A high-affinity PADI2 antibody and its preparation method

Abstract

The present invention relates to the field of antibodies. Specifically, the present invention provides a high-affinity PADI2 antibody and a method for preparing the same. Through antibody screening experiments, the present invention screened and obtained eight high-affinity PADI2 antibodies. These antibodies specifically bind to the PADI2 protein with high affinity. Experimental results demonstrated that the antibodies exhibited excellent binding ability in ELISA, Western blot, and cell-based assays, and are potentially useful for the detection, diagnosis, and treatment of PADI2-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular, to a high-affinity PADI2 antibody and a preparation method thereof. Background Art

[0002] PADI2 (Peptidyl Arginine Deiminase 2) is a calcium-dependent enzyme expressed in mammals that is primarily responsible for deiminating arginine residues in proteins to citrulline. This process, known as citrullination or deimination, is an important post-translational modification. PADI2 plays a role in various physiological and pathological processes, including gene expression regulation, epigenetic modification, and the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Therefore, high-affinity antibodies targeting PADI2 have important application value in disease diagnosis and treatment research.

[0003] Currently, the detection of PADI2 mainly relies on the development and application of specific antibodies. These antibodies are widely used in the following research and detection methods:

[0004] Immunohistochemistry (IHC): Anti-PADI2 antibodies were used to detect PADI2 expression in tissue sections to study its distribution and changes in different tissues and disease states.

[0005] Western Blot: Anti-PADI2 antibody was used to detect the expression level of PADI2 in protein samples and evaluate its changes under different experimental conditions.

[0006] Enzyme-linked immunosorbent assay (ELISA): uses anti-PADI2 antibodies to quantitatively detect the concentration of PADI2 in samples, which is used in clinical diagnosis and scientific research.

[0007] Currently, PADI2 antibodies on the market are primarily provided by well-known bioreagent companies such as Abcam, CST (Cell Signaling Technology), Sigma-Aldrich, and Santa Cruz Biotechnology. These antibodies are primarily used in research tools such as Western blot (WB), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FACS). However, due to the inconsistent quality of existing PADI2 antibodies and their poor sensitivity, specificity, and stability, no mature products are suitable for clinical testing and treatment. Specific shortcomings are as follows:

[0008] 1. The low affinity results in insufficient detection sensitivity and inability to effectively identify low-abundance PADI2.

[0009] 2. Lack of specificity: some antibodies may cross-bind to other members of the PADI family (such as PADI4), affecting the accuracy of the experiment.

[0010] 3. Poor stability, may lose activity under different experimental conditions, limiting its application in experiments such as ELISA, Western blot and IHC (immunohistochemistry).

[0011] Although many studies have shown the potential of PADI2 as a disease marker and therapeutic target, there are still relatively limited high-affinity and high-specificity PADI2 antibodies on the market, and there are no mature products that can be used in clinical testing and treatment. Summary of the Invention

[0012] In order to fill the gap in the prior art, the present invention provides a high-affinity and high-specificity PADI2 antibody and a preparation method thereof.

[0013] First, in a first aspect, the present invention provides a PADI2 antibody with high affinity, wherein the antibody is selected from any one of the following:

[0014] (1) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 17, 33, 49, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 18, 34, 50; or

[0015] (2) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 19, 35, 51, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 20, 36, 52; or

[0016] (3) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 21, 37, 53, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 22, 38, 54; or

[0017] (4) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 23, 39, 55, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 24, 40, 56; or

[0018] (5) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 25, 41, 57, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 26, 42, 58; or

[0019] (6) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 27, 43, 59, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 28, 44, 60; or

[0020] (7) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 29, 45, 61, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 30, 46, 62; or

[0021] (8) The antibody comprises heavy chain CDR1, 2, 3 represented by SEQ ID NOs. 31, 47, and 63, and light chain CDR1, 2, 3 represented by SEQ ID NOs. 32, 48, and 64.

