A high affinity padi2 antibody and methods of making the same

Abstract

The present application relates to the field of antibodies, and in particular, the present application provides a high affinity PADI2 antibody and a preparation method thereof. Through antibody screening experiments, eight high affinity PADI2 antibodies are screened, the antibodies can specifically bind to PADI2 protein, and have high affinity. Experimental results show that the antibodies of the present application exhibit excellent binding capacity in ELISA, Western blot and cell experiments, and can be used for detection, diagnosis and treatment of PADI2 related diseases.

Description

[0001] This application is a divisional application of the patent filed on April 14, 2025, with application number 202510457673.8, entitled "A High-Affinity PADI2 Antibody and Its Preparation Method". Technical Field

[0002] This invention relates to the field of antibodies, specifically to a high-affinity PADI2 antibody and its preparation method. Background Technology

[0003] PADI2 (Peptidyl Arginine Deiminase 2) is a calcium-dependent enzyme expressed in mammals, primarily responsible for deiminizing arginine residues in proteins to citrulline. This process, known as citrullination or deiminization, 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 against PADI2 have significant application value in disease diagnosis and treatment research.

[0004] 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:

[0005] Immunohistochemistry (IHC): The expression of PADI2 in tissue sections is detected using anti-PADI2 antibodies to study its distribution and changes in different tissues and disease states.

[0006] Western blotting: The expression level of PADI2 in protein samples is detected by anti-PADI2 antibody to assess its changes under different experimental conditions.

[0007] Enzyme-linked immunosorbent assay (ELISA): This assay uses anti-PADI2 antibodies to quantitatively detect the concentration of PADI2 in a sample, and is used in clinical diagnosis and scientific research.

[0008] Currently, PADI2 antibodies on the market are mainly provided by well-known biopharmaceutical 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, poor sensitivity, specificity, and stability of existing PADI2 antibodies, no mature product has been developed for clinical testing and treatment. Specific shortcomings are as follows:

[0009] 1. Low affinity leads to insufficient detection sensitivity, making it unable to effectively identify low abundance PADI2.

[0010] 2. Insufficient specificity: Some antibodies may cross-bind with other members of the PADI family (such as PADI4), affecting the accuracy of the experiment.

[0011] 3. It has poor stability and may lose its activity under different experimental conditions, which limits its application in experiments such as ELISA, Western blot and IHC (immunohistochemistry).

[0012] Despite numerous studies demonstrating the potential of PADI2 as a disease biomarker and therapeutic target, the availability of high-affinity and high-specificity PADI2 antibodies on the market remains limited, and no mature products are yet available for clinical testing and treatment. Summary of the Invention

[0013] To fill the gap in the prior art, this invention provides a PADI2 antibody with high affinity and high specificity and a method for its preparation.

[0014] Firstly, in a first aspect, the present invention provides a PADI2 antibody with high affinity, said antibody being selected from any of the following:

[0015] (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

[0016] (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

[0017] (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

[0018] (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

[0019] (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

[0020] (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

[0021] (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

[0022] (8) The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO.31, 47, 63, and light chain CDR1, 2, 3 as shown in SEQ ID NO.32, 48, 64.

[0023] In a preferred embodiment, the antibody is selected from any of the following:

[0024] (1) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.1 and 2, respectively; or

[0025] (2) The heavy chain and light chain of the antibody are shown in SEQ ID NO.3 and 4, respectively; or

[0026] (3) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 5 and 6, respectively; or

[0027] (4) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.7 and 8, respectively; or

[0028] (5) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 9 and 10, respectively; or

[0029] (6) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 11 and 12, respectively; or

[0030] (7) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 13 and 14, respectively; or

[0031] (8) The heavy chain and light chain of the antibody are shown in SEQ ID NO.15 and 16, respectively.

[0032] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned PADI2 antibody.

[0033] In a third aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0034] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule or expression vector.

[0035] In a fifth aspect, the present invention provides an engineered bacterium, the engineered bacterium comprising the aforementioned nucleic acid molecule or expression vector.

