ANTIGEN AND ANTIBODIES PREPARED BASED ON PADI4 SERVING AS TUMOR MARKER, AND APPLICATION THEREOF
20220268777 · 2022-08-25
Inventors
- Xiaotian CHANG (Jinan, CN)
- Xueyan LV (Jinan, CN)
- Dongxia YANG (JINAN, CN)
- Yanqiu XING (JINAN, CN)
- Yuxia GAO (Jinan, CN)
- Shili SHAO (Jinan, CN)
- Feng LIU (Jinan, CN)
- Junchao FENG (Jinan, CN)
- Jun XING (Jinan, CN)
- Lin LI (Jinan, CN)
Cpc classification
C12N9/78
CHEMISTRY; METALLURGY
G01N2333/978
PHYSICS
C07K2317/33
CHEMISTRY; METALLURGY
International classification
Abstract
An antigen and antibodies prepared based on peptidylarginine deiminase (PADI4) serving as a tumor marker, and application thereof are disclosed. The antigen has an amino acid sequence shown as SEQ ID NO. 1. Specific antibodies prepared by using the antigen are also disclosed. The protein PADI4 monoclonal antibodies include a protein PADI4 monoclonal antibody coated onto an ELISA plate and a biotin-labeled protein PADI4 monoclonal antibody. A kit prepared by using the antibodies of the present disclosure can effectively and stably determine a protein PADI4 level of a human serum, and has a broad spectrum, detectability, and high test repeatability.
Claims
1. A tumor antigen associated with a peptidylarginine deiminase 4 protein, having an amino acid sequence shown as SEQ ID NO: 1.
2. The tumor antigen according to claim 1, wherein the antigen is expressed by nucleic acid having a nucleotide sequence shown as SEQ ID NO: 2.
3. An antibody against the tumor antigen of claim 1 is antibody 135-B9 or antibody 197-A5.
4. The antibody according to claim 3, the antibody 135-B9 comprises one heavy chain having an amino acid sequence shown as SEQ ID NO: 3 and one light chain having an amino acid sequence shown as SEQ ID NO: 5.
5. The antibody according to claim 4, wherein the heavy chain of the antibody 135-B9 is expressed by a nucleic acid having a nucleotide sequence shown as SEQ ID NO: 4, and the light chain of the antibody 135-B9 is expressed by a nucleic acid having a nucleotide sequence shown as SEQ ID NO: 6.
6. The antibody according to claim 3, wherein the antibody 197-A5 comprises two heavy chains having amino acid sequences shown as SEQ ID NO: 7 and SEQ ID NO: 9, respectively, and one light chain having an amino acid sequence shown as SEQ ID NO: 11.
7. The antibody according to claim 6, wherein the two heavy chains of the antibody 197-A5 are expressed by two nucleic acids having nucleotide sequences shown as SEQ ID NO: 8 and SEQ ID NO: 10, respectively, and the light chain of the antibody 197-A5 is expressed by a nucleic acid having a nucleotide sequence shown as SEQ ID NO: 12.
8. A kit for detecting a tumor comprising an antibody, a tumor antigen, buffers and visualized reagents, wherein the antibody is antibody 135-B9 or antibody 197-A5; the tumor antigen having an amino acid sequence shown as SEQ ID NO: 1.
9. The kit according to claim 8, wherein the antigen is expressed by nucleic acid having a nucleotide sequence shown as SEQ ID NO: 2.
10. The kit according to claim 8, wherein the antibody 135-B9 comprises one heavy chain having an amino acid sequence shown as SEQ ID NO: 3 and one light chain having an amino acid sequence shown as SEQ ID NO: 5.
11. The kit according to claim 8, wherein the antibody 197-A5 comprises two heavy chains having amino acid sequences shown as SEQ ID NO: 7 and SEQ ID NO: 9, respectively, and one light chain having an amino acid sequence shown as SEQ ID NO: 11.
12. The kit according to claim 10, wherein the heavy chain of the antibody 135-B9 is expressed by a nucleic acid having a nucleotide sequence shown as SEQ ID NO: 4, and the light chain of the antibody 135-B9 is expressed by a nucleic acid having a nucleotide sequence shown as SEQ ID NO: 6.
13. The kit according to claim 11, wherein the two heavy chains of the antibody 197-A5 are expressed by two nucleic acids having nucleotide sequences shown as SEQ ID NO: 8 and SEQ ID NO: 10, respectively, and the light chain of the antibody 197-A5 is expressed by a nucleic acid having a nucleotide sequence shown as SEQ ID NO: 12.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0055] The technical solutions of the present disclosure will be further described below with reference to the embodiments and drawings, but the scope of protection of the present disclosure is not limited thereto.
Sources of Reagents
[0056] 96-well ELISA plates with item No. 3599 were purchased from Corning;
[0057] antigen proteins PADI4 were artificially synthesized;
[0058] health human serums were obtained from the health examination people of Shandong
[0059] Cancer Hospital;
[0060] horseradish peroxidase-streptavidin with item No. 016-030-084 was purchased from Jackson;
[0061] washing solutions and stop solutions were purchased from AtaGenix; and
[0062] horseradish peroxidase chromogenic substrates were TMB chromogenic solutions and purchased from AtaGenix.
Example 1
[0063] Preparation of a Protein PADI4 Monoclonal Antibody
[0064] (1) Preparation of an Antigen
[0065] a) Codon Optimization and Gene Synthesis;
[0066] the protein PADI4 contains a total of 663 amino acids (AAs), has a molecular weight of 74.47 KDa, does not have a signal peptide and transmembrane helix, and has a high hydrophobicity region at 265-271 AAs. It is found from results of comprehensive homology comparison that a soluble protein is required to serve as an antigen. 1-260 AAs (a hydrophobic region is removed, and the homology is low) of a soluble protein PADI4 expressed in an Escherichia coli system was used to immunize mice to prepare a monoclonal antibody. A vector was pET28b, the C-terminus was bound to a 6 His tag on the vector, and a restriction site was NcoI/XhoI.
