluted goat anti-rabbit IgG (h+l) -HRP was added and incubated at 37 ℃ for 1 hour. After the secondary antibody incubation was completed, the plate was washed three times with 0.05% PBST, 50. Mu.L TMB was added, incubated at room temperature for 5-10 minutes in the absence of light, 100uL 1M concentrated sulfuric acid was added for termination, and the absorbance at 450nm was read, as shown in FIG. 3, wherein sino-Ab (10809-MM 04, yiqiao Shenzhou) was the positive control.

(4) Antibody affinity assay

According to ELISA results, a proper chip is selected, and a manual mode is adopted to search the combined Ru value of the antigen-antibody under a certain concentration. The method comprises the steps of adopting an anti-mouse antibody chip coupled with a Protein G chip or a CM5 chip, selecting a Multi-CYCLE KINETICS AFFINITY using GST Capture kit program, and setting sample injection and dissociation time, flow rate, regeneration solution, startup circulation times, analyte concentration gradient and sample position. In Biacore Insight Evaluation Software, click "PREDEFINED" finds the corresponding analysis method (kinetic/affinity-Antibody/general-Multi-CYCLE KINETICS AFFINITY using GST Capture kit) (corresponding to the experimental method), double-click or click the lower right "Open" button for analysis. The analysis software automatically performs data fitting and outputs the results as shown in table 5.

Table 5DSC2 antibody affinity assay

Antibodies to	Substances to be 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

(5) Antibody epitope competition and pairing

According to the affinity measurement result, anti-huamn IgG Fc is fixed on a CM5 chip, then one antibody 1 is captured, then redundant Fc is blocked, then antibody 1 is taken as negative, and then antibody 2 and DSC2 premix are added for competition analysis, wherein the signal value of the method is that different epitope binding exists, and competition is avoided. The results are shown in Table 6 for two antibodies R2 and R3 (specific sequences of these two antibodies are shown in tables 1-4) with different epitopes.

TABLE 6 mouse and Rabbit anti epitope analysis

Antibody name

Positive and negative

Negative of

R3

Antibody name

Positive and negative

Negative of

R2

29

-5

12

R3

131

-3

113

Example 3

The embodiment discloses an application of a human desmoglein 2 (DSC 2/Desmocollin-2) monoclonal antibody in an immunoassay tool, wherein the immunoassay tool comprises a detection kit.

1. Human desmoglein 2 (DSC 2/Desmocollin-2) quantitative detection kit (double antibody sandwich assay).

Detection principle the detection principle of the quantitative detection kit (double antibody sandwich quantitative method) of human desmoglein 2 (DSC 2/Desmocollin-2) is to use a pair of antibodies capable of binding to at least two different epitopes on human desmoglein 2 (DSC 2/Desmocollin-2). The capture antibodies were pre-coated on the microwell surface of the microwell plate and selectively bound to human desmoglein 2 (DSC 2/Desmocollin-2). After washing, horseradish peroxidase-labeled secondary antibody was added and bound to the second site of human desmoglein 2 (DSC 2/Desmocollin-2) to form a sandwich complex. Finally, TMB substrate is directly used for color development, and the color depth in the microwell plate is positively correlated with the concentration of human desmoglein 2 (DSC 2/Desmocollin-2). The concentration of human desmoglein 2 (DSC 2/Desmocollin-2) can be quantitatively detected by reading at 450nm with an enzyme-labeled instrument.

R2-R3 kit

Including capture antibody R2, detection antibody horseradish peroxidase labeled R3, NEST detachable Elisa plate, dilution buffer, etc.

Linear assay human desmoglein 2 (DSC 2/Desmocollin-2) calibrator at concentrations of 0ng/ml, 0.078ng/ml, 0.156ng/ml, 0.312ng/ml, 0.625ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml, and 96-well plates were added and each concentration was assayed 3 times in parallel. After 30min of reaction, the intensity of the reaction signal is detected by an enzyme-labeled instrument, and an example of the corrected absorbance signal value corresponding to each calibrator is obtained, as shown in Table 7.

Table 7 absorbance signal values corresponding to calibrator

A calibration curve was drawn by linear fitting the calibrator concentration to its corresponding absorbance signal value, as shown in fig. 4, with R ² =0.99, indicating that absorbance signal values are highly linearly related to the detected concentration over the range of 0.078-5 ng/ml.

3. Preparation method of horseradish peroxidase labeled R3

HRP-labeled kit Proteintech-PK20001 was used.

The marking is carried out according to the following steps:

1) Taking out the HRP-labeled kit from the minus 20 ℃ and balancing for 30 minutes at room temperature, and fully thawing and uniformly mixing the reaction starting solution and the reaction stopping solution.

2) To 10. Mu.l of the antibody or other protein molecule solution to be labeled, 1. Mu.l of the reaction initiating solution was added, and the mixture was repeatedly blown several times with a pipette to mix well, thereby avoiding the generation of bubbles. For example, the labeling of 400. Mu. g R3 (30. Mu.l) was added to 170. Mu.l of 1 XPBS, and after mixing, 20. Mu.l of starting solution was added, and the mixture was blown.

3) The cover of horseradish peroxidase tube (bottle) was opened, the above-mentioned activated antibody or other protein solution was directly added to the tube (bottle), and the mixture was repeatedly blown several times with a pipette to mix well, avoiding the generation of bubbles, and left at room temperature for 3 hours.

4) The reaction terminating solution was added to the horseradish peroxidase reaction tube (bottle) in a ratio of 1. Mu.l of the reaction terminating solution per 10. Mu.l of the antibody or other protein solution, and the mixture was left at room temperature for 1 hour.