[0022] In a preferred embodiment, the antibody is selected from any one of the following:

[0023] (1) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 1 and 2, respectively; or

[0024] (2) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 3 and 4, respectively; or

[0025] (3) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 5 and 6, respectively; or

[0026] (4) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 7 and 8, respectively; or

[0027] (5) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 9 and 10, respectively; or

[0028] (6) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 11 and 12, respectively; or

[0029] (7) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 13 and 14, respectively; or

[0030] (8) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 15 and 16, respectively.

[0031] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned PADI2 antibody.

[0032] In a third aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0033] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or expression vector.

[0034] In a fifth aspect, the present invention provides an engineered bacterium comprising the aforementioned nucleic acid molecule or expression vector.

[0035] In a sixth aspect, the present invention provides a method for producing the PADI2 antibody, characterized in that it comprises culturing the aforementioned recombinant cells or engineered bacteria to produce the antibody.

[0036] In a seventh aspect, the present invention provides a PADI2 detection kit, comprising the PADI2 antibody.

[0037] In an eighth aspect, the present invention provides use of the antibody or kit in basic medical research for non-diagnostic / therapeutic purposes, wherein the basic medical research is Western Blot, immunohistochemistry and flow cytometry analysis.

[0038] Compared with the prior art, the technical solution of the present invention has the following significant improvements :

[0039] 1. High affinity: The eight PADI2 antibodies screened in the present invention all exhibited high affinity. Affinity measurement data showed that these antibodies were able to efficiently recognize and bind to PADI2, and had higher detection sensitivity in experimental applications.

[0040] 2. Excellent Specificity: The eight PADI2 antibodies identified in this study underwent rigorous cross-reactivity testing during the screening process and are shown to specifically recognize PADI2 without cross-reacting with PADI4 or other homologous proteins. This property makes them suitable for various medical research and clinical applications, avoiding false-positive results due to nonspecific binding.

[0041] 3. Applicable to multiple detection methods: The eight PADI2 antibodies screened in the present invention were verified in multiple experimental platforms such as ELISA, Western blot (WB), immunofluorescence (IF), and flow cytometry (FACS), and all showed good binding performance.

[0042] 4. Potential clinical application value: Because PADI2 is of great significance in the research of various diseases, these high-affinity antibodies can be used for disease mechanism research, serum biomarker detection, and potential diagnostic reagent development. Combined with a highly sensitive detection platform, they could become biomarker detection tools for PADI2-related diseases in the future, enabling early diagnosis and disease progression monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0044] Figure 1 ELISA affinity ranking of supernatants from wells of 44 sub-strains;

[0045] Figure 2 Protein gradient detection for 22 cell lines;

[0046] Figure 3 Cross-identification verification of supernatants from 11 sub-strains;

[0047] Figure 4 To re-cross-validate the supernatants of 44 wells using Mouse PADI2, RAT PADI2, and PADIs proteins;

[0048] Figure 5 Protein gradient assays were re-supplemented for 31 cell lines;

[0049] Figure 6 Subtype detection was performed for antibodies produced by 10 ascites cell lines;

[0050] Figure 7 The results of antibody titer test. DETAILED DESCRIPTION

[0051] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0052] The reagents involved in the following examples are as follows:

[0053] Reagents factory Item No. HumanPADI2 Cusabio CSB-MP896493HUd7 MousePADI2 Cusabio CSB-MP3642MO RATPADI2 Cusabio CSB-MP017377RA HumanPADI1 Cusabio CSB-MP891543HU HumanPADI3 Cusabio CSB-MP891552HU HumanPADI4 Cusabio CSB-MP890757HU HumanPADI6 Cusabio CSB-MP744226HU Goat anti-mouse secondary antibody-HRP Cusabio CSB-PA573747 Goat anti-human secondary antibody-HRP Cusabio CSB-PA160506 avidin-HRP Jackson 016-030-084 DMEM Hyclone SH30022.01 Dual antibody Hyclone SV30010 Fetal bovine serum ExCell FSS500 HT Sigma H0137-10VL HAT Sigma H0262-10VL DMSO Sigma D2650 Freund's adjuvant Sigma F5506, F5881 Rapid immune adjuvant Invivogen 10253-45-01 Subtype detection kit Frdbio FRD90100P8Rd Subtype detection kit Proteintech PK20002