[0036] In a sixth aspect, the present invention provides a method for producing the aforementioned PADI2 antibody, characterized in that it includes culturing the aforementioned recombinant cells or engineered bacteria to produce antibodies.

[0037] In a seventh aspect, the present invention provides a PADI2 detection kit, the kit comprising the aforementioned PADI2 antibody.

[0038] In an eighth aspect, the present invention provides the use of the antibody or kit in basic medical research for non-diagnostic / therapeutic purposes, wherein the basic medical research is Western blotting, immunohistochemistry, and flow cytometry.

[0039] Compared with the prior art, the technical solution of the present invention has the following significant advantages. :

[0040] 1. High affinity: The eight PADI2 antibodies screened in this invention all exhibit high affinity. Affinity measurement data show that these antibodies can efficiently recognize and bind to PADI2, and have higher detection sensitivity in experimental applications.

[0041] 2. Excellent specificity: The eight PADI2 antibodies screened in this invention underwent rigorous cross-reactivity testing during the screening process, demonstrating their specific recognition of PADI2 without cross-reactivity with PADI4 or other homologous proteins. This characteristic makes them suitable for various medical research projects, giving them greater clinical application value and avoiding false positive results caused by non-specific binding.

[0042] 3. Applicable to multiple detection methods: The eight PADI2 antibodies screened in this invention have been validated on multiple experimental platforms such as ELISA, Western blot (WB), immunofluorescence (IF), and flow cytometry (FACS), and all have shown good binding performance.

[0043] 4. Potential Clinical Application Value: Given the significant role of PADI2 in research on various diseases, these high-affinity antibodies can be used for disease mechanism studies, serum biomarker detection, and the development of potential diagnostic reagents. 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. Attached Figure Description

[0044] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0045] Figure 1 ELISA affinity ranking of the supernatant from the wells of 44 sub-identical strains;

[0046] Figure 2 Protein gradient detection for 22 cell lines;

[0047] Figure 3 Cross-identification and verification of the supernatant from the wells of 11 sub-identified plants;

[0048] Figure 4 To re-validate the supernatants from the 44 wells of the fixation strain using Mouse PADI2, RAT PADI2, and PADIs proteins;

[0049] Figure 5 Protein gradient assays were re-applied to 31 cell lines;

[0050] Figure 6 Subtype detection was performed on antibodies produced by 10 ascites-injected cell lines;

[0051] Figure 7 This is the result of antibody titer testing. Detailed Implementation

[0052] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0053] The reagents involved in the following examples are as follows:

[0054]

[0055] The biomaterials involved in the following embodiments are as follows:

[0056]

[0057] PADI2 positive / negative cell lines refer to the following: The coding sequence (CDS) of the human PADI2 gene (Gene ID: 11240) was obtained through chemical synthesis. The human PADI2 CDS sequence was cloned into an MSCV vector, and the MSCV virus containing the human PADI2 gene CDS sequence was packaged using the PCL-10A1 packaging plasmid. The virus was used to transduce 293T cell lines, and positive cell clones were sorted by flow cytometry using GFP on the vector as a sorting marker to obtain PADI2 positive 293T cell lines. Wild-type 293T cells served as a negative control cell line.

[0058] Example 1: Immunization of mice

[0059] 1.1 Immunization grouping and immunization schedule

[0060] Antigen: Human PADI2: Catalog No. CSB-MP896493H Ud7, Batch No. DA05994a1g0CX, Label HisTag

[0061] Mouse breed: PADI2-KO mouse

[0062] Quantity: 6, labeled 1-6

[0063] Adjuvants: Freund's adjuvant or rapid immune adjuvant

[0064] First immunization: June 17, 2024, 50μg / animal

[0065] Second immunization: 50μg / animal on July 1, 2024

[0066] Third immunization: 50 μg / animal on July 15, 2024; serum titer was measured on July 23, 2024 after the third immunization.

[0067] Fourth immunization: 75μg or 100μg / animal on July 29, 2024; serum titer was measured on August 12, 2024 after the fourth immunization.

[0068] Fifth immunization: 50 μg / animal on August 12, 2024; serum titer was measured on August 20, 2024 after the fifth immunization.

