CN111735948A - Application of PADI4 in preparation of tumor diagnosis kit - Google Patents

Abstract

The invention discloses a tumor diagnosis kit, comprising: the kit comprises an enzyme label plate coated with a protein PADI4 monoclonal antibody, a control sample, a washing solution, a stop solution, a diluent, horseradish peroxidase-labeled streptavidin and a horseradish peroxidase chromogenic substrate; the protein PADI4 monoclonal antibody comprises an antibody 135-B9 and an antibody 197-A5; the protein PADI4 detection kit can effectively and stably determine the level of the protein PADI4 in human serum, and has broad spectrum, detectability and good test repeatability.

Description

Application of PADI4 in preparation of tumor diagnosis kit

Technical Field

The invention relates to application of PADI4 in preparation of a tumor diagnosis kit, in particular to application of peptidyl arginine deiminase4 as a tumor marker in preparation of a tumor clinical diagnosis kit, and belongs to the technical field of molecular biological detection.

Background

Peptidyl arginine deiminase (PAD or PADI) is an enzyme in human tissue, and currently, five PAD enzymes (i.e., PAD1, 2, 3, 4, and 6) are co-found. These enzymes are encoded by a cluster of genes located in the lp36 region of human chromosome and have different tissue distribution. It can perform post-translational modification (post-translational modification) on other tissue proteins in the presence of calcium ions. This enzyme catalyzes the amino group of arginine (arginin) in the polypeptide chain to form a carbonyl group, thereby converting arginine to citrulline (citrulline). Citrulline is an unnatural amino acid. This process of arginine conversion to citrulline in a polypeptide catalyzed by PAD is called citrullination (citrullination). After the protein is citrullinated, the structure of the protein is changed, so that the enzyme activity, the metabolic activity, the regulation function and the structural function of the protein are changed. Citrullination is therefore a post-translational modification of proteins as important as phosphorylation, acetylation, glycosylation, methylation, ubiquitination.

In recent years, through researches on immunology, cellular biochemistry and molecular genetics, PAD4 (peptidyl arginine deiminase 4peptidyl deiminase4 or PADI4) is proved to play an important role in the pathogenesis of human rheumatoid arthritis, and can be used as an adenocarcinoma marker for preparing adenocarcinoma clinical diagnostic reagents. Screening technologies such as tissue chips and the like find that the expression level of the PADI4 (peptidyl arginine deiminase 4) is remarkably increased in various malignant tumor tissues and blood of patients (breast cancer, lung cancer, kidney cancer, bladder cancer, colon cancer, uterine cancer, ovarian cancer and the like), and is low or not expressed in various benign tumors, chronic inflammations (gastric smooth muscle tumor, uterine myoma, cervical polyps, cholecystitis, cervicitis, synovitis and the like) and healthy people. These results show that PADI4 is not only closely related to the tumor pathogenesis process, but also expressed in various malignant tumor tissues, and is a tumor marker with broad spectrum and detectability.

Chinese patent document CN101101290A (application No. 200610070392.4) discloses a tumor serum marker consisting of peptidyl arginine deiminase 4; wherein: the tumor refers to one of breast cancer, liver cancer, kidney cancer, ovarian cancer, prostatic cancer and bladder cancer. It also discloses the application of the tumor serum marker in the preparation of clinical diagnostic reagents for detecting tumors. The clinical tumor diagnosis reagent or kit prepared by the tumor serum marker provided by the invention can detect only one index to realize the preliminary determination of whether a detected person has malignant tumor, complete the health general investigation in shorter detection time with less cost and provide reliable basis for clinical diagnosis. However, no kit for detecting PADI4 in serum of a patient is established at home at present, and the main technical obstacles are that the antigen is human PADI4 protein, an insect baculovirus expression system is adopted for recombinant expression, a Twin Strep label is added at the N end for purification, the cell adopts sf9 cell, and soluble protein is required to be obtained as the antigen. Through expression and purification tests, the PADI4 is expressed in Sf9 cells, the optimal expression condition is that 30ul virus plasmid (M.O.I. -1) infects Sf9 cells for 3 days, the technical obstacle is that the yield is lower after expression and purification, the yield is only 0.34mg/L, the purity is only 80%, the antigen amount required by the preparation of the monoclonal antibody is at least 3.5mg, and therefore, a larger volume is required to amplify to obtain enough antigen; meanwhile, the preparation of the antibody needs to be carried out by immunization, fusion, subcloning, screening and strain determination, and in the antibody production stage, because the homology between the PADI4 protein and the PADI2 protein is high, the removal of cross reaction in screening is also a main problem which hinders the development of the technology.

Therefore, the PADI4 detection kit with high sensitivity, high specificity and high stability is developed and used for detecting PADI4 in the serum of cancers such as breast cancer, lung cancer, kidney cancer, bladder cancer, colon cancer, uterine cancer, ovarian cancer and the like, and the kit has important significance for screening, diagnosing and treating the tumors.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of the PADI4 (peptidyl arginine deiminase 4) in preparing the tumor diagnosis kit, and the detection kit has high repeatability, good stability, broad spectrum and detectability and can be used for clinical diagnosis of tumors.

In order to achieve the purpose, the invention adopts the technical scheme that:

a tumor diagnosis kit comprising:

the kit comprises an enzyme label plate coated with a protein PADI4 monoclonal antibody, a control sample, a washing solution, a stop solution, a diluent, horseradish peroxidase labeled streptavidin (HRP-SA) and a horseradish peroxidase chromogenic substrate;

the protein PADI4 monoclonal antibody comprises an antibody 135-B9 and an antibody 197-A5;

the antibody 135-B9 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO.3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 5;

the antibody 197-A5 comprises two heavy chains and a light chain, wherein the amino acid sequences of the heavy chains are respectively shown as SEQ ID NO.7 and SEQ ID NO.9, and the amino acid sequence of the light chain is shown as SEQ ID NO. 11.

According to the invention, the enzyme label plate is a 96-hole enzyme label plate.

Preferably according to the invention, the control sample comprises a positive control which is an artificially synthesized PADI4 protein.

Preferably, according to the invention, the diluent is 5% FBS-PBST: adding 10ml FBS (fetal bovine serum) into 90ml PBST solution, and mixing well to obtain the final product.

Preferably according to the invention, the wash solution is a PBST solution: adding Tween-20 into PBS solution at a volume percentage of 0.1%, and mixing uniformly, wherein the pH of the PBS solution is 7.4.

According to the invention, the stop solution is preferably 2mol/L hydrochloric acid solution.

According to the invention, the horse radish peroxidase chromogenic substrate comprises a chromogenic solution A and a chromogenic solution B, wherein the chromogenic solution A is prepared by diluting 30% hydrogen peroxide by a citric acid buffer solution by 1000 times, and the chromogenic solution B is prepared by adding tetramethyl benzidine into a citric acid buffer solution containing 20% dimethyl sulfoxide by mass according to the proportion of 0.4 mg/ml.

Preferably, the antibody 135-B9 is coated on an enzyme label plate, and the antibody 197-A5 is the antibody 197-A5 labeled by biotin.

The preparation method of the tumor diagnosis kit comprises the following steps:

(1) preparing an antigen with an amino acid sequence shown as SEQ ID NO. 1;

(2) preparing an antibody 135-B9; the antibody 135-B9 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO.3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 5;

(3) preparing an antibody 197-A5, and then labeling with biotin to prepare a biotin-labeled antibody 197-A5; the antibody 197-A5 comprises two heavy chains and a light chain, wherein the amino acid sequences of the heavy chains are respectively shown as SEQ ID NO.7 and SEQ ID NO.9, and the amino acid sequence of the light chain is shown as SEQ ID NO. 11;

(4) and (3) coating the antibody 135-B9 prepared in the step (2) on an enzyme label plate, and then assembling the kit to prepare the tumor diagnosis kit.