5) After termination, an equal volume of product protection solution is added, fully and uniformly mixed, and the mixture is preserved at-20 ℃. (this step is not required when 50% glycerol is already present in the antibody or other protein solution to be labelled), the concentration date is labelled and stored in a-20℃freezer.

Note that:

a) The antibody to be labeled should have a potency above 1:20.

B) Preferably, the antibody buffer is PBS (0.01M, pH 7.4), and preferably contains no glycerol, sodium azide, or amino substances (including glycine, tris, etc.). But small amounts of amino substances (< 0.05M) and small amounts of sodium azide (< 0.02%) do not have a serious effect on the labeling results. If the antibody to be labeled contains a substance at a concentration higher than above, sufficient dialysis with the above PBS buffer solution is required.

C) The dosage of the antibody is 0.5-5 mg/ml. For specific amounts, reference is made to table 8 below:

TABLE 8 molecular markers and volume relationships for antibodies and other proteins

D) The amount of antibody may be adjusted as required by the experiment. The smaller the amount of antibody, the more efficient the HRP labeling within the corresponding HRP dose range.

E) The HRP is used immediately after being dissolved, and split charging is not recommended after the HRP is dissolved.

The application method of the R2-R3 kit comprises the following steps:

4.1 preparation of reagents

(1) All reagents were equilibrated to room temperature (18-25 ℃) prior to use.

(2) The capture antibody R2 solution was diluted to 2. Mu.g/ml with 1 XPBS for the R2 protein.

(3) The DSC2 standard protein should be diluted with 1% casein in PBS and prepared for use.

DSC2 standards were first diluted to 10ng/ml stock using 1% casein in PBS (or directly using kit standards), followed by two-fold serial gradient dilution, see Table 9 below.

TABLE 9 dilution concentration set-up

(4) The detection antibody R3-HRP should be diluted to 0.25 μg/ml with 1% casein in PBS prior to use.

4.2 Procedure

(1) A solution of the capture antibody R2 (2. Mu.g/ml) was added to the appropriate wells at 100. Mu.l/well. The hole cover was covered and incubated at 4℃for 16 hours.

(2) The solution in the wells was discarded and washed 4 times with 1 XPBST. The plate was inverted and blotted dry with a clean paper towel.

(3) To each well 200 μl 1% casein in PBS was added. The lid was covered and closed at 37 ℃ for 2 hours.

(4) Washing according to the step (2).

(5) The standard and test samples were added to the appropriate wells at 100. Mu.l/well. The hole cover was covered and incubated with gentle shaking at 25℃for 2.0 hours.

(6) Washing according to the step (2).

(7) To each well was added a solution of detection antibody horseradish peroxidase-labeled R3 in a volume of 100. Mu.l/well. The hole cover was covered and incubated with gentle shaking at 25℃for 1 hour.

(8) Washing twice according to step (2).

(9) TMB substrate solution was added to each well at a volume of 100. Mu.l/well. Incubate with gentle shaking in a dark environment at 25 ℃ for 30 minutes.

(10) The color development was stopped by adding 50. Mu.l of 0.2M sulfuric acid to each well. Immediately after this, the sample was read with a microplate reader at 450 nm.

Example 4 evaluation of Performance of human desmoglein 2 (DSC 2/Desmocollin-2) H3-R3 kit

1. Sensitivity of

The minimum detection value of the R2-R3 kit of the embodiment 3 is 0.078ng/ml, which shows that the kit has higher sensitivity.

2. Accuracy of

The recovery rate of the R2-R3 kit of example 3 was 97-111%, indicating that the accuracy of the kit was high.

3. Linear range

The R2-R3 kit of the embodiment 3 has a correlation coefficient of more than or equal to 0.99 within the range of 0.078ng/ml to 5 ng/ml.

In conclusion, the kit disclosed by the invention has the advantages of good performance, 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 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; the antibody binds to human DSC2 protein.

2. The antibody according to claim 1, wherein the framework region of the light chain variable region and/or the heavy chain variable region is a human, mouse, or rabbit framework region.

3. The antibody according to claim 2, wherein 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 according to claim 3, wherein 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 according to claim 4, wherein 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 according to claim 5, wherein 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 according to claim 6, wherein 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 of claim 7, wherein 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 of claim 8, wherein 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 according to claim 9, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4.

11. The antibody according to any one of claims 1-10, wherein 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, wherein the antibody is scFv.

13. The antibody according to claim 11, wherein the antibody is a full-length antibody.

14. The antibody of claim 13, wherein the amino acid sequence of the light chain of the antibody is 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.

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

16. The isolated nucleic acid according to claim 15, wherein 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.

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 according to claim 17, wherein the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

19. The recombinant expression vector as described in claim 18, wherein 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, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

21. The transformant according to claim 20, wherein the host cell is a yeast cell or a mammalian cell.

22. The transformant according to claim 21, wherein the host cell is HEK293 cell or CHO cell.

23. The transformant according to any one of claims 20-22, wherein the host cell is a 293F cell.

24. A method for preparing an antibody for detecting human DSC2 protein, characterized in that the method comprises 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 comprises the antibody as described in any one of claims 1-14.

27. The kit of claim 26, wherein the kit further comprises 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 of claim 27, wherein 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; wherein the capture antibody is the antibody of any one of claims 1-14.

29. The kit of claim 28, wherein 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, 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.

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

31. The kit according to claim 30, wherein 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.

32. The kit according to any one of claims 28-31, wherein 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 comprises 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 according to claim 33, wherein the mass ratio of the capture antibody to the detection antibody is (1-10):1.

35. The method according to claim 34, wherein the mass ratio of the capture antibody to the detection antibody is (3-9):1.

36. The method of claim 35, wherein the mass ratio of the capture antibody to the detection antibody is 8:1.