[0054] The biological materials involved in the following examples are as follows:

[0055] Sample type name factory Item No. batch quantity mice PADI2-KO mice / / 6 Positive cells* HEK293T-hPADI2 / / 24.6.3 5 Negative cells* HEK293T / / 24.5.26 3 sticks

[0056] *PADI2-positive / negative cell lines are obtained by chemically synthesizing the coding sequence (CDS) of the human PADI2 gene (Gene ID: 11240). The human PADI2 CDS sequence is cloned into the MSCV vector and packaged with the PCL-10A1 packaging plasmid to produce the MSCV virus containing the human PADI2 CDS sequence. PADI2-positive 293T cell lines are obtained by transducing the virus into 293T cells, and positive cell clones are isolated by flow cytometry using the GFP marker on the vector. Wild-type 293T cells serve as the negative control cell line.

[0057] Example 1 Mouse immunization

[0058] 1.1 Immunization Grouping and Immunization Schedule Antigen: Human PADI2: Catalog No. CSB-MP896493H Ud7, Batch No. DA05994a1g0CX, Label His Tag Mouse Breed: PADI2-KO mice

[0059] Quantity: 6 pieces, marked as 1-6

[0060] Adjuvant: Freund's adjuvant or rapid immunoadjuvant

[0061] First immunization: June 17, 2024, 50 μg / animal

[0062] Second immunization: July 1, 2024, 50 μg / animal

[0063] Third immunization: 50 μg / animal on July 15, 2024; serum titer after the third immunization will be tested on July 23, 2024; Fourth immunization: 75 μg or 100 μg / animal on July 29, 2024; serum titer after the fourth immunization will be tested on August 12, 2024;

[0064] Fifth immunization: 50 μg / mouse on August 12, 2024; the serum titer after the fifth immunization was detected on August 20, 2024; Sixth immunization: 50 μg / mouse on August 26, 2024; the serum titer after the sixth immunization was detected on September 30, 2024.

[0065] 1.2 Mouse immunization process

[0066] Immunization with Freund's Adjuvant: Dilute the antigen with PBS and mix it with Freund's adjuvant at a ratio of 1:1 (total volume 0.5 mL). Emulsify at 4°C for 3-5 minutes. Use Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for subsequent booster immunizations. Transfer the emulsified antigen into a 1 mL syringe. Remove any air bubbles from the syringe. Remove the mice from the cage and place them in a custom holder. Administer multiple subcutaneous injections on the back of the mice.

[0067] b. Rapid Immunization with Adjuvant: Dilute the antigen with PBS and mix it evenly with the rapid immunotherapy adjuvant at a ratio of 1:1 (total volume 0.2 mL). Transfer the mixed antigen and adjuvant into a 1 mL syringe. Remove any air bubbles from the syringe. Remove the mice from the cage and place them in a specially designed holder. Administer multiple subcutaneous injections into the upper leg muscles.

[0068] c. Immunization cycle: The first five immunizations were administered 2 weeks apart, and the sixth immunization was administered 1.5 months apart. (Note: Mouse No. 2 died on August 7, 2024)

[0069] 1.3PADI2 titer detection process

[0070] a. Coating: Dilute the antigen to 2 μg / mL using coating buffer CB, add 100 μL / well of the ELISA plate, and incubate at 4°C overnight. b. Blocking: Remove the ELISA plate, pat dry the liquid in the wells, block with 5% skim milk (dissolved in PBS), add 200 μL / well of the ELISA plate, incubate at 37°C for 2 hours, and wash the plate three times with TBS.

[0071] c. Sample addition: Dilute the immune serum in PBS at serial dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 (pre-immune serum was used as a negative control). Add 100 μL to each well and incubate at 37°C for 1 hour. Wash the plate three times with TBS.

[0072] d. Add secondary antibody: goat anti-mouse secondary antibody-HRP (1:10,000 enzyme dilution), 100 μL / well, incubate at 37°C for 40 min, and wash the plate five times with TBS.