According to the invention, the marking in the step (3) preferably comprises the following specific steps:

1) dissolving Biotin (Biotin) with N, N-Dimethylformamide (DMF) to obtain Biotin solution with concentration of 20 mg/ml;

2) dissolving the antibody 197-A5 in a PBS buffer solution, and adjusting the pH to 8.5 by using a Carbonate Buffer Solution (CBS) to prepare an antibody 197-A5 solution with the concentration of 1-10 mg/ml;

3) mixing the biotin solution prepared in the step 1) and the antibody 197-A5 prepared in the step 2) according to the proportion of adding 5 mul of biotin solution to each mg of antibody, and stirring for 2 hours at room temperature in a dark place;

4) collecting liquid (a mixed solution of an antibody buffer solution and a biotin buffer solution), dialyzing by using a PBS buffer solution, and replacing the PBS buffer solution for 3-4 times in the process.

According to the invention, the coating of the step (4) preferably comprises the following specific steps:

and (2) diluting the prepared 135-B9 monoclonal antibody to 4 mu g/ml by adopting a direct adsorption method and using PBS buffer solution with the pH value of 9.6, adding the diluted monoclonal antibody to a 96-hole ELISA plate according to the adding amount of 100 mu l/hole, standing for 2 hours at the temperature of 37 ℃, washing by using a washing solution, and drying by drying to obtain the 96-hole ELISA plate coated with the 135-B9 monoclonal antibody.

Introduction to the principle

The tumor marker has important significance for diagnosis, treatment, curative effect judgment, relapse monitoring and prognosis observation of tumors. The blood tumor markers usually used by hospital inspection departments are more than 20, but the sensitivity and specificity of the markers are not satisfactory, the drug treatment effect and the operation treatment effect can be monitored without the tumor markers, and the early tumor screening effect is not high. Moreover, to date, no tumor marker has been available for detecting most malignancies. The kit has high specificity by using the PADI4 as a tumor marker. The existing tumor markers are also highly expressed in the blood of about 10% of healthy people and chronic patients. PADI4 is expressed at significantly higher levels in various malignant tissues and in the blood of patients, and is either underexpressed or not expressed in various benign tumors, chronic inflammation and healthy people.

The avidin used in the application is a basic glycoprotein extracted from ovalbumin, has a molecular weight of 68kDa, and consists of 4 subunitsComposition, having very high affinity for biotin with binding constant up to 1015M^-1(ii) a Biotin is easily combined with proteins such as antibodies and the like through covalent bonds, so that horseradish peroxidase labeled streptavidin molecules combined with enzyme react with biotin molecules combined with specific antibodies, the multi-stage amplification effect is achieved, and the enzymes generate catalysis effect to generate color when meeting corresponding chromogenic substrates, so that the aim of detecting target antigens or antibody molecules is fulfilled.

Advantageous effects

The invention carries out immunization through specific antigen to obtain an antibody 135-B9 and an antibody 197-A5, and establishes a high-sensitivity biotin-avidin-enzyme-linked immunosorbent assay kit through the high amplification effect between biotin and horseradish peroxidase labeled streptavidin on the basis of a conventional enzyme-linked immunosorbent assay to determine the expression level of the protein PADI4 in a sample to be detected. The protein PADI4 detection kit can effectively and stably determine the tumor protein PADI4 level in human serum, and has high specificity and good test repeatability.

Drawings

FIG. 1 is a PAGE image of denatured and non-denatured gels of the two antibodies after purification of the tumor protein monoclonal antibodies 135-B9 and 197-A5;

FIG. 2 shows the results of SDS-PAGE and Western blot analysis of antigen recognized by the tumor protein monoclonal antibody 135-B9;

FIG. 3 is a graph showing the expression level of PADI4 in absorption peaks at 450nm of human serum of breast cancer, post-operation breast cancer, breast fibrosis patients and healthy persons, measured by ELISA;

FIG. 4 is a graph showing the expression level of PADI4 in the absorbance peaks at 450nm of human serum of patients with liver cancer, post-operation liver cancer, hepatic cavernous hemangioma and healthy persons, measured by ELISA;

FIG. 5 is a graph showing the expression level of PADI4 in ovarian cancer, ovarian cancer post-operative patients, and in the absorbance peak at 450nm of healthy human serum, measured by ELISA;

FIG. 6 is a graph showing the detection of the expression level of PADI4 in the absorbance peak at 450nm of human serum of patients with prostate cancer, post-prostate cancer, prostate hyperplasia and healthy persons by ELISA;

FIG. 7 is a graph of antibody pair combination 135(4ug/ml) to 197(0.5ug/ml) against a standard curve;

FIG. 8 is a graph of antibody pair combination 135(4ug/ml) to 197(0.25ug/ml) against a standard curve;

FIG. 9 is a graph of antibody pair combination 135(2ug/ml) to 197(0.25ug/ml) against a standard curve;

FIG. 10 is a graph of antibody pair combination 135(1ug/ml) to 197(0.25ug/ml) against a standard curve;

FIG. 11 is a graph of antibody pair combination 135(4ug/ml) to 197(0.125ug/ml) against a standard curve;

FIG. 12 is a graph of antibody pair combination 135(2ug/ml) to 197(0.125ug/ml) against a standard curve;

FIG. 13 is a graph of antibody pair combination 135(1ug/ml) to 197(0.125ug/ml) against a standard curve;

FIG. 14 is a graph of the standard curve for antibody pairing combination 135-197 biotin;

FIG. 15 is a graph of the standard curve corresponding to antibody pair combination 135-122 biotin;

detailed description of the preferred embodiments

The technical solutions of the present invention are further described below with reference to the following examples and the drawings of the specification, but the scope of the present invention is not limited thereto.

Sources of reagents

96-well enzyme label plate: brand Corning, cargo number: 3599;

PADI4 antigen protein, artificial synthesis

Human serum healthy: from tumor hospital health check-up population of Shandong province;

horseradish peroxidase-streptavidin: brand Jackson, cat no: 016, 030, 084;

the cleaning solution and the stop solution are purchased from Pujian Biotechnology GmbH;

horse radish peroxidase chromogenic substrate: TMB color developing solution purchased from Pujian Biotechnology Ltd;

example 1

Preparation of monoclonal antibody against the protein PADI 4:

(1) antigen preparation:

a) codon optimization and gene synthesis;

the protein PADI4 has 663AAs in total, the molecular weight is 74.47KDa, no signal peptide and no transmembrane helix, and a region with stronger hydrophobicity is arranged at 265-271 AAs. The result of comprehensive homology comparison needs soluble protein as antigen, 1-260AAs (hydrophobic region removed and lower homology) of soluble expression protein PADI4 of escherichia coli system is used for immunizing a mouse to prepare the monoclonal antibody, pET28b is adopted as a carrier, the C end is connected with a 6His tag on the carrier, and the enzyme cutting site is NcoI/XhoI.

b) Plasmid extraction (kit name: endo-free plasma Mini Kit I (50), OMEGA bio-tek):

1) taking 4mL of overnight cultured bacterial liquid, centrifuging at 12000rpm for 1min, and collecting thalli;

2) adding 250 μ L of Solutioni to resuspend the cells;

3) adding 250 μ L Solution II, slightly inverting for 4-6 times to fully crack thallus, standing at room temperature for 2 min;

4) adding 350 μ L Solution III, immediately reversing and mixing uniformly for several times until white flocculent precipitate appears, and centrifuging at 12,000rpm for 10 min;

5) transferring the supernatant collected in the last step into an adsorption column, centrifuging at 12,000rpm for 1min, and pouring off the waste liquid in the collection tube;

6) adding 500 μ L Buffer HB into the adsorption column, centrifuging at 12,000rpm for 1min, and pouring off the waste liquid in the collection tube;

7) adding 700 mu L of DNA Wash Buffer into the adsorption column, centrifuging at 12,000rpm for 1min, pouring off waste liquid, and repeatedly washing once;

8) centrifuging the empty column at 12,000rpm for 2 min;

9) the adsorption column was placed in a sterile centrifuge tube, 50. mu.L of sterile water was added, the tube was left at room temperature for 2min, centrifuged at 12,000rpm for 1min, the plasmid solution was collected in the centrifuge tube, and the procedure was repeated 1 time.

c) Plasmid transformation DH10Bac

1) DH10Bac competent cells (purchased from Invitrogen) were thawed on ice;