[0067] b) Plasmid Extraction (a Kit: Endo-Free Plasmid Mini Kit I (50), OMEGA Bio-Tek): [0068] 1) 4 mL of overnight cultured bacteria solution was taken and centrifuged at 12,000 rpm for 1 min, and thallus were collected; [0069] 2) 250 μL of Solution I was added to resuspend cells; [0070] 3) 250 μL of Solution II was added, and the thallus were completely lysed by means of gentle up-down reversal for 4 to 6 times and placed at room temperature for 2 min; [0071] 4) 350 μL of Solution III was added, and the mixture was immediately uniformly mixed by means of up-down reversal for several times until white flocculent precipitates appeared and centrifuged at 12,000 rpm for 10 min; [0072] 5) the supernate collected at the previous step was transferred into an adsorption column and centrifuged at 12,000 rpm for 1 min, and a waste liquid in a collection tube was removed; [0073] 6) 500 μL of Buffer HB was added into the adsorption column, the mixture was centrifuged at 12,000 rpm for 1 min, and a waste liquid in the collection tube was removed; [0074] 7) 700 μL of DNA Wash Buffer was added into the adsorption column, the mixture was centrifuged at 12,000 rpm for 1 min, a waste liquid was removed, and the mixture was washed again; [0075] 8) the empty column was centrifuged at 12,000 rpm for 2 min; and [0076] 9) the adsorption column was placed into an aseptic centrifuge tube, 50 μL of aseptic water was added, the centrifuge tube was placed at room temperature for 2 min and centrifuged at 12,000 rpm for 1 min, and a plasmid solution was collected into a new centrifuge tube and subjected to the previous processing again.
[0077] c) Transformation of DH10Bac by Plasmids [0078] 1) DH10Bac competent cells (purchased from Invitrogen) were defrosted on ices; [0079] 2) 100 uL of efficient DH10Bac competent cells was poured into a pre-cooled 1.5 mL tube; [0080] 3) 3 μL of plasmid DAN prepared at step b) was added to the cells, and the mixture was mixed gently; [0081] 4) the cells were incubated on ices for 30 min; [0082] 5) the cells were subjected to heat shock at 42° C. for 90 s; [0083] 6) the cells were placed on ices immediately for 2 min; [0084] 7) 900 μL of nonreactive LB medium was added; [0085] 8) the cells were subjected to shake culture at 180 rpm at 37° C. for 4 h; [0086] 9) 10 μL, 20 μL, and 30 μL of cells were respectively taken and coated onto LB plates according to different concentrations, three LB plates were coated for each concentration, and each of the LB plates contained 50 μg/mL of Kan, 7 μg/mL of Gentamicin, 10 μg/mL of tetracycline, 100 μg/mL of X-gal, and 40 μg/mL of IPTG; and [0087] 10) the cells were placed into an incubator and cultured in the dark at 37° C. for 48 h;
[0088] d) Blue-White Screening and Verification [0089] 1) the white clones cultured at step c) were picked up, streaked onto a new LB plate (containing 50 μg/mL of Kan, 7 μg/mL of Gentamicin, 10 μg/mL of tetracycline, 100 μg/mL of X-gal, and 40 μg/mL of IPTG), and cultured in the dark at 37° C. for 48 h; [0090] 2) 16 clones with white spots were respectively inoculated into 5 mL of solution containing 50 μg/mL of Kan, 7 μg/mL of Gentamici, and 10 μg/mL of tetracycline, and cultured at 200 rpm at 37° C. overnight; [0091] 3) recombinant Bacmid was verified by PCR, and the No. 1 to No. 16 monoclonal strains were verified; [0092] 4) recombinant plasmid DNA of a positive clone identified by PCR was extracted by using a kit (Endo-free plasmid Mini Kit I (50), OMEGA bio-tek);
[0093] e) Preparation of the P1 Generation of Recombinant Baculovirus (the Transfection of Sf9 Cells was Performed on a 6-Well Plate) [0094] 1) 9×10.sup.5 Sf9 cells were inoculated onto a 6-well plate, each well contained 2 mL of SFX medium containing an antibiotic, and the cells were cultured at 27° C. for 1 h; [0095] 2) preparation of transfection reagents: 2 μg of purified recombinant plasmid DNA was added into 100 μL of SFX medium without an antibiotic; a Cellfectin reagent was mixed uniformly and completely by means of up-down reversal for 5 to 10 times; 6 μL of Cellfectin reagent was sucked and added into 100 μL of SFX medium without an antibiotic; the Cellfectin reagent was added into the medium (with a total volume of about 210 μL) containing the plasmid DNA; and the mixture was gently mixed at room temperature for 3 min and incubated at room temperature for 15 min; [0096] 3) during the incubation of the DNA/transfection reagent mixture, the medium was removed from the cells, the cells were washed with 2 mL of medium without an antibiotic and a serum, and the washing medium was removed; [0097] 4) 0.8 mL of medium without an antibiotic and a serum was added into each tube containing the mixture and gently mixed with the mixture, and the mixture was added into the wells containing the cells; [0098] 5) the cells were cultured at 27° C. for 5 h; [0099] 6) the culture solution was removed, and 5 mL of complete medium (containing 50 units/mL of penicillin, 50 μg/mL of streptomycin, and 5% of serum) was added to the cells; [0100] 7) the cells were cultured at 27° C. for 1 week and centrifuged at 1,000 rpm/min for 5 min, and a supernatant was taken as a P1 virus strain (culture medium);
[0101] f) P1 Generation Expression Detection [0102] 1) the cells obtained by centrifugation were added into 10 mL of PBS and subjected to ultrasonic treatment for a total of 3 min, with each ultrasonic time of 2 s and an interval of 2 s; the cells were centrifuged at 12,000 rpm for 2 min to separate a supernate (native) from precipitates (denatured), and the precipitates were dissolved into 8Murea+PBS; [0103] 2) 12 μL of collected virus supernate (culture medium), the supernate (native), and the precipitates (denatured) were respectively sampled and subjected to SDS-PAGE (12%, 80 v spacer gel, 20 min, 120 v spacer gel, 45 min) to verify the expression;