[0073] e. Color development: Add TMB substrate, 90 μL / well, and incubate at 37°C in the dark for 5-20 min.

[0074] f. Stop: Add 50 μL / well of stop solution and read the titer using a microplate reader (wavelength 450 nm). The maximum dilution with a positive reaction is the serum titer of the immunized mouse. Repeat the test using the same method after each test.

[0075] 1.4 Serum Western Blot Detection Process

[0076] Immune serum sample (10 μL) was mixed with 2× loading buffer (10 μL). 20 μL of the sample and loading buffer mixture was slowly added to the sample tank using a pipette. The lysate was loaded at 20 μg, and the in vitro expressed PADI2 protein was loaded at 50 ng and 400 ng. At the same time, 10 μL of marker (commercial molecular weight reference) was loaded.

[0077] Western Blot Assay:

[0078] a. Gel preparation: Prepare 12% separation gel and 6% stacking gel for later use;

[0079] b. Electrophoresis: Turn on the power supply and run the stacking gel at 80V for 30 minutes. Adjust the voltage to 120V and run the gel until the bromophenol blue loading buffer migrates to the bottom of the gel. Turn off the power supply.

[0080] c. Transfer: After taking the gel, remove the upper concentrated gel and immerse the separation gel in transfer buffer. Soak the PVDF membrane in isopropanol for 1 minute and then transfer it to the transfer buffer. Immerse the filter paper in the transfer buffer (cut the PVDF membrane and filter paper into the same size as the gel). Rinse the graphite electrode with transfer buffer, lay three pieces of filter paper, and drop a little transfer buffer. Lay the separation gel and drop a little transfer buffer. Lay the membrane and drop a little transfer buffer. Finally, lay three pieces of filter paper, drop a little transfer buffer, and use a coating rod to drive out bubbles. Cover the electrode, adjust the voltage to the maximum, and transfer the membrane at 1.5mA / cm2 gel volume for 1.5h (the load voltage should not exceed 1V / cm 2 );

[0081] d. Blocking: Remove the membrane and rinse with PBST for 5 minutes (shake on a horizontal shaker). Remove the membrane and immerse it in blocking solution at 37°C for 2 hours or at 4°C overnight (the blocking solution is 5% skim milk powder).

[0082] e. Incubate with primary antibody: Remove the membrane and wash three times with PBST for 10 minutes each time (shake on a horizontal shaker). Remove the membrane and soak in primary antibody diluent diluted with 2.5% skim milk powder at 25°C for 1 hour.

[0083] f. Incubation with secondary antibody (anti-mouse IgG): Remove the membrane and wash three times with PBST for 10 minutes each time (shake on a horizontal shaker). Remove the membrane and soak in secondary antibody diluent (1:50,000) diluted with 5% skim milk powder at 25°C for 1 hour.

[0084] g.ECL chemiluminescent X-ray film development and imaging.

[0085] 1.5 Test results:

[0086] The results of the three-immune serum titer test of 6 mice showed that: in the case of a slight background of pre-immune serum, the overall immune serum titer was low, and the mouse immune serum titer was ranked as follows: No. 1> No. 4=No. 2> No. 3=No. 6=No. 5.

[0087] The mice were vaccinated with increasing doses over four vaccinations: 75 μg / mouse for four mice (Nos. 1, 3, 4, and 6) and 100 μg / mouse for two mice (Nos. 5 and 2). Based on the titer of the serum from the four vaccinations, mice Nos. 3 and 4 were discarded, and mice Nos. 1, 6, and 5 were vaccinated with 50 μg of the rapid immunization adjuvant. The titer of mouse No. 1 was the best. Western blot analysis of the serum from the four vaccinations of mouse No. 1 at a 1 / 500 dilution showed distinct target bands for the HU, MO, and RA-PADI2 recombinant proteins. In contrast to HEK293T cells, no target bands were observed in HEK293T-PADI2 cells.