2) 100ul of high efficiency DH10Bac competent cells were poured into a pre-cooled 1.5ml tube;

3) adding 3. mu.l of plasmid DNA prepared in step b) to the cells and mixing gently;

4) incubate for 30 minutes on ice;

5) heat shock at 42 degrees for 90 seconds;

6) immediately placing on ice for 2 minutes;

7) adding 900 mul of LB culture medium without resistance;

8) culturing for 4 hours at 37 ℃ and 180rpm by shaking;

9) coating LB plate with different concentrations of 10 mul, 20 mul and 30 mul, and coating three LB plates with each concentration, wherein the LB plate comprises Kan 50 mug/ml, Gentamicin 7 mug/ml, tetracyline 10 mug/ml, X-gal 100 mug/ml and IPTG 40 mug/ml;

10) culturing in a 37-degree incubator for 48 hours in a dark place;

d) blue-white spot screening and verification

1) Picking the white clone cultured in the step c), re-streaking on a fresh LB plate (50. mu.g/ml Kan, 7. mu.g/ml Gentamicin, 10. mu.g/ml tetracyline, 100. mu.g/ml X-gal, 40. mu.g/ml IPTG), and culturing at 37 ℃ in the dark for 48 h;

2) inoculating 16 white spot clones into 5ml of liquid containing 50 mu g/ml Kan, 7 mu g/ml Gentamici and 10 mu g/ml Mettracycline respectively, and culturing at 37 ℃ and 200rpm overnight;

3) PCR (polymerase chain reaction) verification and recombination Bacmid, and selecting No. 1-16 monoclonal strains for verification;

4) the positive clone identified by PCR is subjected to kit extraction to obtain recombinant plasmid DNA (kit name: endo-freeplasmid Mini Kit I (50), OMEGA bio-tek);

e) preparation of recombinant baculovirus P1 generation (transfection of Sf9 cells in 6-well plates)

1) 9 × 10 inoculum on 6-well plates containing 2ml of SFX medium containing antibiotics per well⁵Sf9 cells, cultured at 27 ℃ for 1 hour;

2) preparation of transfection reagents: add 2. mu.g of purified recombinant plasmid DNA to 100. mu.l of antibiotic-free SFX medium; completely mixing the Cellffectin reagent, and turning and mixing for 5-10 times; aspirate 6 μ l of Cellffectin reagent and add to 100 μ l of antibiotic-free SFX medium; adding Cellffectin reagent to the culture medium containing plasmid DNA (total volume about 210 u l); gently mixing at room temperature for 3min, and incubating at room temperature for 15 min;

3) during incubation of the DNA/transfection reagent mixture, the medium was removed from the cells and washed with 2ml of antibiotic-free and serum-free medium, and the wash medium was discarded;

4) adding 0.8ml of antibiotic-free and serum-free culture medium into each tube containing the mixture, gently mixing uniformly, and adding the mixture into the holes containing the cells;

5) culturing at 27 ℃ for 5 hours;

6) the medium was removed and 5ml of whole medium (50 units/ml penicillin and 50. mu.g/ml streptomycin, 5% serum) was added to the cells;

7) after culturing at 27 ℃ for 1 week, centrifuging at 1000rpm/min for 5 minutes, and taking the supernatant as P1 virus strain (culturmedium);

f) p1 stands for Dada test

1) Adding 10ml of PBS buffer solution into the cells obtained by centrifugation, and carrying out ultrasonic treatment for 2s at intervals of 2s for 3 min; centrifuging at 12000rpm for 2min to separate supernatant (native) and precipitate (precipitated), and dissolving the precipitate with 8Murea + PBS;

2) collecting virus supernatant (culture medium), supernatant (native) and precipitate (denated), and performing SDS-PAGE (12%, 80v concentrated gel, 20min, 120v separation gel, 45min) on 12 μ l sample to verify expression;

g) preparation of P2 Generation Virus

1) P1 represents that the test result shows that the target protein is expressed, and the P2 generation virus preparation is continued;

2) a200 ml volume of cells was prepared and a 200. mu.l volume of P1 passage virus was added.

3) Culturing the cells in a 27-degree humid incubator for 1 week, centrifuging at 500 Xg for 5 minutes to remove cells and debris, and collecting the obtained supernatant, namely the P2 generation virus;

h) MOI testing

1) 30ml of cell supernatant (2 × 10) in good cell state was added to a six-well plate⁵Cells/ml);

2) adding 30 μ l, 150 μ l and 300ul of P2 generation virus solution, culturing for 48h and 72h, and sampling 1.5ml for expression test and identification;

3) carrying out ultrasonic treatment on the collected sample for 2s at intervals of 2s for 3 min; centrifuging at 12000rpm for 2min to separate supernatant (native) and precipitate (precipitated), and dissolving the precipitate with 8Murea + PBS;

4) SDS-PGAE was identified as above;

i)200ml purification test

1) According to the MOI test result, selecting 30 mu l P2 virus infection for 72h as a purification test condition;

2) culturing 200ml cells until the growth condition is good, and detecting the cell density to be 3 × 10⁶Adding 30 mu l of P2 generation virus, culturing for 72h, and collecting a sample;

3)12000rpm, centrifuging for 30min and collecting supernatant;

4) and (3) purification: affinity purification of STREP tag by using Strep-tactin resin;

(2) preparation of monoclonal antibody:

1) animal immunization: immunizing a mouse by using the prepared antigen for four times, and performing impact immunization before fusion for 1-2 times;

2) cell fusion and screening: and selecting mice with better serum immune results, fusing for 1-2 times, performing 5-6 rounds of Elisa screening (needing detection cross reaction) on each fusion, obtaining monoclonal antibodies in the first round, establishing strains, producing ascites, and pairing. If no matched antibody is obtained, performing the second round of cell fusion, establishing a cell strain obtained by the second round of cell fusion, producing ascites, and then matching with the cell strain obtained by the first round. Thereby obtaining two hybridoma cell strains with the capability of stably secreting the monoclonal antibody of the protein PADI4, which are respectively named as hybridoma cell strain 135-A1-B9 and hybridoma cell strain 197-C11-A5;

3) ascites production and purification: injecting the cultured hybridoma cells into abdominal cavity of mouse, producing ascites after 1-2 weeks, purifying the ascites by protein A/G, and purifying to obtain protein PADI4 monoclonal antibodies 135-B9 and 197-A5;

FIG. 1 is a PAGE graph showing denatured and non-denatured gels of the tumor proteins after the monoclonal antibodies 135-B9 and 197-A5 were purified, wherein lane 1 shows SDS-PAGE results of the heavy and light chains of the monoclonal antibody 135-B9 after denaturation, lane 2 shows SDS-PAGE results of the heavy and light chains of the monoclonal antibody 197-A5 after denaturation, lane 3 shows SDS-PAGE results before the monoclonal antibody 135-B9 is denatured, and lane 4 shows SDS-PAGE results before the monoclonal antibody 197-A5 is denatured. FIG. 2 shows the results of Westernblot analysis of antigen recognition by monoclonal antibody 135-B9, wherein lane 1 shows the results of SDS-PAGE analysis of 0.5. mu.g antigen recognition by monoclonal antibody 135-B9, and lane 2 shows the results of SDS-PAGE analysis of 0.25. mu.g antigen recognition by monoclonal antibody 135-B9;

4) and (3) detecting the antibody titer after purification: detecting titer by indirect ELISA method, if the titer is more than 1:64000, if not, re-culturing cell strain corresponding to the antibody, and injecting the cell into mouse to produce ascites purified antibody;

5) the purified antibody was labeled (Biotin)

i) Biotin was dissolved in DMF at a concentration of 20 mg/ml;

ii) the antibody was dissolved in PBS and adjusted to pH8.5 using CBS to a final concentration of 1-10 mg/ml;

iii) adding 5 mul biotin solution to each mg of antibody, and stirring for 2 hours at room temperature in a dark place;

iv) collect the reaction using PBS dialysis overnight, midway through the change of PBS 3-4 times.

Biotinylated monoclonal antibody 197-A5

1) Biotin was dissolved in DMF at a concentration of 20 mg/ml.

