High-affinity PADI2 antibody and preparation method thereof

Abstract

The invention relates to the field of antibodies, and particularly provides a high-affinity PADI2 antibody and a preparation method thereof. According to the invention, eight high-affinity PADI2 antibodies are obtained by screening through an antibody pair screening experiment, and the antibodies can be specifically combined with PADI2 protein and have high affinity. Experimental results show that the antibody disclosed by the invention shows excellent binding capacity in ELISA (Enzyme-Linked Immunosorbent Assay), Western blot and cell experiments, and can be used for detecting, diagnosing and treating PADI2 related diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibodies. Specifically, the present invention relates to a high-affinity PADI2 antibody and a method for preparing the same. Background Art

[0002] PADI2 (Peptidyl Arginine Deiminase 2) is a calcium-dependent enzyme expressed in mammals, mainly responsible for deiminating arginine residues in proteins to citrulline. This process is called citrullination or deimination, which is an important post-translational modification. PADI2 plays a role in a variety of 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 important application values 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): Using anti-PADI2 antibodies to detect the expression of PADI2 in tissue sections to study its distribution and changes in different tissues and disease states.

[0005] Western Blot: Detecting the expression level of PADI2 in protein samples through anti-PADI2 antibodies to evaluate its changes under different experimental conditions.

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

[0007] Currently, the PADI2 antibodies on the market are mainly provided by well-known biological reagent companies such as Abcam, CST (Cell Signaling Technology), Sigma-Aldrich, and Santa Cruz Biotechnology. These antibodies are mainly used in research tools such as Western blot (WB), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FACS). However, due to the uneven quality of existing PADI2 antibodies, with poor sensitivity, specificity, and stability, there is no mature product that can be applied to the field of clinical detection and treatment. The specific defects are as follows:

[0008] 1. The affinity is relatively low, resulting 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 experiments.

[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 multiple studies have shown the potential of PADI2 as a disease biomarker and therapeutic target, high - affinity and high - specificity PADI2 antibodies are still relatively limited on the market, and there is no mature product that can be applied to the fields of clinical detection and treatment. Summary of the Invention

[0012] To fill the gaps in the existing technology, the present invention provides a high - affinity and high - specificity PADI2 antibody and its preparation method.

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

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

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

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

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

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

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

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

[0021] (8) The antibody comprises heavy chain CDR1, 2, 3 having sequences as shown in SEQ ID NO.31, 47, 63, and light chain CDR1, 2, 3 having sequences as shown in SEQ ID NO.32, 48, 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 as shown in SEQ ID NO.1 and 2 respectively; or

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

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

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

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

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

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

[0030] (8) The heavy chain and light chain of the antibody are as 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, and the expression vector comprises the aforementioned nucleic acid molecule.

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

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

[0035] In a sixth aspect, the present invention provides a method for producing the PADI2 antibody described above, which is characterized by comprising culturing the aforementioned recombinant cell or genetically engineered bacterium to produce the antibody.

[0036] In a seventh aspect, the present invention provides a PADI2 detection kit, which comprises the PADI2 antibody described above.

[0037] In an eighth aspect, the present invention provides the use of the antibody or kit described above 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 remarkable improvements :

[0039] 1. High affinity: The 8 PADI2 antibodies screened in the present invention all exhibit high affinity. The determination data of the affinity show that these antibodies can efficiently recognize and bind to PADI2, and have higher detection sensitivity in experimental applications.

[0040] 2. Good specificity: The 8 PADI2 antibodies screened in the present invention have undergone strict cross-reaction tests during the screening process, can specifically recognize PADI2, and will not cross-react with PADI4 or other homologous proteins. This characteristic makes it suitable for various medical research and has more clinical application value, avoiding false positive results caused by non-specific binding.

[0041] 3. Suitable for multiple detection methods: The 8 PADI2 antibodies screened in the present invention have been verified in multiple experimental platforms such as ELISA, Western blot (WB), immunofluorescence (IF), and flow cytometry (FACS), and all exhibit good binding performance.

[0042] 4. Have potential clinical application value: Since 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 the development of potential diagnostic reagents. Combined with a high-sensitivity detection platform, it can become a biomarker detection tool for PADI2-related diseases in the future, for early diagnosis and disease course monitoring. Description of the Drawings

[0043] The 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 to the present invention. In the drawings:

[0044] Figure 1 To rank the ELISA affinities of the supernatants of 44 sub-defined strains of holes;

[0045] Figure 2 To detect the protein gradients of 22 cell lines;

[0046] Figure 3 To verify the cross-recognition of the supernatants of 11 sub-defined strains of holes;

[0047] Figure 4 To re-cross-verify the supernatants of 44 defined strains of holes with Mouse PADI2, RAT PADI2, and PADIs proteins;

[0048] Figure 5 To re-supplement the protein gradient detection for 31 cell lines;

[0049] Figure 6 To detect the subtypes of the antibodies produced by 10 ascites-producing cell lines;

[0050] Figure 7 For the antibody titer detection results. Specific implementation manner

[0051] The following is a description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present invention and are not intended to limit the present invention.