[0069] Sixth immunization: 50 μg / animal on August 26, 2024; serum titer was measured on September 30, 2024 after the sixth immunization.

[0070] 1.2 Mouse immunization procedure

[0071] a. Freund's adjuvant immunization: Dilute the antigen with PBS and mix it with Freund's adjuvant at a volume 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 air bubbles from the syringe, remove the mice from the cage and place them in a specially designed frame. Administer multiple subcutaneous injections on the back of the mice.

[0072] b. Rapid immune adjuvant immunization: Dilute the antigen with PBS and mix it with the rapid immune adjuvant at a volume ratio of 1:1 (total volume 0.2 mL). Transfer the antigen mixed with the adjuvant into a 1 mL syringe, remove air bubbles from the syringe, take the mice to be immunized from the cage and place them in a specially designed fixation frame. Perform multiple subcutaneous injections in the muscles of the mouse's legs.

[0073] c. Immunization cycle: The first five immunizations were spaced 2 weeks apart, and the sixth immunization was spaced 1.5 months apart. (Note: Mouse No. 2 died on August 7, 2024)

[0074] 1.3 PADI 2 valence testing procedure

[0075] a. Coating: Dilute the antigen to 2 μg / mL with coating buffer CB, add 100 μL / well to the microplate, and incubate overnight at 4°C.

[0076] b. Blocking: Remove the microplate and blot dry the liquid in the wells. Block with 5% skim milk (dissolved in PBS). Add 200 μL / well to the microplate and incubate at 37°C for 2 hours. Wash the plate 3 times with TBS.

[0077] c. Sample addition: Serially dilute the immune serum with PBS at ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000 (using pre-immunization serum as a negative control), 100 μL per well, and incubate at 37°C for 1 h. Wash the plate 3 times with TBS.

[0078] d. Add secondary antibody: goat anti-mouse secondary antibody-HRP (1:10000 enzyme dilution), 100 μL / well, incubate at 37℃ for 40 min, wash the plate 5 times with TBS;

[0079] e. Color development: Add TMB substrate, 90 μL / well, incubate at 37°C in the dark for 5-20 min.

[0080] f. Termination: Add stop solution, 50 μL / well, and read the value using a microplate reader (wavelength 450 nm). The highest dilution with a positive reaction is the serum titer of the immunized mice. Repeat the test using the same method after each test.

[0081] 1.4 Serum Western Blot Detection Procedure

[0082] The 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 well using a pipette. 20 μg of lysis buffer was loaded, and 50 ng and 400 ng of PADI2 protein expressed in vitro were loaded. At the same time, 10 µL of marker (commercial molecular weight reference) was loaded.

[0083] Western Blot Analysis:

[0084] a. Gel preparation: Prepare 12% separating gel and 6% stacking gel for later use;

[0085] b. Electrophoresis: Turn on the power and run the stacking gel at 80V for 30 minutes. Adjust the voltage to 120V and continue electrophoresis until the bromophenol blue loading buffer migrates to the bottom of the gel. Turn off the power.

[0086] c. Transfer: After collecting the gel, remove the top layer of stacking gel and immerse the separating gel in the transfer buffer. Immerse the PVDF membrane in isopropanol for 1 minute and then transfer it to the transfer buffer. Immerse the filter paper in the transfer buffer as well (cut both the PVDF membrane and filter paper to the same size as the gel). Rinse the graphite electrode with the transfer buffer, place three sheets of filter paper, and add a small amount of transfer buffer. Place the separating gel on top and add a small amount of transfer buffer. Place the membrane on top and add a small amount of transfer buffer again. Finally, place three sheets of filter paper, add a small amount of transfer buffer, and use a coating stick to remove air bubbles. Cover the electrode, adjust the voltage to maximum, and transfer the gel at 1.5 mA / cm² for 1.5 hours (the load voltage should not exceed 1 V / cm²). 2 );

[0087] d. Blocking: Remove the membrane and rinse with PBST for 5 min (shaking on a horizontal shaker). Remove the membrane and immerse it in blocking solution at 37°C for 2 h or 4°C overnight (blocking solution is 5% skim milk powder);

[0088] e. Incubation with primary antibody: Remove the membrane and wash three times with PBST for 10 minutes each time (using a horizontal shaker). Remove the membrane and immerse it in a primary antibody dilution buffer diluted with 2.5% skim milk powder at 25°C for 1 hour.