2) The antibody was dissolved in PBS and adjusted to pH8.5 using CBS to a final concentration of 1-10 mg/ml.

3) 5ul of biotin solution was added per mg of antibody, and the mixture was stirred at room temperature for 2 hours in the dark.

4) The collected reaction was dialyzed overnight against PBS, and PBS was changed 3 to 4 times in the middle.

The components are assembled into a tumor diagnosis kit in a conventional manner.

Coating a 96-well enzyme label plate:

and (2) diluting the prepared 135-B9 monoclonal antibody to 4 mu g/ml by adopting a direct adsorption method and using PBS buffer solution with the pH value of 9.6, adding the diluted monoclonal antibody to a 96-hole ELISA plate according to the adding amount of 100 mu l/hole, standing for 2 hours at the temperature of 37 ℃, washing by using a washing solution, and drying by drying to obtain the 96-hole ELISA plate coated with the 135-B9 monoclonal antibody.

Example 2

The procedure for using the kit described in example 1 was as follows:

1) the antibody immobilized plate returns to room temperature for equilibrium.

2) Standard sample dilutions (PADI4) were added, diluted with dilutions from 10ng/ml to 0.015625ng/ml for a total of 7 gradients, 100. mu.l per well, the final well being the final blank, 100. mu.l of dilution was added, and 100. mu.l of sample was measured at 37 ℃, 1.5 h.

3) The plates were washed 5 times with PBST 300. mu.l and blotted dry.

4) 197-C11-A5-Antibody-Bio was added and diluted to 2ug/ml with diluent for use, 100. mu.l per well, 37 ℃ for 1 h.

5) The plates were washed 5 times with PBST 300. mu.l and blotted dry.

6) Streptavidin-HRP was added and diluted 1:2000 with diluent for use, 100. mu.l per well, 37 ℃ for 30 min.

7) The plates were washed with PBST 300ul 5 times and the liquid was patted dry.

8) TMB developed 100. mu.l per well for 10min at 37 ℃.

9) Stop with 2M HCl, 50. mu.l per well.

3. And (3) detection results:

within 15min after the stop solution is added, detecting the optical density OD value of each detection hole by using an enzyme-linked immunosorbent assay under the condition of the wavelength of 450nm, wherein the detection standard of the detection kit is as follows: and judging the detection sample to be positive when the OD value of the serum to be detected is more than the OD value, or judging the detection sample to be negative. Through detection, the coefficient of variation CV value of the coated 96-well enzyme label plate prepared by the invention is less than 20%, test tests are carried out on the detection kits of the same batch and different batches, and the test results show that the CV values of the kits in batches and among batches are less than 20%, which indicates that the PADI4 detection kit has high precision.

Example 3 clinical sample testing using the PADI4 test kit of the invention:

example 1: 112 cases of breast cancer, 86 cases after breast cancer surgery, 77 cases of hepatocellular carcinoma, 24 cases after hepatocellular carcinoma surgery, 64 cases of esophageal cancer, 24 cases after esophageal cancer surgery, 94 cases of gastric cancer, 43 cases after gastric cancer surgery, 2 cases of colon cancer, 15 cases after colon cancer surgery, 19 cases of rectal cancer, 28 cases after rectal cancer surgery, 21 cases of pancreatic cancer, 6 cases after pancreatic cancer surgery, 29 cases of ovarian cancer, 11 cases after ovarian cancer surgery, and 160 cases of a normal control group matched with gender and age were tested, and the results are shown in table 1:

TABLE 1 tumor patients and patients post-operative and normal PADI4 level test data

All test data are obtained by 3 times of repeated tests, and the PADl4 has very high expression in the serum of various malignant tumor patients with breast cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer and ovarian cancer compared with a healthy control group through a t test, and the difference has statistical significance (P < 0.05). The expression of PADl4 in the serum of patients after operations such as breast cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer and the like is obviously reduced.

Example 2: the blood of a tumor patient is detected by an ELISA method, and the PADI4 and the citrullinated antithrombin product thereof are found to be obviously expressed in the blood of patients with breast cancer, lung cancer, kidney cancer, bladder cancer, colon cancer, uterine cancer, ovarian cancer and the like, but are not expressed or are expressed in a low amount in normal people. As shown in fig. 3, the expression level of PADI4 was measured in the serum of various tumor patients as well as postoperative and healthy persons by ELISA. Each bar in the histogram represents the absorption peak at 450nm of a patient's or healthy human serum. The result shows that compared with the serum of patients after benign tumors and tumor resection operations and the serum of normal human, the expression level of the PADI4 in the serum of patients with malignant tumors such as breast cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, bladder cancer and the like is obviously increased.

Comparative example 1

To further illustrate the outstanding effect of the technical solution obtained in the present invention in detecting the expression level of the protein PADI4, the effect of other antibodies obtained by screening under the same conditions in the development process of the present invention was selected to be compared with the effect of the antibodies described in the present application:

(1) establishment of a double-antibody sandwich ELISA detection system: and (2) producing antibodies from 26 monoclonal cell strains obtained by screening, selecting 21 strains of antibodies with better titer for biotin labeling after purification, adopting a chessboard titration experiment, detecting whether the 26 strains of monoclonal antibodies can be matched with the 18 strains of biotin labeled antibodies by a double-antibody sandwich ELISA method, taking the PADI4 protein as a standard protein and the PADI2 protein as a negative control, selecting 9 strains of capture antibodies and 9 strains of biotin labeled antibodies, and carrying out next endogenous detection.

(2) Paired antibody detection of endogenous samples: a chessboard titration experiment is adopted, 9 monoclonal antibodies and 9 biotin-labeled antibodies which are detected and screened by a double-antibody sandwich ELISA method are paired, the screened antibodies are respectively used as capture antibodies to coat an ELISA plate, the biotin-labeled antibodies are used as detection antibodies, the PADI4 protein is used as a standard protein, the PADI2 protein is used as a negative control protein, and positive serum and negative serum provided by the Paris A are used as endogenous samples to be paired and verified respectively. The next optimization experiment was performed by selecting the 10 antibody pairs that gave the greatest P/N values (as shown in table 2 below) and the most sensitive responses by the checkerboard titration experiment.

TABLE 2P/N values of positive and negative samples tested by each group of antibody pairs

(3) Preliminary screening of optimal antibody pairs: the 10 groups of antibody pairs (see table 2) determined in the previous step were tested for graded dilutions of PADI2 protein, PADI4 protein, positive sera, negative sera, with the following results:

table 310 group of antibodies on the OD value of the negative control protein

Plate-1	1	2	3	4	5	6	7	8	9	10
											PAID2	Antibody 135	Antibody 135	Antibody 135	Antibody 135	Antibody 135	Antibody 135	Antibody 197	Antibody 197	Antibody 200	Antibody 200
A	0.04	0.05	0.04	0.03	0.04	0.06	0.06	0.06	0.32	0.30
											B	0.03	0.03	0.03	0.03	0.05	0.03	0.05	0.05	0.30	0.30
C	0.03	0.03	0.03	0.02	0.05	0.03	0.05	0.06	0.29	0.30
											D	0.03	0.03	0.03	0.03	0.05	0.03	0.05	0.05	0.33	0.28
E	0.04	0.03	0.03	0.03	0.05	0.03	0.05	0.05	0.29	0.29
											F	0.05	0.03	0.03	0.03	0.05	0.03	0.05	0.06	0.27	0.27
G	0.03	0.03	0.03	0.03	0.05	0.03	0.05	0.06	0.27	0.25
											H	0.03	0.04	0.04	0.03	0.06	0.04	0.06	0.06	0.26	0.21
	Biotin-139	Biotin-148	Biotin-197	Biotin-65	Biotin-122	Biotin-137	Biotin-135	Biotin-200	Biotin-148	Biotin-197

The data in table 3 indicate that none of the group 10 antibody pairs recognized the negative control protein PADI2, and there was no cross-reaction.