[0104] g) Preparation of the P2 Generation of the Virus [0105] 1) results of the P1 generation expression detection showed that the target protein was expressed, and the P2 generation of the virus was prepared; [0106] 2) 200 mL of the cells was prepared, and 200 μL of the P1 generation of the virus was added; [0107] 3) the cells were placed into a humid incubator and cultured at 27° C. for 1 week, and centrifuged at 500×g for 5 min, and the cells and fragments were removed, and a supernate was collected as the P2 generation of the virus;
[0108] h) MOI Measurement [0109] 1) 30 mL of cell supernate (2×10.sup.5 cells/mL) in good conditions was added into a 6-well plate; [0110] 2) 30 μL, 150 μL, and 300 uL of P2 generation of the virus solution were respectively added to the cells, the cells were cultured for 48 h and 72 h, respectively, and 1.5 mL of cells was sampled for the expression detection and identification; [0111] 3) the collected sample was subjected to ultrasonic treatment for a total of 3 min, with each ultrasonic time of 2 s and an interval of 2 s; the sample was centrifuged at 12,000 rpm for 2 min to separate a supernate (native) from precipitates (denatured), and the precipitates were dissolved into 8Murea+PBS; and [0112] 4) SDS-PGAE identification was performed similarly;
[0113] 200 mL Purification Testing [0114] (1) according to a result of the MOI measurement, 30 μL of P2 generation of the virus was taken to infect the cells for 72 h, which was the condition of purification testing; [0115] (2) 200 mL of cells were cultured until the cells grew in good conditions, after the density of the cells reached 3×10.sup.6, 30 μL of P2 generation of the virus was added, the cells were cultured for 72 h, and a sample was collected; [0116] (3) the sample was centrifuged at 12,000 rpm for 30 min, and a supernate was collected; and [0117] (4) purification: STREP-tag affinity purification was performed by using a Strep-tactin resin; [0118] (5) Preparation of a monoclonal antibody: [0119] 1) animal immunization: the antigen prepared at the previous step was used to immunize mice for 4 times, before fusion, the immunity was impacted, and the fusion was performed for 1 to 2 times; [0120] 2) cell fusion and screening: mice with high serum immunity were selected for cell fusion, the fusion was performed for 1 to 2 times, 5 to 6 rounds of ELISA screening were performed for each fusion (a cross-reaction was detected), in the first round, monoclonal antibodies were obtained, a strain was established, ascites was produced, and the antibody were paired. If no paired antibodies were obtained, the second round of cell fusion was performed, a cell strain obtained in the second round of fusion was established, ascites was produced, and then the cell strain was paired with the cell strain obtained in the first round. Thus, two hybridoma cell strain capable of stably secreting protein PADI4 monoclonal antibodies were obtained, and they were respectively named hybridoma cell strain 135-A1-B9 and hybridoma cell strain 197-C11-A5; [0121] 3) ascites production and purification: the cultured hybridoma cells were injected into abdominal cavities of the mice, after 1 to 2 weeks, ascites was produced, and the ascites was purified by protein A/G to prepare the protein PADI4 monoclonal antibodies 135-B9 and 197-A5;
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[0125] Biotin was dissolved into DMF until the concentration was 20 mg/mL;
[0126] ii) the antibody was dissolved into PBS, and a pH value was regulated to 8.5 with CBS until the final concentration was 1 to 10 mg/mL;
[0127] iii) every 1 mg of antibody was added with 5 μL of biotin solution, and the mixture was stirred in the dark at room temperature for 2 h; and
[0128] iv) a reactant was collected and dialyzed with PBS overnight, and PBS was replaced for 3 to 4 times during dialysis.
[0129] Biotinylated Monoclonal Antibody 197-A5 [0130] 1) Biotin was dissolved into DMF until the concentration was 20 mg/mL; [0131] 2) the antibody was dissolved into PBS, and a pH value was regulated to 8.5 with CBS until the final concentration was 1 to 10 mg/mL; [0132] 3) every 1 mg of antibody was added with 5 uL of biotin solution, and the mixture was stirred in the dark at room temperature for 2 h; and [0133] 4) a reactant was collected and dialyzed with PBS overnight, and PBS was replaced for 3 to 4 times during dialysis.
[0134] The above-mentioned components were assembled into a kit for diagnosing a tumor in a conventional manner.
[0135] Coating of 96-Well ELISA Plate:
[0136] the monoclonal antibody 135-B9 prepared at the previous step was diluted to 4 μg/mL with PBS with a pH value of 9.6 by a direct adsorption method, the diluted monoclonal antibody 135-B9 was added into a 96-well ELISA plate according to an addition amount of 100 μL/well, and the ELISA plate was placed at 37° C. for 2 h, washed with a washing solution, and spin-dried to prepare a 96-well ELISA plate coated with the monoclonal antibody 135-B9.
Example 2
[0137] Steps of using the kit of Example 1 were as follows: [0138] 1) the plate coated with the antibody was resuscitated and balanced at room temperature; [0139] 2) standard samples (PADI4) were diluted at seven gradients from 10 ng/mL to 0.015625 ng/mL with diluents, 100 μL of diluted sample was added into each well, the last well was a blank control and added with 100 μL of diluent, and the plate was placed at 37° C. for 1.5 h; [0140] 3) the plate was washed with 300 μL of PBST for 5 times, and was patted dry; [0141] 4) 197-C11-A5-Antibody-Bio was diluted to 2 ug/mL with a diluent, 100 μL of diluted 197-C11-A5-Antibody-Bio was added into each well, and the plate was placed at 37° C. for 1 h; [0142] 5) the plate was washed with 300 μL of PBST for 5 times, and was patted dry; [0143] 6) Streptavidin-HRP was diluted to 1:2,000 with a diluent, 100 μL of diluted Streptavidin-HRP was added into each well, and the plate was placed at 37° C. for 30 min; [0144] 7) the plate was washed with 300 uL of PBST for 5 times, and was patted dry; [0145] 8) 100 μL of TMB chromogenic substrate was added into each well, and a chromogenic reaction was performed at 37° C. for 10 min; and [0146] 9) 50 μL of 2M hydrochloric acid was added into each well to stop the reaction.