[0089] f. Incubation with secondary antibody (anti-mouse IgG): Remove the membrane and wash three times with PBST for 10 min each time (using a horizontal shaker). Remove the membrane and immerse it in a secondary antibody dilution buffer (1:50000) diluted with 5% skim milk powder at 25°C for 1 h.

[0090] g. ECL (Electroluminescent Chromatography) X-ray imaging.

[0091] 1.5 Test Results:

[0092] The results of serum titer testing of 6 mice after three immunizations showed that, under the condition of slight background in pre-immunization serum, the overall immune serum titer was low. The ranking of mouse immune serum titers was: No. 1 > No. 4 = No. 2 > No. 3 = No. 6 = No. 5.

[0093] The immunization dose for the fourth immunization in mice was increased. Four mice (numbers 1, 3, 4, and 6) received 75 μg / mouse, and two mice (numbers 5 and 2) received 100 μg / mouse. Based on the serum titer results of the fourth immunization, mice 3 and 4 were discontinued, while mice 1, 6, and 5 underwent a fifth immunization with 50 μg of rapid immunoadjuvant. Mice 1 showed the best titer. Western blot analysis of the 1 / 500 dilution of serum from mice 1 showed clear target bands for HU, MO, and RA-PADI2 recombinant proteins; however, HEK293T-PADI2 cells showed no clear target bands compared to HEK293T cells.

[0094] The titer results of the five-immune vaccinations in mice 1, 6, and 5 showed that compared with the four-immune vaccinations, the titers of all three mice were significantly increased, with mice 5 and 6 showing the most significant increases. The titer ranking was: 1 > 6 > 5. Western blot analysis of the serum from mice 1, 6, and 5 after the five-immune vaccinations (1 / 500 dilution) showed that, while the HU-PADI2 recombinant protein had a clear target band, the serum from mice 1 and 5 showed a clear target band (approximately 75 kDa) in HEK293T-PADI2 cell samples, but no clear target band in HEK293T and MCF-7 samples; no clear target band was detected in the serum from mouse 6.

[0095] The titer results of the six-immunization assays on mice (samples 1, 6, and 5) showed that compared with the five-immunization assay, mouse 5 showed an improved titer, while mice 1 and 6 showed no significant improvement. Slight background was observed in the detection of his-tag irrelevant proteins. The mouse titer ranking was: 5 > 1 = 6. Based on the titer results, mice 1 and 5 were selected for fusion.

[0096] Example 2: Screening of spleen cell fusion between mice No. 1 and No. 5

[0097] 2.1 Cell Fusion

[0098] Take 50 μg of PADI2 protein, dilute it in PBS to 100 μL, mix it with rapid immune adjuvant at a volume ratio of 1:1, and administer it intraperitoneally to mice 1 and 5.

[0099] Mice 1 and 5, after the shock, were euthanized by removing their eyes, bleeding, and completely cervically dislocating. Blood was collected and separated, and the serum was used as a positive control serum for antibody testing. The spleen was aseptically removed, ground into single cells (splenic cells), washed twice with serum-free DMEM, and then prepared for fusion.

[0100] Take 9 ml of the fusion pool, add an equal volume of 75% ethanol and soak for 10 min, rinse twice with sterile deionized water, and rinse twice with BTX fusion buffer. Take myeloma cells in logarithmic growth phase (SP2 / 0) and prepared spleen cells, mix them at a 1:1 ratio, and perform electrofusion. After completing the electrofusion program, let stand for 2-3 min, then collect the cells in 40 mL of HAT selection medium and incubate at 37℃ with 5% CO2 for 1 h. Add HAT selection medium, plate the cells in 96-well plates, and incubate at 37℃ with 5% CO2. On days 5-6, completely replace the medium with HT complete medium and incubate at 37℃ with 5% CO2 for 24-48 h. Analyze the supernatant afterward.