[0088] The results of pentaimmune titer testing of mice No. 1, 6, and 5 showed that compared with the tetraimmune titer, the titers of all three mice were significantly higher, with the increases being particularly pronounced for mice No. 5 and 6. The titer ranking of mice was: No. 1 > No. 6 > No. 5. Western blot analysis of sera from mice No. 1, 6, and 5 at a 1 / 500 dilution showed that, while the HU-PADI2 recombinant protein had a distinct target band, sera from mice No. 1 and 5 had a distinct target band (around 75 kD) in HEK293T-PADI2 cell samples, but no significant target band was detected in HEK293T or MCF-7 samples. No significant target band was detected in the serum from mouse No. 6.

[0089] The titer test results for six immunizations of mice, No. 1, No. 6, and No. 5, showed that compared to the five immunizations, the titer of mouse No. 5 was significantly higher, while the titers of mice No. 1 and No. 6 remained unchanged. There was also minimal background in the detection of His-tag-unrelated proteins. The titer ranking of mice was: No. 5 > No. 1 = No. 6. Based on the titer test results, mice No. 1 and No. 5 were selected for fusion.

[0090] Example 21 and 5 mouse spleen cell fusion screening

[0091] 2.1 Cell fusion

[0092] 50 μg of PADI2 protein was diluted to 100 μL with PBS, mixed with rapid immune adjuvant at a volume ratio of 1:1, and injected intraperitoneally into mice No. 1 and No. 5.

[0093] After the shock, mice #1 and #5 were taken, their eyes enucleated, and blood was drawn to death by complete cervical distension. Blood was collected and separated, and the serum was used as a positive control serum for antibody testing. The spleens were aseptically removed and ground into single cells (splenocytes), washed twice with serum-free DMEM, and then prepared for fusion.

[0094] Remove 9ml of the fusion pool and add an equal volume of 75% ethanol to soak for 10 minutes. Rinse twice with sterile deionized water and twice with BTX fusion buffer. Take myeloma cells (SP2 / 0) in the logarithmic growth phase and mix with the prepared spleen cells in a 1:1 ratio before electrofusion. After the electrofusion procedure is completed, let it rest for 2-3 minutes, collect the cells in 40mL of HAT selection medium, place them in a 37℃ 5% CO2 incubator, incubate for 1 hour, supplement with HAT selection medium, plate the cells in a 96-well culture plate, and culture in a 37℃ 5% CO2 incubator. On the 5th-6th day, replace all the medium with HT complete medium. After culturing in a 37℃ 5% CO2 incubator for 24-48 hours, the supernatant is tested.

[0095] 2.2 ELISA screening of fusion supernatant

[0096] Mice #1 and #5 were fused across 60 plates and screened using His-tagged PADI2 protein for primary ELISA. Based on the screening results, 378 original wells with an OD >= 2 were selected and confirmed by ELISA using HU-PADI2 protein at 2μg / ml, 0.5μg / ml, and 0.2μg / ml, along with a His-tagged unrelated protein. Based on the test results, 60 strains with a signal value of 1 or higher when coated with PADI2-2μg / ml, and 0.25 or higher when coated with PADI2-0.5μg / ml and PADI2-0.2μg / ml, were selected for subcloning.

[0097] Example 3 Screening of subclones

[0098] Take the cells that need to be subcloned and gently blow to make a suspended single cell, add it to a counting plate for counting and calculate the cell density; take 100 μL of the cells that need to be subcloned and dilute to 1×10 3 / mL; take 50-100μL of cells and add them to 5-10mL of culture medium, mix well, and plate 100μL / well into a 96-well plate containing easyclone, ensuring that each well contains one cell; culture for 5-7 days and take the supernatant for ELISA detection.

[0099] The results of the initial ELISA screening of 60 subclonal cell lines showed that the five monoclonal cells with the highest ELISA readings were selected for confirmation screening. Cell lines with fewer than five monoclonal cells or no monoclonal cells were supplemented with polyclonal cells for confirmation screening. (Among them, five lines had no positive monoclonal cells, and a total of 291 lines were selected for confirmation screening.)