[0147] 3. Test Results:
[0148] within 15 min after the stop solution was added, the optical density (OD) of each detection well was measured by using an ELISA analyzer at a wavelength of 450 nm, and a test standard for the kit is that: if OD of a serum to be measured is greater than a certain threshold, the sample is determined to be positive, otherwise, the sample is determined to be negative. Tests found that a coefficient of variation (CV) of the coated 96-well ELISA plate prepared in the present disclosure is less than 20%. Kits of the same batch and different batches were tested, and test results showed that CV of the kits of the same batch and different batches were all less than 20%, which indicates that the PADI4 detection kit of the present disclosure has high accuracy.
Example 3 Clinical Sample Test Using the PADI4 Detection Kit of the Present Disclosure
[0149] Case 1: 112 cases with breast cancer, 86 postoperative cases with breast cancer, 77 cases with hepatocellular carcinoma, 24 postoperative cases with hepatocellular carcinoma, 64 cases with esophageal cancer, 24 postoperative cases with esophageal cancer, 94 cases with stomach cancer, 43 postoperative cases with stomach cancer, 21 cases with colon cancer, 15 postoperative cases with colon cancer, 19 cases with rectal cancer, 28 postoperative cases with rectal cancer, 21 cases with pancreatic cancer, 6 postoperative cases with pancreatic cancer, 29 cases with ovarian cancer, 11 postoperative cases with ovarian cancer, and 160 normal control cases of matched gender and age were tested, and results are shown in Table 1:
TABLE-US-00001 TABLE 1 Detection data of PADI4 levels of subjects with tumor, postoperative subjects, and normal subjects Item Number of Samples Serum OD (X ± SD) Object (cases) (ng/mL) Breast cancer 112 0.4093 ± 0.0233 After breast cancer surgery 86 0.2552 ± 0.022 Health 42 0.1801 ± 0.0211 Hepatocellular carcinoma 77 0.318 ± 0.0281 After hepatocellular 24 0.212 ± 0.0222 carcinoma surgery Health 42 0.0979 ± 0.0155 Esophageal cancer 64 0.4444 ± 0.0289 After esophageal cancer 24 0.2126 ± 0.0244 surgery Health 44 0.2403 ± 0.0603 Stomach cancer 94 0.4189 ± 0.0287 After stomach cancer 43 0.3641 ± 0.0565 surgery Health 38 0.2512 ± 0.0233 Colon cancer 21 0.3417 ± 0.0292 After colon cancer surgery 15 0.1678 ± 0.0224 Health 17 0.1404 ± 0.0299 Rectal cancer 19 0.2632 ± 0.0377 After rectal cancer surgery 28 0.1997 ± 0.026 Health 17 0.2012 ± 0.0378 Pancreatic cancer 21 0.3769 ± 0.0565 After pancreatic cancer 6 0.229 ± 0.0874 surgery Health 60 0.2365 ± 0.0198 Ovarian cancer 29 0.5053 ± 0.0477 After ovarian cancer 11 0.3941 ± 0.0351 surgery Health 23 0.1488 ± 0.0148
[0150] All the test data were obtained from 3 repeated tests. The tests found that the PADI4 expression levels of the serums of the subjects with a variety of malignant tumors, such as breast cancer, hepatocellular carcinoma, esophageal cancer, stomach cancer, colorectal cancer, pancreatic cancer, and ovarian cancer, are higher than those of the serums of the health controls, and differences are statistically significant (P<0.05). The PADI4 expression levels of the serums of the postoperative subjects with breast cancer, hepatocellular carcinoma, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, etc. are significantly decreased.
[0151] Case 2: blood of the subjects with tumor was tested by an ELISA method, and it is found that PADI4 and its product, i.e. citrullinated antithrombin, are significantly expressed in the blood of the subjects with breast cancer, lung cancer, kidney cancer, bladder cancer, colon cancer, uterus cancer, ovarian cancer, etc., but are not or lowly expressed in the normal subjects.
[0152] Control 1
[0153] In order to further described the outstanding effect of the technical solutions obtained by the present disclosure in detecting a protein PADI4 expression level, other antibodies obtained by screening under the same conditions during the development process of the present disclosure were selected as effect controls of the antibodies of the present disclosure. [0154] (1) Establishment of a double-antibody sandwich ELISA detection system: 26 monoclonal cell stains obtained by screening were used to produce antibodies, after purification, 21 strains of antibodies with high potency were selected for biotin labeling, a square titration experiment was performed, whether 26 strains of monoclonal antibodies can be paired with 18 strains of biotin-labeled antibodies was tested by a double-antibody sandwich ELISA method, the protein PADI4 was used as a standard protein, the protein PADI2 was used as a negative control, and 9 strains of capture antibodies and 9 strains of biotin-labeled antibodies were selected for the following endogenesis test. [0155] (2) Endogenous sample detection using paired antibodies: a square titration experiment was performed, whether the screened 9 strains of monoclonal antibodies can be paired with the 9 strains of biotin-labeled antibodies was tested by the double-antibody sandwich ELISA method, these screened antibodies were used as capture antibodies and coated onto ELISA plates, the biotin-labeled antibodies were used as detection antibodies, the protein PADI4 was used as a standard protein, the protein PADI2 was used as a negative control protein, positive serums and negative serums provided by owners were used as endogenous samples and respectively subjected to pairwise coupling verification. Through a square titration experiment, the most responsive 10 groups of antibody pairs with the maximum P/N (as shown in Table 2) were selected for the following optimization experiment.