[0101] 2.2 Fusion supernatant ELISA screening

[0102] Sixty plates were fused from mice 1 and 5. ELISA was used for initial screening using PADI2 protein with a His tag. Based on the screening results, 378 original wells with an OD>=2 were selected. ELISA confirmation screening was then performed using HU-PADI2 protein coated at concentrations of 2 μg / ml, 0.5 μg / ml, and 0.2 μg / ml, along with His tag-independent proteins. Based on the test results, 60 strains with a signal value above 1 for PADI2-2 μg / ml, and signal values ​​above 0.25 for PADI2-0.5 μg / ml and PADI2-0.2 μg / ml were selected for subcloning.

[0103] Example 3: Screening of subclones

[0104] Take the cells to be subcloned, gently pipette them to form a suspension of single cells, add them to a counting chamber, and calculate the cell density; take 100 μL of the cells to be subcloned and dilute to 1 × 10⁻⁶. 3 Cells / mL; Add 50-100 μL of cells to 5-10 mL of culture medium and mix well. Spread 100 μL / well into a 96-well plate containing easyclone, ensuring that each well contains one cell. Culture for 5-7 days and collect the supernatant for ELISA detection.

[0105] The initial ELISA screening results of 60 subclonal cell lines showed that the top 5 single clones with the highest ELISA readings from each cell line were selected for confirmatory screening. For cell lines with fewer than 5 single clones or no single clones, supplementary polyclonal clones were used for confirmatory screening. (Of these, 5 cell lines had no positive single clones, and a total of 291 cell lines were selected for confirmatory screening in designated wells).

[0106] In the ELISA confirmatory screening results of 291 well supernatants from 60 subclonal cell lines:

[0107] ①The supernatant of 44 cell lines was coated with 0.2μg / ml HU-PADI2 and the OD value was detected was >1. The top 1 cell line was selected for seeding and cell expansion culture.

[0108] ② Discard the 5 cell lines that did not have single clones;

[0109] ③ The supernatant of 10 cell lines was coated with 0.2 μg / ml HU-PADI2. If the OD value was <1, the cell lines were discarded.

[0110] ④ One his tag-irrelevant protein was discarded due to background interference.

[0111] Further ELISA affinity ranking (supernatant gradient) was performed on the supernatants of 44 sub-drug strains. The supernatants were coated with HU-PADI2 at 2 μg / ml, and the cell supernatants were diluted 4-fold starting at 1 / 2 for 7 gradients. The results were then analyzed. Figure 1 The top 22 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 was diluted to 0.025 μg / ml (2.5 ng / well). A total of 11 strains had an OD value greater than 1. These 11 strains were selected for cross-validation of Mouse PADI2, RAT PADI2 and PADI series proteins.

[0112] Cross-identification verification of supernatant from 11 sub-determined plants:

[0113] Protein detection:

[0114]

[0115] ELISA test results show ( Figure 3 The mouse PADI2, RAT PADI2, and PADIs proteins were coated at 2 μg / ml. Only cell lines 4A7D9, 50H2D4, and 59C3G2 did not recognize PADI1, PADI3, PADI4, and PADI6 proteins; other cell lines showed strong signals. Cross-validation was performed again on the supernatants of 44 cell lines using Mouse PADI2, RAT PADI2, and PADIs proteins. It was found that 31 out of the 44 cell lines did not recognize PADI1, PADI3, PADI4, and PADI6 proteins. Figure 4 ). These 31 cell lines were selected for re-administered protein gradient assays. The supernatant from the 31 cell lines that did not recognize PADIs was analyzed ( Figure 5 When the antigen was coated at 0.025 μg / ml, 10 strains had an OD>1; 3 strains had an OD>0.9. (Of the 3 strains with OD>1, none recognized mice or rats; of the 1 strain with OD>0.9, one recognized mice and rats, and two recognized only mice.) Nine strains with OD>1 (excluding the 48C11E11 strain which did not recognize mice or rats) and one strain with OD>0.9 (recognizing mice and rats, 43C10H11) were selected for ascites fluid injection to prepare antibodies.