Table 410 group antibody pairs test Positive control protein OD values

Plate-2	1	2	3	4	5	6	7	8	9	10
											PAID4	Antibody 135	Antibody 135	Antibody 135	Antibody 135	Antibody 135	Antibody 135	Antibody 197	Antibody 197	Antibody 200	Antibody 200
A	4.12	4.15	4.32	2.4	3.17	4.23	4.13	4.26	3.843	3.93
											B	3.85	3.92	3.86	1.43	1.96	3.21	3.95	3.95	3.62	2.93
C	2.49	2.77	2.83	0.65	0.86	2.98	3.53	3.08	2.53	2.06
											D	1.66	1.97	1.79	0.39	0.48	1.89	2.47	2.58	1.41	1.05
E	1.10	1.19	1.19	0.19	0.27	1.12	1.69	1.58	0.81	0.70
											F	0.65	0.67	0.65	0.11	0.16	0.57	0.92	0.85	0.47	0.44
G	0.38	0.39	0.37	0.06	0.10	0.34	0.46	0.54	0.32	0.37
											H	0.04	0.04	0.04	0.03	0.04	0.02	0.04	0.04	0.18	0.17
	Biotin-139	Biotin-148	Biotin-197	Biotin-65	Biotin-122	Biotin-137	Biotin-135	Biotin-200	Biotin-148	Biotin-197

The data in table 4 illustrate that the 10 antibody pairs recognize the positive control protein PADI 4.

Table 510 sets of antibody pairs for testing positive and negative serum OD values

From the data in table 5, 5 groups of antibody pairs with positive serum OD/negative serum OD greater than 3 fold were selected ( columns 1, 2, 3, 5 and 6) for further optimization of the conditions for antibody capture and detection: the capture antibody 135 was set to three concentration gradients (1/2/4ug/ml), 5 detection antibodies were set to 3 concentration gradients (0.5/1/2ug/ml) respectively for detection of PADI4 protein (dilution at a concentration of 0.1ug/ml-0.0015625ug/ml fold), negative serum (dilution multiple 1/2/4 fold) and positive serum (dilution multiple 1/2/4 fold), and the plates were as shown in table 6:

TABLE 6

The data are shown in Table 7 (black bold for PADI4 data-standard; column 3, 4, 7 and 8, D, E, F line negative serum data (1/2/4 fold dilution); remaining black non-bold for positive serum data):

TABLE 7

As can be seen from the data in Table 7, the OD value of the shaded area data decreases with the increase of the dilution factor of the serum; the corresponding antibody pair combinations were 135(4ug/ml) to 197(0.5ug/ml), and the corresponding standards are shown in Table 8 below and FIG. 7:

TABLE 8

(4) The antibody pairing combination 135(4ug/ml) -197(0.5ug/ml) was continued to be condition optimized to determine the optimal working concentration of antibody pair:

optimizing conditions: the capture antibody 135 is set to three concentration gradients (4/2/1ug/ml), the detection antibody 197 is set to 3 concentration gradients (0.5/1/2ug/ml) respectively to detect the PADI4 protein (concentration 50ng/ml-0.78125ng/ml double-ratio dilution);

and (4) optimizing the conditions on the basis of the first step: the capture antibody 135 is set to 1 concentration gradient (4ug/ml), the detection antibody 197 is set to 2 concentration gradients (0.25/0.125ug/ml) respectively to detect the PADI4 protein (concentration 50ng/ml-0.78125ng/ml double-ratio dilution);

and on the basis of the second step, continuously carrying out condition optimization: setting three concentration gradients (4/2/1ug/ml) for the capture antibody 135, setting 2 concentration gradients (0.25/0.125ug/ml) for the detection antibody 197, and performing PADI4 protein detection (concentration 50ng/ml-0.78125ng/ml double-ratio dilution);

the data of each condition combination are shown in tables 9 to 14 and fig. 8 to 13:

TABLE 9

Watch 10

TABLE 11

TABLE 12

Watch 13

TABLE 14

The above six antibody pair data (R) were compared in combination²Values, OD value for same antigen concentration, detection antibody concentration value (sensitivity)), antibody for optimal working concentration as follows: antibody 135(4ug/ml) -PADI4 protein (50ng/ml-0.78125ng/ml) -197-bio (0.125 ug/ml).

To summarize:

displaying through the cloth board and the detected data: from the OD values of the positive serum, the negative serum and the blank control at 3 dilution times (1 ×, 2 ×, 4 ×), the ratio of the OD value of the positive serum to the OD value of the negative serum at each dilution time (minus the blank background value) was calculated, and the results are shown in table 15:

watch 15

Through comprehensive comparison of the data, each antibody pair respectively detects the PADI4 protein and the PADI2 protein (de-cross screening), and positive serum and negative serum are screened for OD values to determine five groups of antibody pairs; the five groups of antibody pairs are better for 135-197biotin and 135-122biotin by comparing the positive serum/negative serum ratio of the antibody pairs (the ratio is high and the negative discrimination is good) under the condition of detecting the same serum dilution, and the corresponding markers are shown in the table 16-table 17 and the figure 14-figure 15:

TABLE 16

TABLE 17

Comparison of the sensitivity (OD values for the same antigen concentration) and correlation of the standards (R) between the two pairs of antibodies²) The 135-197biotin antibody pair performed significantly better than the 135-122 biotin.