TABLE-US-00002 TABLE 2 P/N of positive samples to negative samples that are tested by each group of antibody pair 1 2 3 4 5 Antibody Antibody Antibody Antibody Antibody P/N 26 28 31 65 116 Biotin-31 0.1 0.7 2.6 1.4 −6.8 Biotin-65 0.2 1.3 −9.2 0.0 −0.2 Biotin-122 −5.0 1.9 −11.9 −0.3 −0.1 Biotin-135 3.0 3.1 2.1 2.5 1.3 Biotin-137 −1.1 −0.2 1.4 0.6 0.2 Biotin-139 2.4 3.8 1.4 2.6 0.9 Biotin-148 3.9 1.1 2.0 0.8 0.6 Biotin-197 −3.3 −8.9 1.9 1.8 0.4 Biotin-200 3.3 2.3 3.3 3.9 2.6 6 7 8 9 Antibody Antibody Antibody Antibody P/N 122 135 197 200 Biotin-31 1.5 1.6 1.8 0.7 Biotin-65 0.3 6.0 2.1 249.1 Biotin-122 0.4 4.7 3.1 6.9 Biotin-135 2.8 0.6 3.1 0.8 Biotin-137 7.8 2.5 0.3 3.2 Biotin-139 2.0 2.7 0.6 3.0 Biotin-148 2.0 3.0 2.1 4.9 Biotin-197 0.8 2.9 1.2 3.5 Biotin-200 3.6 0.4 5.3 4.1
[0156] (3) Preliminary screening of optimal antibody pair: the 10 groups of antibody pairs (see Table 2) determined at the previous step were used to test the protein PADI2, the protein PADI4, the positive serums, and the negative serum that were subjected to gradient dilution, and results are shown below:
TABLE-US-00003 TABLE 3 OD of the negative control protein tested by the 10 groups of antibody pairs Plate-1 1 2 3 4 5 PADI2 Antibody Antibody Antibody Antibody Antibody 135 135 135 135 135 A 0.04 0.05 0.04 0.03 0.04 B 0.03 0.03 0.03 0.03 0.05 C 0.03 0.03 0.03 0.02 0.05 D 0.03 0.03 0.03 0.03 0.05 E 0.04 0.03 0.03 0.03 0.05 F 0.05 0.03 0.03 0.03 0.05 G 0.03 0.03 0.03 0.03 0.05 H 0.03 0.04 0.04 0.03 0.06 Biotin-139 Biotin-148 Biotin-197 Biotin-65 Biotin-122 Plate-1 6 7 8 9 10 PADI2 Antibody Antibody Antibody Antibody Antibody 135 197 197 200 200 A 0.06 0.06 0.06 0.32 0.30 B 0.03 0.05 0.05 0.30 0.30 C 0.03 0.05 0.06 0.29 0.30 D 0.03 0.05 0.05 0.33 0.28 E 0.03 0.05 0.05 0.29 0.29 F 0.03 0.05 0.06 0.27 0.27 G 0.03 0.05 0.06 0.27 0.25 H 0.04 0.06 0.06 0.26 0.21 Biotin-137 Biotin-135 Biotin-200 Biotin-148 Biotin-197
[0157] The data in Table 3 indicate that neither of the 10 groups of antibody pairs recognize the negative control protein PADI2, and there is no cross reaction.
TABLE-US-00004 TABLE 4 OD of the positive control protein tested by the 10 groups of antibody pair Plate-2 1 2 3 4 5 PADI4 Antibody Antibody Antibody Antibody Antibody 135 135 135 135 135 A 4.12 4.15 4.32 2.4 3.17 B 3.85 3.92 3.86 1.43 1.96 C 2.49 2.77 2.83 0.65 0.86 D 1.66 1.97 1.79 0.39 0.48 E 1.10 1.19 1.19 0.19 0.27 F 0.65 0.67 0.65 0.11 0.16 G 0.38 0.39 0.37 0.06 0.10 H 0.04 0.04 0.04 0.03 0.04 Biotin-139 Biotin-148 Biotin-197 Biotin-65 Biotin-122 Plate-2 6 7 8 9 10 PADI4 Antibody Antibody Antibody Antibody Antibody 135 197 197 200 200 A 4.23 4.13 4.26 3.843 3.93 B 3.21 3.95 3.95 3.62 2.93 C 2.98 3.53 3.08 2.53 2.06 D 1.89 2.47 2.58 1.41 1.05 E 1.12 1.69 1.58 0.81 0.70 F 0.57 0.92 0.85 0.47 0.44 G 0.34 0.46 0.54 0.32 0.37 H 0.02 0.04 0.04 0.18 0.17 Biotin-137 Biotin-135 Biotin-200 Biotin-148 Biotin-197
[0158] The data in Table 4 indicate that all the 10 groups of antibody pairs can recognize the positive control protein PADI4.
TABLE-US-00005 TABLE 5 OD of the positive serums and negative serums that are tested by the 10 groups of antibody pair Plate-3 1 2 3 4 5 + Serum Antibody Antibody Antibody Antibody Antibody 135 135 135 135 135 A 1.11 1.24 1.63 0.24 0.43 B 1.33 1.34 1.37 0.26 0.41 C 1.09 1.24 1.19 0.23 0.38 D 0.79 0.81 0.84 0.18 0.28 E 0.54 0.58 0.62 0.13 0.20 F 0.31 0.31 0.35 0.09 0.14 G 0.19 0.18 0.22 0.06 0.12 H 0.04 0.04 0.04 0.03 0.06 Biotin-139 Biotin-148 Biotin-197 Biotin-65 Biotin-122 Plate-3 6 7 8 9 10 + Serum Antibody Antibody Antibody Antibody Antibody 135 197 197 200 200 A 2.08 0.52 0.63 0.54 0.61 B 1.98 0.76 0.88 0.80 0.77 C 1.67 0.68 0.76 0.58 0.68 D 1.18 0.53 0.68 0.50 0.55 E 0.80 0.31 0.47 0.36 0.45 F 0.50 0.24 0.36 0.30 0.34 G 0.30 0.13 0.24 0.29 0.28 H 0.03 0.04 0.04 0.20 0.21 Biotin-137 Biotin-135 Biotin-200 Biotin-148 Biotin-197 Plate-4 1 2 3 4 5 − Serum Antibody Antibody Antibody Antibody Antibody 135 135 135 135 135 A 0.34 0.45 0.37 0.06 0.09 B 0.34 0.45 0.35 0.06 0.10 C 0.30 0.39 0.32 0.08 0.11 D 0.19 0.29 0.20 0.06 0.10 E 0.12 0.20 0.13 0.05 0.07 F 0.09 0.13 0.10 0.05 0.09 G 0.08 0.08 0.08 0.04 0.07 H 0.06 0.05 0.04 0.04 0.06 Biotin-139 Biotin-148 Biotin-197 Biotin-65 Biotin-122 Plate-4 6 7 8 9 10 − Serum Antibody Antibody Antibody Antibody Antibody 135 197 197 200 200 A 0.36 0.45 0.34 0.28 0.29 B 0.33 0.39 0.41 0.29 0.30 C 0.28 0.38 0.37 0.39 0.34 D 0.24 0.26 0.30 0.35 0.32 E 0.14 0.20 0.22 0.29 0.29 F 0.08 0.14 0.19 0.32 0.26 G 0.06 0.11 0.12 0.29 0.26 H 0.04 0.06 0.06 0.30 0.25 Biotin-137 Biotin-135 Biotin-200 Biotin-148 Biotin-197