[0100] In the ELISA confirmation screen test results of the supernatants of 291 fixed-line wells of 60 subcloned cells:

[0101] ① The supernatants of 44 cell lines were coated with HU-PADI2 at 0.2 μg / ml and the OD was > 1. The top 1 cell line was selected for seed preservation and cell culture expansion;

[0102] ②5 cell lines without monoclonal clones were discarded;

[0103] ③ The supernatants of 10 cell lines were coated with HU-PADI2 at 0.2 μg / ml and the OD was < 1, and the cell lines were discarded;

[0104] ④ One strain with his-tagged irrelevant protein had background and was discarded.

[0105] The supernatants of 44 strains were further sorted by ELISA affinity (supernatant gradient), HU-PADI2 was coated at 2 μg / ml, and the cell supernatants were diluted 4-fold starting from 1 / 2 in 7 gradients. Figure 1) Top22 cell lines (3D12E1, 4A7D9, 41D3E12, 41B5E11, 42A2G8, 48C1E4, 48C7F4, 48A12D4, 50H2D4, 50H4G1, 51E10D11, 53B11H4, 54G1E7, 54B6E11, 55D2H2, 55D4G7, 55B6D7, 57H8F11, 58B5G1, 59C3G2, 59H10D1, 60F7C8) were selected for protein gradient detection. The results showed that ( Figure 2 ), the antigen coating was diluted to 0.025 μg / ml (2.5 ng / well), and a total of 11 strains had OD values ​​greater than 1. These 11 strains were selected for cross-validation of Mouse PADI2, RAT PADI2 and PADIs series proteins.

[0106] Cross-identification verification of supernatants from 11 wells with one sub-strain:

[0107] Detection protein:

[0108] name Item No. batch Label HumanPADI2 CSB-MP896493HUd7 DA06085k1g0CX-1 HisTag PADI1 CSB-MP891543HU DD06009k1g0CX HisTag PADI3 CSB-MP891552HU DD06022k1g0CX HisTag PADI4 CSB-MP890757HU DD06020k1g0CX HisTag PADI 6 CSB-MP744226HU DD06022k1g0CX HisTag RatPADI2 CSB-MP3642MO DD06042k1g0zn-2 HisTag MousePADI2 CSB-MP017377RA DD05767k1g0HP-3 HisTag

[0109] ELISA test results showed that ( Figure 3 ), Mouse PADI2, RAT PADI2, and PADIs proteins were coated at 2 μg / ml. Only 4A7D9, 50H2D4, and 59C3G2 did not recognize PADI1, PADI3, PADI4, and PADI6 proteins, while other cell lines had strong signals. The supernatants of 44 strains were cross-validated using Mouse PADI2, RAT PADI2, and PADIs proteins. It was found that 31 of the 44 strains did not recognize PADI1, PADI3, PADI4, and PADI6 proteins ( Figure 4 ). These 31 cell lines were selected to be re-supplemented with protein gradient test. In the supernatant test of 31 cell lines that do not recognize PADIs ( Figure 5 ) When coated with 0.025 μg / ml of antigen, 10 strains had an OD > 1; 3 strains had an OD > 0.9. (Three strains with an OD > 1 did not recognize mice or rats; one strain with an OD > 0.9 recognized mice and rats, and two strains only recognized mice.) Nine strains with an OD > 1 (excluding one strain, 48C11E11, which did not recognize mice or rats) and one strain, 43C10H11, with an OD > 0.9 (which recognized mice and rats), were selected for ascites fluid extraction and antibody preparation, totaling 10 strains.

[0110] Example 4 Preparation of Ascites and Antibody Detection

[0111] The antibody subtypes produced by 10 ascites cell lines were detected using a kit (Frdbio kit / Proteintech kit). The results showed that ( Figure 6 ), among which 6 cell lines (4A7D9, 42A2G8, 50H2D4, 51E10D11, 59H10D1, 59C3G2) were of IgG2b and Igκ subtypes; 2 cell lines (12A4G6, 57B4B2) were of IgG1 and Igκ subtypes; 1 cell line (50G9E6) was of IgG2b and Igλ subtypes; 1 cell line (43C10H11) was of IgG2C and Igκ subtypes.