SEQUENCE LISTING

<110> Shandong New Biotech Co Ltd

Application of <120> PADI4 in preparation of tumor diagnosis kit

<160>12

<170>PatentIn version 3.5

<210>1

<211>695

<212>PRT

<213> Artificial Synthesis

<400>1

Met Gly Ala Ser Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

20 25 30

Met Ala Gln Gly Thr Leu Ile Arg Val Thr Pro Glu Gln Pro Thr His

35 40 45

Ala Val Cys Val Leu Gly Thr Leu Thr Gln Leu Asp Ile Cys Ser Ser

50 55 60

Ala Pro Glu Asp Cys Thr Ser Phe Ser Ile Asn Ala Ser Pro Gly Val

65 70 75 80

Val Val Asp Ile Ala His Gly Pro Pro Ala Lys Lys Lys Ser Thr Gly

85 90 95

Ser Ser Thr Trp Pro Leu Asp Pro Gly Val Glu Val Thr Leu Thr Met

100 105 110

Lys Val Ala Ser Gly Ser Thr Gly Asp Gln Lys Val Gln Ile Ser Tyr

115 120 125

Tyr Gly Pro Lys Thr Pro Pro Val Lys Ala Leu Leu Tyr Leu Thr Gly

130 135 140

Val Glu Ile Ser Leu Cys Ala Asp Ile Thr Arg Thr Gly Lys Val Lys

145 150 155 160

Pro Thr Arg Ala Val Lys Asp Gln Arg Thr Trp Thr Trp Gly Pro Cys

165 170 175

Gly Gln Gly Ala Ile Leu Leu Val Asn Cys Asp Arg Asp Asn Leu Glu

180 185 190

Ser Ser Ala Met Asp Cys Glu Asp Asp Glu Val Leu Asp Ser Glu Asp

195 200 205

Leu Gln Asp Met Ser Leu Met Thr Leu Ser Thr Lys Thr Pro Lys Asp

210 215 220

Phe Phe Thr Asn His Thr Leu Val Leu His Val Ala Arg Ser Glu Met

225 230 235 240

Asp Lys Val Arg Val Phe Gln Ala Thr Arg Gly Lys Leu Ser Ser Lys

245 250 255

Cys Ser Val Val Leu Gly Pro Lys Trp Pro Ser His Tyr Leu Met Val

260 265 270

Pro Gly Gly Lys His Asn Met Asp Phe Tyr Val Glu Ala Leu Ala Phe

275 280 285

Pro Asp Thr Asp Phe Pro Gly Leu Ile Thr Leu Thr Ile Ser Leu Leu

290 295 300

Asp Thr Ser Asn Leu Glu Leu Pro Glu Ala Val Val Phe Gln Asp Ser

305 310 315 320

Val Val Phe Arg Val Ala Pro Trp Ile Met Thr Pro Asn Thr Gln Pro

325 330 335

Pro Gln Glu Val Tyr Ala Cys Ser Ile Phe Glu Asn Glu Asp Phe Leu

340 345 350

Lys Ser Val Thr Thr Leu Ala Met Lys Ala Lys Cys Lys Leu Thr Ile

355 360 365

Cys Pro Glu Glu Glu Asn Met Asp Asp Gln Trp Met Gln Asp Glu Met

370 375 380

Glu Ile Gly Tyr Ile Gln Ala Pro His Lys Thr Leu Pro Val Val Phe

385 390 395 400

Asp Ser Pro Arg Asn Arg Gly Leu Lys Glu Phe Pro Ile Lys Arg Val

405 410 415

Met Gly Pro Asp Phe Gly Tyr Val Thr Arg Gly Pro Gln Thr Gly Gly

420 425 430

Ile Ser Gly Leu Asp Ser Phe Gly Asn Leu Glu Val Ser Pro Pro Val

435 440 445

Thr Val Arg Gly Lys Glu Tyr Pro Leu Gly Arg Ile Leu Phe Gly Asp

450 455 460

Ser Cys Tyr Pro Ser Asn Asp Ser Arg Gln Met His Gln Ala Leu Gln

465 470 475 480

Asp Phe Leu Ser Ala Gln Gln Val Gln Ala Pro Val Lys Leu Tyr Ser

485 490 495

Asp Trp Leu Ser Val Gly His Val Asp Glu Phe Leu Ser Phe Val Pro

500505 510

Ala Pro Asp Arg Lys Gly Phe Arg Leu Leu Leu Ala Ser Pro Arg Ser

515 520 525

Cys Tyr Lys Leu Phe Gln Glu Gln Gln Asn Glu Gly His Gly Glu Ala

530 535 540

Leu Leu Phe Glu Gly Ile Lys Lys Lys Lys Gln Gln Lys Ile Lys Asn

545 550 555 560

Ile Leu Ser Asn Lys Thr Leu Arg Glu His Asn Ser Phe Val Glu Arg

565 570 575

Cys Ile Asp Trp Asn Arg Glu Leu Leu Lys Arg Glu Leu Gly Leu Ala

580 585 590

Glu Ser Asp Ile Ile Asp Ile Pro Gln Leu Phe Lys Leu Lys Glu Phe

595 600 605

Ser Lys Ala Glu Ala Phe Phe Pro Asn Met Val Asn Met Leu Val Leu

610 615 620

Gly Lys His Leu Gly Ile Pro Lys Pro Phe Gly Pro Val Ile Asn Gly

625 630 635 640

Arg Cys Cys Leu Glu Glu Lys Val Cys Ser Leu Leu Glu Pro Leu Gly

645 650 655

Leu Gln Cys Thr Phe Ile Asn Asp Phe Phe Thr Tyr His Ile Arg His

660665 670

Gly Glu Val His Cys Gly Thr Asn Val Arg Arg Lys Pro Phe Ser Phe

675 680 685

Lys Trp Trp Asn Met Val Pro

690 695

<210>2

<211>2109

<212>DNA

<213> Artificial Synthesis

<400>2

gaattcgccg ccaccatggg cgcttcctgg agccaccccc agttcgagaa gggtggtggc 60

agcggtggcg gcagcggagg ttcagcttgg tcccaccctc agttcgaaaa gatggcccag 120

ggtactctga tccgtgtcac ccccgagcag cctacccacg ccgtctgcgt gctgggtacc 180

ctgactcagc tggacatctg ctccagcgct cctgaggact gcacttcctt cagcatcaac 240

gcttcccctg gtgtcgtcgt ggacatcgcc cacggtcctc ccgccaagaa gaagtccacc 300

ggtagcagca cttggcctct ggaccctggt gtggaggtca ccctgactat gaaggtggct 360

tccggcagca ccggcgacca gaaggtccag atcagctact acggtcctaa gacccccccc 420

gtgaaggctc tgctgtacct gactggcgtc gagatcagcc tgtgcgctga catcacccgt 480

actggcaagg tgaagcctac ccgtgctgtg aaggaccagc gcacttggac ctggggccct 540

tgcggtcagg gtgctatcct gctggtcaac tgcgaccgtg acaacctgga gtcctccgct 600

atggactgcg aagacgacga agtgctggac agcgaggacc tgcaggacat gtccctgatg 660

actctgagca ctaagactcc caaggacttc ttcaccaacc acaccctggt cctgcacgtc 720

gcccgcagcg agatggacaa ggtgcgcgtc ttccaggcta cccgcggtaa actgtcctcc 780

aagtgcagcg tcgtgctggg ccctaagtgg ccttcccact acctgatggt ccccggtggt 840

aaacacaaca tggacttcta cgtcgaggcc ctggccttcc ctgacactga cttccctggt 900

ctgatcactc tgaccatctc cctgctggac actagcaacc tggaactgcc cgaggccgtg 960

gtgttccagg acagcgtcgt gttccgtgtg gctccctgga tcatgacccc caacacccag 1020

ccccctcagg aagtgtacgc ttgcagcatc ttcgaaaacg aggacttcct gaagagcgtc 1080

actaccctgg ctatgaaggc caagtgcaag ctgaccatct gccctgagga agagaacatg 1140

gacgaccagt ggatgcagga cgaaatggag atcggctaca tccaggctcc tcacaagact 1200

ctgcccgtcg tgttcgacag cccccgtaac cgcggtctga aggaatttcc catcaagcgc 1260

gtgatgggcc ctgacttcgg ttacgtgacc cgtggtcccc agactggcgg tatctccggc 1320

ctggacagct tcggcaacct ggaggtctcc ccccctgtga ctgtgcgcgg caaggagtac 1380

cccctgggtc gcatcctgtt cggcgactcc tgctaccctt ccaacgactc ccgtcagatg 1440

caccaggctc tgcaggactt cctgagcgcc cagcaggtgc aggctcctgt gaagctgtac 1500

agcgactggc tgagcgtcgg tcacgtcgac gagttcctgt ccttcgtgcc tgcccctgac 1560

cgcaagggct tccgcctgct gctggccagc cccagatcct gctacaagct gttccaggag 1620

cagcagaacg aaggccacgg tgaggctctg ctgttcgaag gcatcaagaa gaagaagcag 1680

cagaagatca agaacatcct gagcaacaag actctgcgcg aacacaactc cttcgtcgaa 1740

cgttgcatcg actggaaccg tgaactgctg aagcgcgagc tgggcctggc cgagagcgac 1800

atcatcgaca tccctcagct gttcaagctg aaggaatttt ctaaggccga agcctttttc 1860

cctaacatgg tgaacatgct ggtgctgggt aaacacctgg gtatccctaa gcccttcggt 1920

cccgtcatca acggccgctg ctgcctggag gagaaggtct gctccctgct ggagcccctg 1980

ggtctgcagt gcaccttcat caacgacttc ttcacttacc acatccgtca cggcgaagtg 2040

cactgcggta ccaacgtgcg ccgcaagcct ttctccttca agtggtggaa catggtgcct 2100

taaaagctt 2109

<210>3

<211>469

<212>PRT

<213> Artificial Synthesis

<400>3

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Ile Leu Gln

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

35 40 45

Ser Thr Ser Gly Met Cys Val Gly Trp Ile Arg Gln Pro Ser Gly Lys

50 55 60

Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr

65 70 75 80

Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser

8590 95

Asn Gln Val Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala

100 105 110

Thr Tyr Tyr Cys Ala Arg Tyr Tyr Arg Phe Asp Glu Gly Phe Asp Tyr

115 120 125

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly Lys

465

<210>4

<211>1419

<212>DNA

<213> Artificial Synthesis

<400>4

gccgccacca tgaagcacct gtggttcttt ctgctgctgg tggccgctcc tagatgggtg 60

ctgagcgagg tgaagctgca ggagtcagga cctgggatat tgcagccctc ccagaccctc 120

agtctgactt gttctttctc tgggttttca ctgagcactt ctggtatgtg tgtaggctgg 180

attcgtcagc cttcagggaa gggtctggag tggctggcac acatttggtg ggatgatgac 240