[0159] According to the data in Table 5, 5 groups of antibody pairs (Columns 1, 2, 3, 5, and 6) with positive serum OD/negative serum OD of greater than 3 were selected for the following optimization of the capture antibody and detection antibodies: three concentration gradients (1/2/4 ug/mL) were set for the capture antibody 135, and three concentration gradients (0.5/1/2 ug/mL) were respectively set for the 5 detection antibodies to test the protein PADI4 (subjected to doubling dilution from 0.1 ug/mL to 0.0015625 ug/mL), the negative serums (dilution multiple: 1/2/4 multiples), and the positive serums (dilution multiple: 1/2/4 multiples), and arrangements are shown in Table 6:
TABLE-US-00006 TABLE 6 Plate-1 1 2 3 4 5 6 A Antibody Antibody Antibody Antibody Antibody Antibody B 135 135 135 135 135 135 C 2 ug/ml 2 ug/ml 2 ug/ml 2 ug/ml 2 ug/ml 2 ug/ml D E F G H Plate-1 7 8 9 10 11 12 A Antibody Antibody Antibody Antibody Antibody Antibody B 135 135 135 135 135 135 C 2 ug/ml 2 ug/ml 4 ug/ml 4 ug/ml 4 ug/ml 4 ug/ml D E F G H Plate-2 1 2 3 4 5 6 7 8 A Antibody Antibody Antibody Antibody Antibody Antibody Antibody Antibody B 135 135 135 135 135 135 135 135 C 2 ug/ml 2 ug/ml 2 ug/ml 2 ug/ml 4 ug/ml 4 ug/ml 4 ug/ml 4 ug/ml D E F G H Plate-1 1 2 3 4 5 6 A PADI4 PADI4 + Serum + Serum PADI4 PADI4 0.1 ug/ml 0.1 ug/ml 1:1 1:1 0.1 ug/ml 0.1 ug/ml B PADI4 PADI4 + Serum + Serum PADI4 PADI4 0.05 ug/ml 0.05 ug/ml 1:2 1:2 0.05 ug/ml 0.05 ug/ml C PADI4 PADI4 + Serum + Serum PADI4 PADI4 0.025 ug/ml 0.025 ug/ml 1:4 1:4 0.025 ug/ml 0.025 ug/ml D PADI4 PADI4 PADI4 PADI4 0.0125 ug/ml 0.0125 ug/ml PBS PBS 0.0125 ug/ml 0.0125 ug/ml E PADI4 PADI4 − Serum − Serum PADI4 PADI4 0.00625 ug/ml 0.00625 ug/ml 1:1 1:1 0.00625 ug/ml 0.00625 ug/ml F PADI4 PADI4 − Serum − Serum PADI4 PADI4 0.003125 ug/ml 0.003125 ug/ml 1:2 1:2 0.003125 ug/ml 0.003125 ug/ml G PADI4 PADI4 − Serum − Serum PADI4 PADI4 0.0015625 ug/ml 0.0015625 ug/ml 1:4 1:4 0.0015625 ug/ml 0.0015625 ug/ml H PBS PBS PBS PBS PBS PBS Plate-1 7 8 9 10 11 12 A + Serum + Serum PADI4 PADI4 + Serum + Serum 1:1 1:1 0.1 ug/ml 0.1 ug/ml 1:1 1:1 B + Serum + Serum PADI4 PADI4 + Serum + Serum 1:2 1:2 0.05 ug/ml 0.05 ug/ml 1:2 1:2 C + Serum + Serum PADI4 PADI4 + Serum + Serum 1:4 1:4 0.025 ug/ml 0.025 ug/ml 1:4 1:4 PADI4 PADI4 D PBS PBS 0.0125 ug/ml 0.0125 ug/ml PBS PBS E − Serum − Serum PADI4 PADI4 − Serum − Serum 1:1 1:1 0.00625 ug/ml 0.00625 ug/ml 1:1 1:1 F − Serum − Serum PADI4 PADI4 − Serum − Serum 1:2 1:2 0.003125 ug/ml 0.003125 ug/ml 1:2 1:2 G − Serum − Serum PADI4 PADI4 − Serum − Serum 1:4 1:4 0.0015625 ug/ml 0.0015625 ug/ml 1:4 1:4 H PBS PBS PBS PBS PBS PBS Plate-2 1 2 3 4 5 6 7 8 A PADI4 PADI4 + Serum + Serum PADI4 PADI4 + Serum + Serum 0.1 ug/ml 0.1 ug/ml 1:1 1:1 0.1 ug/ml 0.1 ug/ml 1:1 1:1 B PADI4 PADI4 + Serum + Serum PADI4 PADI4 + Serum + Serum 0.05 ug/ml 0.05 ug/ml 1:2 1:2 0.05 ug/ml 0.05 ug/ml 1:2 1:2 C PADI4 PADI4 + Serum + Serum PADI4 PADI4 + Serum + Serum 0.025 ug/ml 0.025 ug/ml 1:4 1:4 0.025 ug/ml 0.025 ug/ml 1:4 1:4 D PADI4 PADI4 PBS PBS PADI4 PADI4 PBS PBS 0.0125 ug/ml 0.0125 ug/ml 0.0125 ug/ml 0.0125 ug/ml E PADI4 PADI4 − Serum − Serum PADI4 PADI4 − Serum − Serum 0.00625 ug/ml 0.00625 ug/ml 1:1 1:1 0.00625 ug/ml 0.00625 ug/ml 1:1 1:1 F PADI4 PADI4 − Serum − Serum PADI4 PADI4 − Serum − Serum 0.003125 ug/ml 0.003125 ug/ml 1:2 1:2 0.003125 ug/ml 0.003125 ug/ml 1:2 1:2 G PADI4 PADI4 − Serum − Serum PADI4 PADI4 − Serum − Serum 0.0015625 ug/ml 0.0015625 ug/ml 1:4 1:4 0.0015625 ug/ml 0.0015625 ug/ml 1:4 1:4 H PBS PBS PBS PBS PBS PBS PBS PBS Plate-1 1 2 3 4 5 6 7 8 9 10 11 12 A Biotin-139 2 ug/ml Biotin-148 2 ug/ml Biotin-197 0.5 ug/ml B C D E F G H Plate-2 1 2 3 4 5 6 7 8 A Biotin-122 2 ug/ml Biotin-137 0.5 ug/ml B C D E F G H