[0112] 4.1 Preparation of ascites:

[0113] a. One week in advance, mice were intraperitoneally injected with incomplete adjuvant (0.5 mL / mouse);

[0114] b. Collect the passaged cells by centrifugation and resuspend them in 1 mL of DMEM to a concentration of 1 × 10 6 / mL, cells were drawn up with a sterile syringe and injected into the peritoneal cavity of mice;

[0115] c. After an interval of 6 days, observe the mice daily for ascites production. If the abdomen is noticeably swollen and feels tense when touched, collect ascites using a sterile syringe and ascites needle.

[0116] d. Centrifuge at 10,000 rpm for 5 min to remove cellular components and other precipitates, collect the supernatant, and store at -20°C.

[0117] 4.2 Antibody purification and labeling:

[0118] a. Take affinity chromatography Protein G column and Protein A column, wash with water for 10 times column bed volume, and then rinse with sodium acetate buffer for 10 times column bed volume;

[0119] b. Take ascites, centrifuge at 12000 rpm for 10 min at 4°C, collect the supernatant, filter, and mix with sodium acetate buffer;

[0120] c. Load the Protein G column at a rate of 0.5 ml / min, collect the breakthrough, and after loading, continue rinsing with sodium acetate buffer until the G250 assay is colorless.

[0121] d. Rinse the column bed with glacial acetic acid elution buffer, collect the elution peak, quickly adjust the pH of the elution peak to neutral with saturated sodium carbonate, and wash with water 10 times the column bed volume;

[0122] e. Seal the column with 10 ml of NaCl-sodium azide buffer and place at 4°C;

[0123] f. The elution peak was concentrated by ultrafiltration to a volume equal to that of serum, and placed in a dialysis bag for overnight dialyzation;

[0124] g. After 12 hours, change the medium (5L PBS);

[0125] h. Remove the sample, centrifuge at 12,000 rpm for 10 min at 4°C, collect the supernatant, store at 4°C, perform SDS-PAGE analysis, and send for quality control;

[0126] i. Take 500 μg of purified antibodies and label them with biotin. Perform ELISA on the antibodies and biotin-labeled antibodies.

[0127] The results of antibody titer test showed that among the 10 antibodies, except for 43C10H11 which had a low titer, the titers of other antibodies were relatively high ( Figure 7 Among the 10 biotinylated antibodies, 21E10G7 had a relatively low titer, while the other antibodies had relatively high titers, and their titers were generally better than those of non-biotinylated antibodies.

[0128] Example 5 Monoclonal Antibody Sequencing

[0129] Eight cell lines with the best antibody effects were selected and sequenced by Wuhan Jinkairui Biological Company. The resulting sequencing results were used for antibody sequence analysis. The sequence information is as follows:

[0130]

[0131]

[0132] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A PADI2 antibody with high affinity, characterized in that The antibody comprises heavy chain CDR1, 2, 3 represented by SEQ ID NOs. 17, 33, and 49, and light chain CDR1, 2, 3 represented by SEQ ID NOs. 18, 34, and 50.

2. The PADI2 antibody according to claim 1, wherein The heavy chain and light chain of the antibody are shown in SEQ ID NO. 1 and 2, respectively.

3. A nucleic acid molecule encoding the PADI2 antibody according to claim 1 or 2.

4. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that The method comprises the nucleic acid molecule according to claim 3 or the expression vector according to claim 4.

6. An engineered bacterium, characterized in that: The method comprises the nucleic acid molecule according to claim 3 or the expression vector according to claim 4.

7. A method for producing the PADI2 antibody according to claim 1 or 2, characterized in that: The method comprises culturing the recombinant cell according to claim 5 or the engineered bacteria according to claim 6 to produce antibodies.

8. A PADI2 detection kit, characterized in that The invention comprises the PADI2 antibody according to claim 1 or 2.

9. Use of the antibody according to claim 1 or 2, or the kit according to claim 8 in basic medical research for non-diagnostic / therapeutic purposes, wherein: The basic medical research includes Western Blot, immunohistochemistry and flow cytometry analysis.

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