aagcgctata acccagccct taagagccga ctgacaatct ccaaggatac ctccagcaac 300

caggttttcc tcaagatcgc cagtgtggac actgcagata ctgccacata ctactgtgct 360

cgatactata ggttcgacga agggtttgac tactggggcc aaggcaccac tctcacagtc 420

tcctcagcta gcaccaaggg accttctgtg ttccctctgg ctccttcttc taagtccact 480

tccggtggta cagcagctct gggttgtctg gtgaaggatt acttcccaga accagtgact 540

gtgtcctgga actccggagc tctgacttct ggagtgcata ctttcccagc agtgctgcaa 600

tctagcggac tgtactctct gtcttccgtg gtgactgtgc cttcttcttc cctggggact 660

caaacttaca tctgcaacgt gaaccacaag ccctccaaca ccaaggtgga caagaaggtg 720

gagccaaaga gctgcgataa gacccacacc tgtccacctt gtccagctcc agaactgctg 780

ggtgggcctt ctgtgtttct gttcccacct aagccaaagg ataccctgat gatctctagg 840

accccagaag tgacctgtgt ggtcgtcgat gtgtctcatg aagaccctga agtgaagttc 900

aactggtacg tggacggggt ggaagtgcat aacgcaaaga ccaagcccag ggaagagcaa 960

tacaactcca cctacagggt ggtctccgtc ctgacagtcc tgcatcagga ttggctgaac 1020

ggcaaggagt acaagtgcaa ggtctccaat aaagccctgc ctgcccctat cgagaaaacc 1080

attagcaaag ccaaaggcca gcccagggag ccccaggtct atacactgcc ccccagcagg 1140

gaggagatga caaaaaatca ggtcagcctg acatgcctgg tcaaaggctt ttatcccagc 1200

gacattgccg tcgagtggga gtccaatggc cagcccgaga ataattataa aacaacaccc 1260

cccgtcctgg acagcgacgg cagctttttt ctgtatagca aactgacagt cgataaaagc 1320

aggtggcagc agggcaatgt cttttcctgc agcgtcatgc acgaggccct gcacaatcac 1380

tatactcaga aaagcctgag cctgtccccc gggaaatga 1419

<210>5

<211>239

<212>PRT

<213> Artificial Synthesis

<400>5

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Ile Thr Gln Asp Glu Leu Ser Asn Pro

20 25 30

Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser

35 40 45

Leu Leu His Ser Asp Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>6

<211>729

<212>DNA

<213> Artificial Synthesis

<400>6

gccgccacca tggtgctgca gacccaggtg ttcatctctc tgctgctgtg gatctccggc 60

gcctacggcg atatcgtgat aacccaagat gaactctcca atccagtcac tcttggaaca 120

tcagcttcca tctcctgcag gtctagtaag agtctcctac atagtgatgg catcacttat 180

ttgtattggt atctgcagaa gccaggccag tctcctcagc tcctgattta tcagatgtcc 240

aaccttgcct caggagtccc agacaggttc agtagcagtg ggtcaggaac tgatttcaca 300

ctgagaatca gcagagtgga ggctgaggat gtgggtgttt attactgtgc tcaaaatcta 360

gaacttccgc tcacgttcgg tgctgggacc aagctggagc tgaaacgtac ggtggctgca 420

ccttctgtgt tcatcttccc tccatctgat gagcagctga agtctggaac cgcatctgtc 480

gtctgtctgc tgaacaactt ttaccccagg gaggctaagg tccaatggaa ggtggacaac 540

gccctgcagt ctggtaatag ccaggaaagc gtgaccgaac aggattccaa ggactccacc 600

tactccctgt cctccacact gacactgagc aaagccgact atgaaaagca caaagtgtat 660

gcctgcgagg tcactcatca gggcctgtcc agccccgtga ctaaaagctt taataggggg 720

gagtgctga 729

<210>7

<211>463

<212>PRT

<213> Artificial Synthesis

<400>7

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Ser Leu Val Ala

20 25 30

Pro Ser Gln Asn Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Thr Ser Tyr Ala Val His Trp Phe Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Cys Asn Ser

65 70 75 80

Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr

100 105 110

Tyr Cys Ala Arg Glu Ala Gly Ile Pro Phe Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Leu Thr Val Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

130 135 140

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

145 150 155 160

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

165 170 175

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

180 185 190

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

195 200 205

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

210 215 220

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

225 230 235 240

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

245 250 255

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

260 265 270

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

275 280 285

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

290 295 300

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

305 310 315 320

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

325 330 335

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

340 345 350

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

355 360 365

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

370 375 380

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

385 390 395 400

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

405 410 415

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

420 425 430

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

435 440 445

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460

<210>8

<211>1401

<212>DNA

<213> Artificial Synthesis

<400>8

gccgccacca tgaagcacct gtggttcttt ctgctgctgg tggccgctcc tagatgggtg 60

ctgagcgagg tgaagctgca ggagtcagga cctagcctgg tggctccatc ccagaacctg 120

agcatcacct gcacagtgtc cggcttcagc ctgacctctt acgctgtgca ttggtttagg 180

cagccacctg gcaagggact ggagtggctg ggcgtgatct gggctggagg ctccacaaac 240

tgcaattctg ctctgatgtc ccggctgtct atctccaagg acaacagcaa gtctcaggtg 300

ttcctgaaga tgaactccct gcagaccgac gatacagcca tgtactattg tgcccgcgag 360

gctggcatcc catttgatta ttggggccag ggcaccacac tgaccgtggc tagcaccaag 420

ggaccttctg tgttccctct ggctccttct tctaagtcca cttccggtgg tacagcagct 480

ctgggttgtc tggtgaagga ttacttccca gaaccagtga ctgtgtcctg gaactccgga 540

gctctgactt ctggagtgca tactttccca gcagtgctgc aatctagcgg actgtactct 600

ctgtcttccg tggtgactgt gccttcttct tccctgggga ctcaaactta catctgcaac 660

gtgaaccaca agccctccaa caccaaggtg gacaagaagg tggagccaaa gagctgcgat 720

aagacccaca cctgtccacc ttgtccagct ccagaactgc tgggtgggcc ttctgtgttt 780

ctgttcccac ctaagccaaa ggataccctg atgatctcta ggaccccaga agtgacctgt 840

gtggtcgtcg atgtgtctca tgaagaccct gaagtgaagt tcaactggta cgtggacggg 900

gtggaagtgc ataacgcaaa gaccaagccc agggaagagc aatacaactc cacctacagg 960

gtggtctccg tcctgacagt cctgcatcag gattggctga acggcaagga gtacaagtgc 1020

aaggtctcca ataaagccct gcctgcccct atcgagaaaa ccattagcaa agccaaaggc 1080

cagcccaggg agccccaggt ctatacactg ccccccagca gggaggagat gacaaaaaat 1140

caggtcagcc tgacatgcct ggtcaaaggc ttttatccca gcgacattgc cgtcgagtgg 1200

gagtccaatg gccagcccga gaataattat aaaacaacac cccccgtcct ggacagcgac 1260

ggcagctttt ttctgtatag caaactgaca gtcgataaaa gcaggtggca gcagggcaat 1320

gtcttttcct gcagcgtcat gcacgaggcc ctgcacaatc actatactca gaaaagcctg 1380

agcctgtccc ccgggaaatg a 1401

<210>9

<211>466

<212>PRT

<213> Artificial Synthesis

<400>9

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln LeuGln Glu Ser Gly Pro Glu Leu Glu Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Asn Met Asn Trp Val Lys Gln Arg Asn Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Asn Ile Asp Pro Tyr Asn Gly Gly Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Ser Val Asp Lys Ser Ser Thr

85 90 95

Thr Ala Tyr Met Gln Leu Lys Gly Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Arg Glu Thr Tyr Tyr Gly Asp Ala Met Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Ser Val Thr Val Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly ValHis Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu ProAla Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210>10