[0160] Date are shown in Table 7 (boldface is data-standard curve of PADI4; Rows D, E, and F of Columns 3, 4, 7, and 8 are data of the negative serums (diluted by 1/2/4 multiples); the remaining non-boldface is data of the positive serums):
TABLE-US-00007 TABLE 7 Plate-1 1 2 3 4 5 6 A 4.09 4.14 4.48 4.66 4.11 4.26 B 2.43 2.46 4.30 4.59 2.36 2.61 C 1.48 1.35 4.49 4.67 1.43 1.71 D 0.87 0.91 0.13 0.14 0.86 1.00 E 0.58 0.53 0.95 1.23 0.49 0.55 F 0.33 0.32 0.75 0.91 0.36 0.38 G 0.24 0.21 0.48 0.55 0.25 0.26 H 0.13 0.10 0.11 0.11 0.12 0.15 Plate-1 7 8 9 10 11 12 A 4.70 4.72 3.97 4.47 4.66 4.47 B 5.00 4.64 2.35 2.85 4.59 4.90 C 4.50 4.61 1.29 1.54 4.49 4.55 D 0.14 0.15 0.71 0.67 0.11 0.10 E 1.22 1.05 0.40 0.39 1.04 0.77 F 0.99 0.83 0.26 0.25 0.75 0.63 G 0.65 0.56 0.18 0.17 0.50 0.47 H 0.14 0.12 0.10 0.09 0.10 0.09 Plate-2 1 2 3 4 5 6 7 8 A 2.31 2.32 4.85 4.60 4.53 4.46 5.18 4.45 B 1.28 1.36 4.54 4.34 2.51 2.70 4.52 3.43 C 0.76 0.74 3.17 2.83 1.46 1.34 4.52 4.50 D 0.48 0.44 0.15 0.13 0.89 0.86 0.07 0.08 E 0.28 0.28 0.44 0.34 0.48 0.45 1.06 1.00 F 0.27 0.22 0.31 0.37 0.31 0.29 0.73 0.57 G 0.19 0.19 0.22 0.25 0.21 0.19 0.49 0.51 H 0.15 0.15 0.15 0.14 0.09 0.09 0.07 0.11
[0161] It can be seen from the data in Table 7 that the OD data in the blocks with shadows are decreased with the increase of the serum dilution multiple; and the corresponding antibody pair is 135 (4 μg/mL)-197 (0.5 μg/mL), and a corresponding standard curve is shown in Table 8 and
TABLE-US-00008 TABLE 8 Antibody 135 Antigen: (4 ug/ml) -blank (ug/ml) 2.95 2.92 0.1000000 2.05 2.02 0.0500000 1.16 1.13 0.0250000 0.59 0.56 0.0125000 0.31 0.28 0.0062500 0.17 0.14 0.0031250 0.09 0.06 0.0015625 0.03 0.00 0.0000000 197bio (0.5 ug/ml)
[0162] (4) The antibody pair 135 (4 μg/mL)-197 (0.5 μg/mL) was further optimized to determine the optimal working concentration of the antibody pair:
[0163] (1) optimization: three concentration gradients (4/2/1 μg/mL) were set for the capture antibody 135, three concentration gradients (0.5/1/2 μg/mL) were set for the detection antibody 197, and the protein PADI4 (subjected to doubling dilution from 50 ng/mL to 0.78125 ng/mL) was tested;
[0164] (2) optimization based on (1): one concentration gradient (4 μg/mL) was set for the capture antibody 135, two concentration gradients (0.25/0.125 μg/mL) were set for the detection antibody 197, and the protein PADI4 (subjected to doubling dilution from 50 ng/mL to 0.78125 ng/mL) was tested; and
[0165] (3) optimization based on (2): three concentration gradients (4/2/1 μg/mL) were set for the capture antibody 135, two concentration gradients (0.25/0.125 μg/mL) were set for the detection antibody 197, and the protein PADI4 (subjected to doubling dilution from 50 ng/mL to 0.78125 ng/mL) was tested;
[0166] Date of each condition combination are shown in Table 9 to Table 14 and
TABLE-US-00009 TABLE 9 Antibody Antibody Antibody 135- 135 135 Antigen: 197bio (4 ug/ml) (4 ug/ml) average -blank (ng/ml) 4.42 4.51 4.46 4.35 50 2.83 2.90 2.86 2.75 25 1.61 1.54 1.58 1.46 12.5 0.93 0.85 0.89 0.77 6.25 0.54 0.55 0.54 0.43 3.125 0.29 0.34 0.32 0.20 1.5625 0.24 0.25 0.24 0.13 0.78125 0.12 0.11 0.12 0.00 0.0000000 197bio (0.25 ug/ml)
TABLE-US-00010 TABLE 10 Antibody Antibody Antibody 135- 135 135 Antigen: 197bio (2 ug/ml) (2 ug/ml) average -blank (ng/ml) 3.47 3.40 3.43 3.32 50 1.91 1.34 1.62 1.51 25 0.95 0.98 0.97 0.86 12.5 0.51 0.46 0.49 0.37 6.25 0.38 0.38 0.38 0.27 3.125 0.23 0.25 0.24 0.13 1.5625 0.18 0.19 0.18 0.07 0.78125 0.11 0.12 0.11 0.00 0.0000000 197bio (0.25 ug/ml)
TABLE-US-00011 TABLE 11 Antibody Antibody Antibody 135- 135 135 Antigen: 197bio (1 ug/ml) (1 ug/ml) average -blank (ng/ml) 1.28 1.41 1.35 1.24 50 0.64 0.77 0.71 0.60 25 0.37 0.41 0.39 0.28 12.5 0.23 0.25 0.24 0.13 6.25 0.17 0.19 0.18 0.07 3.125 0.14 0.13 0.14 0.03 1.5625 0.13 0.12 0.12 0.02 0.78125 0.10 0.11 0.11 0.00 0.0000000 197bio (0.25 ug/ml)