[0116] Example 4: Preparation of ascites fluid and antibody detection

[0117] Antibody subtypes produced by 10 ascites cell lines were detected using kits (Frdbio kit / Proteintech kit). The results showed that ( Figure 6Of these, 6 cell lines (4A7D9, 42A2G8, 50H2D4, 51E10D11, 59H10D1, 59C3G2) had subtypes of IgG2b and Igκ; 2 cell lines (12A4G6, 57B4B2) had subtypes of IgG1 and Igκ; 1 cell line (50G9E6) had subtypes of IgG2b and Igλ; and 1 cell line (43C10H11) had subtypes of IgG2C and Igκ.

[0118] 4.1 Preparation of ascites:

[0119] a. One week in advance, inject mice intraperitoneally with an incomplete adjuvant, 0.5 mL / mouse;

[0120] b. Collect passaged cells by centrifugation and resuspend in 1 mL of DMEM at a concentration of 1 × 10⁻⁶. 6 / mL, aspirate cells with a sterile syringe and inject into the peritoneal cavity of mice;

[0121] c. After a 6-day interval, observe the production of ascites in the mice daily. If the abdomen is significantly enlarged and feels tense when touched, ascites can be collected using a sterile syringe ascites needle.

[0122] d. Centrifuge at 10,000 rpm for 5 min to remove cellular components and other precipitates, collect the supernatant, and store at -20 °C.

[0123] 4.2 Antibody purification and labeling:

[0124] a. Take affinity chromatography Protein G column and Protein A column, wash with 10 column bed volume of water, and wash with sodium acetate buffer for 10 column bed volume;

[0125] b. Collect ascites fluid, centrifuge at 12000 rpm for 10 min at 4 ℃, collect the supernatant, filter, and mix with sodium acetate buffer;

[0126] c. Load the Protein G column at a rate of 0.5 ml / min, collect the breakthrough, and after loading, continue washing with sodium acetate buffer until the G250 detection is colorless;

[0127] d. Elute 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 10 column bed volumes of water;

[0128] e. Block the chromatography column with 10 ml NaCl-sodium azide buffer and incubate at 4°C;

[0129] f. Concentrate the elution peak to an equal volume of serum by ultrafiltration, place it in a dialysis bag, and dialyze overnight;

[0130] g. Change the solution once after 12 hours (5L PBS);

[0131] h. Remove the sample, centrifuge at 12000 rpm for 10 min at 4 ℃, collect the supernatant, store it temporarily at 4 ℃, perform SDS-PAGE detection and send it for quality control;

[0132] i. Purify the qualified antibody and label it with 500 μg of biotin. Perform ELISA detection on the antibody and the biotin-labeled antibody.

[0133] Antibody titer test results showed that, except for 43C10H11 which had a relatively low titer, the other antibodies had relatively high titers. Figure 7 Of the 10 biotinylated antibodies, 21E10G7 had a relatively low titer, while the other antibodies had higher titers, and their titers were generally better than those of the unbiotinylated antibodies.

[0134] Example 5: Monoclonal Antibody Sequencing

[0135] Eight cell lines with the best antibody efficacy were selected and sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The sequencing results were then analyzed for antibody sequence information. The sequence information is as follows:

[0136]

[0137] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A PADI2 antibody with high affinity, characterized in that, The antibody comprises heavy chain CDR1, 2, 3 as shown in SEQ ID NO. 31, 47, 63, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 32, 48, 64.

2. A nucleic acid molecule encoding the PADI2 antibody of claim 1.

3. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, It includes the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. An engineered bacterium, characterized in that, It includes the nucleic acid molecule of claim 2 or the expression vector of claim 3.

6. A method for producing the PADI2 antibody according to claim 1, characterized in that, This includes culturing the recombinant cells of claim 4 or the engineered bacteria of claim 5 to produce antibodies.

7. A PADI 2 detection kit, characterized in that, Includes the PADI2 antibody as described in claim 1.

8. The use of the antibody of claim 1 and the kit of claim 7 in basic medical research for non-diagnostic / therapeutic purposes, wherein, The basic medical research mentioned includes Western blotting, immunohistochemistry, and flow cytometry.

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