<211>1410

<212>DNA

<213> Artificial Synthesis

<400>10

gccgccacca tgaagcacct gtggttcttt ctgctgctgg tggccgctcc tagatgggtg 60

ctgagcgagg tgcagctgca ggagtcaggc cctgagctgg agaagcccgg cgctagcgtg 120

aagatcagct gcaaggcctc cggctactcc ttcaccggct acaatatgaa ttgggtgaag 180

cagagaaacg gcaagagcct ggagtggatc ggcaacatcg atccttacaa cggcggcaca 240

aattacaatc agaagttcaa gggcaaggcc accctgtccg tggataagag cagcacaacc 300

gcctacatgc agctgaaggg cctgacatcc gaggactccg ccgtgtactt ttgcgccagg 360

gagacctact acggcgacgc catggattac tggggccagg gcaccagcgt gacagtggct 420

agcaccaagg gaccttctgt gttccctctg gctccttctt ctaagtccac ttccggtggt 480

acagcagctc tgggttgtct ggtgaaggat tacttcccag aaccagtgac tgtgtcctgg 540

aactccggag ctctgacttc tggagtgcat actttcccag cagtgctgca atctagcgga 600

ctgtactctc tgtcttccgt ggtgactgtg ccttcttctt ccctggggac tcaaacttac 660

atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagaaggt ggagccaaag 720

agctgcgata agacccacac ctgtccacct tgtccagctc cagaactgct gggtgggcct 780

tctgtgtttc tgttcccacc taagccaaag gataccctga tgatctctag gaccccagaa 840

gtgacctgtg tggtcgtcga tgtgtctcat gaagaccctg aagtgaagtt caactggtac 900

gtggacgggg tggaagtgca taacgcaaag accaagccca gggaagagca atacaactcc 960

acctacaggg tggtctccgt cctgacagtc ctgcatcagg attggctgaa cggcaaggag 1020

tacaagtgca aggtctccaa taaagccctg cctgccccta tcgagaaaac cattagcaaa 1080

gccaaaggcc agcccaggga gccccaggtc tatacactgc cccccagcag ggaggagatg 1140

acaaaaaatc aggtcagcct gacatgcctg gtcaaaggct tttatcccag cgacattgcc 1200

gtcgagtggg agtccaatgg ccagcccgag aataattata aaacaacacc ccccgtcctg 1260

gacagcgacg gcagcttttt tctgtatagc aaactgacag tcgataaaag caggtggcag 1320

cagggcaatg tcttttcctg cagcgtcatg cacgaggccc tgcacaatca ctatactcag 1380

aaaagcctga gcctgtcccc cgggaaatga 1410

<210>11

<211>238

<212>PRT

<213> Artificial Synthesis

<400>11

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Val

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

35 40 45

Val Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

85 90 95

Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Thr Asn Glu Asp Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>12

<211>726

<212>DNA

<213> Artificial Synthesis

<400>12

gccgccacca tggtgctgca gacccaggtg ttcatctctc tgctgctgtg gatctccggc 60

gcctacggcg acattgtgct gacccaatct ccagcttctt tggttgtgtc tctagggcag 120

agggccacca tatcctgcag agccagtgaa agtgttgata gttatggcaa tagttttatg 180

cactggtatc agcagaaacc aggacagcca cccaaactcc tcatctatcg tgcatccaac 240

ctagaatctg ggatccctgc caggttcagt ggcagtgggt ctaggacaga cttcaccctc 300

accattaatc ctgtggaggc tgatgatgtt gcaacctatt actgtcagca aactaatgag 360

gatccattca cgttcggctc ggggacaaag ttggaaataa aacgtacggt ggctgcacct 420

tctgtgttca tcttccctcc atctgatgag cagctgaagt ctggaaccgc atctgtcgtc 480

tgtctgctga acaactttta ccccagggag gctaaggtcc aatggaaggt ggacaacgcc 540

ctgcagtctg gtaatagcca ggaaagcgtg accgaacagg attccaagga ctccacctac 600

tccctgtcct ccacactgac actgagcaaa gccgactatg aaaagcacaa agtgtatgcc 660

tgcgaggtca ctcatcaggg cctgtccagc cccgtgacta aaagctttaa taggggggag 720

tgctga 726

Claims (10)

1. A tumor diagnosis kit, comprising:

the kit comprises an enzyme label plate coated with a protein PADI4 monoclonal antibody, a control sample, a washing solution, a stop solution, a diluent, horseradish peroxidase labeled streptavidin (HRP-SA) and a horseradish peroxidase chromogenic substrate;

the protein PADI4 monoclonal antibody comprises an antibody 135-B9 and an antibody 197-A5;

the antibody 135-B9 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO.3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 5;

the antibody 197-A5 comprises two heavy chains and a light chain, wherein the amino acid sequences of the heavy chains are respectively shown as SEQ ID NO.7 and SEQ ID NO.9, and the amino acid sequence of the light chain is shown as SEQ ID NO. 11.

2. The tumor diagnostic kit according to claim 1, wherein the microplate is a 96-well microplate.

3. The tumor diagnostic kit of claim 1, wherein the control sample comprises a positive control, wherein the positive control is an artificially synthesized PADI4 protein.

4. The tumor diagnostic kit according to claim 1, wherein the diluent is 5% FBS-PBST: adding 10ml FBS (fetal bovine serum) into 90ml PBST solution, and mixing well to obtain the final product.

5. The tumor diagnostic kit according to claim 1, wherein the washing solution is a PBST solution: adding Tween-20 into PBS solution at a volume percentage of 0.1%, and mixing uniformly, wherein the pH of the PBS solution is 7.4.

6. The kit for diagnosing tumor according to claim 1, wherein the stop solution is a 2mol/L hydrochloric acid solution.

7. The tumor diagnostic kit according to claim 1, wherein the horseradish peroxidase chromogenic substrate comprises a chromogenic solution A prepared by diluting hydrogen peroxide with a citric acid buffer solution at a mass concentration of 30% by 1000 times, and a chromogenic solution B prepared by adding tetramethylbenzidine to a citric acid buffer solution containing dimethyl sulfoxide with a mass concentration of 20% in a ratio of 0.4 mg/ml;

preferably, the antibody 135-B9 is coated on an enzyme label plate, and the antibody 197-A5 is the antibody 197-A5 labeled by biotin.

8. The method for preparing the tumor diagnosis kit of claim 1, comprising the steps of:

(1) preparing an antigen with an amino acid sequence shown as SEQ ID NO. 1;

(2) preparing an antibody 135-B9; the antibody 135-B9 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO.3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 5;

(3) preparing an antibody 197-A5, and then labeling with biotin to prepare a biotin-labeled antibody 197-A5; the antibody 197-A5 comprises two heavy chains and a light chain, wherein the amino acid sequences of the heavy chains are respectively shown as SEQ ID NO.7 and SEQ ID NO.9, and the amino acid sequence of the light chain is shown as SEQ ID NO. 11;

(4) and (3) coating the antibody 135-B9 prepared in the step (2) on an enzyme label plate, and then assembling the kit to prepare the tumor diagnosis kit.

9. The method according to claim 1, wherein the labeling in step (3) comprises the following steps:

1) dissolving Biotin (Biotin) with N, N-Dimethylformamide (DMF) to obtain Biotin solution with concentration of 20 mg/ml;

2) dissolving the antibody 197-A5 in a PBS buffer solution, and adjusting the pH to 8.5 by using a Carbonate Buffer Solution (CBS) to prepare an antibody 197-A5 solution with the concentration of 1-10 mg/ml;

3) mixing the biotin solution prepared in the step 1) and the antibody 197-A5 prepared in the step 2) according to the proportion of adding 5 mul of biotin solution to each mg of antibody, and stirring for 2 hours at room temperature in a dark place;

4) and collecting a mixed solution of the antibody buffer solution and the biotin buffer solution, dialyzing by using a PBS buffer solution, and replacing the PBS buffer solution for 3-4 times in the process.

10. The preparation method according to claim 1, wherein the coating in the step (4) comprises the following specific steps:

and (2) diluting the prepared 135-B9 monoclonal antibody to 4 mu g/ml by adopting a direct adsorption method and using PBS buffer solution with the pH value of 9.6, adding the diluted monoclonal antibody to a 96-hole ELISA plate according to the adding amount of 100 mu l/hole, standing for 2 hours at the temperature of 37 ℃, washing by using a washing solution, and drying by drying to obtain the 96-hole ELISA plate coated with the 135-B9 monoclonal antibody.

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