TABLE-US-00012 TABLE 12 Antibody Antibody Antibody 135- 135 135 Antigen: 197bio (4 ug/ml) (4 ug/ml) average -blank (ng/ml) 3.05 3.19 3.12 3.03 50 1.64 1.61 1.63 1.53 25 0.85 0.89 0.87 0.78 12.5 0.50 0.49 0.50 0.41 6.25 0.29 0.30 0.29 0.20 3.125 0.19 0.19 0.19 0.09 1.5625 0.15 0.15 0.15 0.06 0.78125 0.09 0.09 0.09 0.00 0.0000000 197bio (0.125 ug/ml)
TABLE-US-00013 TABLE 13 Antibody Antibody Antibody 135- 135 135 Antigen: 197bio (2 ug/ml) (2 ug/ml) average -blank (ng/ml) 1.94 1.99 1.96 1.85 50 1.02 0.95 1.62 1.51 25 0.55 0.51 0.97 0.86 12.5 0.27 0.29 0.49 0.37 6.25 0.20 0.20 0.38 0.27 3.125 0.14 0.14 0.24 0.13 1.5625 0.13 0.12 0.18 0.07 0.78125 0.08 0.08 0.11 0.00 0.0000000 197bio (0.125 ug/ml)
TABLE-US-00014 TABLE 14 Antibody Antibody Antibody 135- 135 135 Antigen: 197bio (1 ug/ml) (1 ug/ml) average -blank (ng/ml) 0.74 0.79 0.76 0.68 50 0.35 0.41 0.38 0.29 25 0.21 0.23 0.22 0.14 12.5 0.15 0.17 0.16 0.08 6.25 0.12 0.12 0.12 0.04 3.125 0.10 0.10 0.10 0.01 1.5625 0.09 0.10 0.09 0.01 0.78125 0.08 0.09 0.08 0.00 0.0000000 197bio (0.125 ug/ml)
[0167] It can be found through comprehensive comparison of the data (R.sup.2, OD corresponding to the same antigen concentration, and concentration (sensitivity) of the detection antibody) of the above six antibody pairs that the optimal working concentration of the antibody pair is as follows: Antibody 135 (4 μg/mL)-protein PADI4 (50 ng/mL to 0.78125 ng/mL) -197-bio (0.125 μg/mL).
Conclusions
[0168] The arrangements and the test data show that: according to OD of the positive serums and negative serums at 3 dilution multiples (1×, 2×, 4×), and the blank control, a ratio (minus the blank background value) of the positive serum OD to the negative serum OD at each dilution multiple was calculated, and results are shown in Table 15:
TABLE-US-00015 TABLE 15 135-139biotin 135-148biotin 135~197biotin Dilution + Serum − Serum + Serum − Serum + Serum − Serum multiple OD-blank OD-blank +/− OD-blank OD-blank +/− OD-blank OD-blank +/− 1X 4.435 0.98 4.53 4.565 1 4.565 4.46 0.81 5.51 2X 4.31 0.72 5.99 4.675 0.78 5.99 4.64 0.595 7.79 4X 4.445 0.405 10.98 4.41 0.475 9.28 4.415 0.39 11.32 135-122biotin 135-137biotin Dilution + Serum − Serum + Serum − Serum multiple OD-blank OD-blank +/− OD-blank OD-blank +/− 1X 4.585 0.245 4.34 4.74 0.94 5.04 2X 4.3 0.195 22.00 3.9 0.56 6.96 4X 2.86 0.09 31.70 4.435 0.41 10.81
[0169] According to the comprehensive comparison of the above data, and OD of the protein PADI4, the protein PADI2 (for cross removal screening), the positive serums, and the negative serums that were respectively tested by each antibody pair, five groups of antibody pairs were determined; the five groups of antibody pairs are used to test serums at the same dilution, and through the comparison of the ratios (if the ratio is high, then the discrimination between the positive serum and the negative serum is good) of the positive serums to the negative serums of the antibody pairs, it is found that 135-197biotin and 135-122biotin are better, and the corresponding standard curves are shown in Table 16 and Table 17, and
TABLE-US-00016 TABLE 16 Antibody Antibody 135- 135 Antigen: 197bio (4 ug/ml) -blank (ug/ml) 2.95 2.92 0.1000000 2.05 2.02 0.0500000 1.16 1.13 0.0250000 0.59 0.56 0.0125000 0.31 0.28 0.0062500 0.17 0.14 0.0031250 0.09 0.06 0.0015625 0.03 0.00 0.0000000 197bio (0.5 ug/ml)
TABLE-US-00017 TABLE 17 Antibody Antibody Antibody 135- 135 135 Antigen: 122bio (2 ug/ml) (2 ug/ml) average -blank (ug/ml) 2.31 2.32 2.32 2.17 0.1000000 1.28 1.36 1.32 1.17 0.0500000 0.76 0.74 0.75 0.60 0.0250000 0.48 0.44 0.46 0.31 0.0125000 0.28 0.28 0.28 0.13 0.0062500 0.27 0.22 0.24 0.09 0.0031250 0.19 0.19 0.19 0.04 0.0015625 0.15 0.15 0.15 0.00 0.0000000 122bio 122bio (2 ug/ml) (2 ug/ml)
[0170] It is found through the comparison of the sensitivity (OD corresponding to the same antigen concentration) of the two antibody pairs and the relevance (R.sup.2) of the standard curves that the antibody pair 135-197biotin is significantly better than the antibody pair 135-122biotin.