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

[0053] Reagent Manufacturer Catalog Number 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 Double 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 Quick 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 Manufacturer Catalog Number Batch Quantity Mouse PADI2-KO mouse / / 6 pieces Positive cells* HEK293T-hPADI2 / / 24.6.3 5 pieces Negative cells* HEK293T / / 24.5.26 3 pieces

[0056] *The PADI2 positive / negative cell line refers to: obtaining the coding sequence (Coding Sequence, CDS) of the human PADI2 gene (Gene ID: 11240) by chemical synthesis method, cloning the CDS sequence of human PADI2 into the MSCV vector, and packaging the MSCV virus containing the CDS sequence of the human PADI2 gene using the PCL-10A1 packaging plasmid. Transducing the 293T cell line with the virus, using GFP on the vector as a sorting marker, and sorting out positive cell clones by flow cytometry to obtain the PADI2 positive 293T cell line. Wild-type 293T is used as the negative control cell line.

[0057] Example 1 Mouse immunization

[0058] 1.1 Immunization Grouping and Immunization Schedule Antigen: Human PADI2, Catalog Number CSB-MP896493H Ud7, Batch DA05994a1g0CX, Tag His Tag Mouse Strain: PADI2-KO Mouse

[0059] Quantity: 6 mice, labeled 1 - 6 respectively

[0060] Adjuvant: Freund's Adjuvant or Quick Immunization Adjuvant

[0061] First Immunization: 50 μg / mouse on June 17, 2024

[0062] Second Immunization: 50 μg / mouse on July 1, 2024

[0063] Third Immunization: 50 μg / mouse on July 15, 2024; Serum titer was detected on July 23, 2024 after the third immunization; Fourth Immunization: 75 μg or 100 μg / mouse on July 29, 2024; Serum titer was detected on August 12, 2024 after the fourth immunization;

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

[0065] 1.2 Mouse Immunization Procedure

[0066] a. Immunization with Freund's Adjuvant: Dilute the antigen with PBS and mix it with Freund's Adjuvant at a volume ratio of 1:1 (total volume 0.5 mL), then emulsify it in a 4°C environment 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, expel the air bubbles in the syringe, take out the mouse to be immunized from the cage and place it in a special fixing rack, and perform multiple-point subcutaneous injection on the back of the mouse;

[0067] b. Immunization with Quick Immunization Adjuvant: Dilute the antigen with PBS and mix it evenly with Quick Immunization 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, expel the air bubbles in the syringe, take out the mouse to be immunized from the cage and place it in a special fixing rack, and perform multiple-point subcutaneous injection on the leg muscles of the mouse;

[0068] c. Immunization Cycle: The interval between each of the first five immunizations is 2 weeks, and the interval for the sixth immunization is 1.5 months. (Note: Mouse No. 2 died on August 7, 2024)

[0069] 1.3 PADI2 Titer Detection Procedure

[0070] a. Coating: Dilute the antigen to 2 μg / mL with coating buffer CB, add 100 μL per well to the ELISA plate, and incubate overnight at 4°C. b. Blocking: Take out the ELISA plate, pat dry the liquid in the wells, block with 5% skim milk (dissolved in PBS), add 200 μL per well to the ELISA plate, incubate at 37°C for 2 h, and wash the plate 3 times with TBS;

[0071] c. Sample addition: Dilute the immune serum serially at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 with PBS (using pre-immune serum as a negative control), add 100 μL per well, and incubate at 37°C for 1 h. Wash the plate 3 times with TBS.

[0072] d. Secondary antibody addition: Goat anti-mouse secondary antibody-HRP (diluted 1:10000 with enzyme dilution), add 100 μL per well, incubate at 37°C for 40 min, and wash the plate 5 times with TBS;

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

[0074] f. Termination: Add stop solution, 50 μL per well, read the absorbance with an ELISA reader (wavelength 450 nm). The maximum dilution with a positive reaction is the serum titer of the immunized mice. Repeat the measurement once using the same method after each detection.

[0075] 1.4 Serum Western Blot detection procedure

[0076] Mix the immune serum sample (10 μL) with 2× loading buffer (10 μL), slowly add 20 μL of the sample and Loading buffer mixture to the sample well using a pipette. The loading amount of the lysate is 20 μg, the loading amount of the in vitro expressed PADI2 protein is 50 ng and 400 ng, and at the same time, load 10 μL of the marker (commercial molecular weight reference).

[0077] Western Blot detection:

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

[0079] b. Electrophoresis: Turn on the power supply, run the stacking gel at 80 V for 30 min, adjust the voltage to 120 V and electrophorese until the bromophenol blue loading buffer migrates to the bottom of the gel, then turn off the power supply;

[0080] c. Transfer membrane: After removing the upper stacking gel, immerse the separating gel in the transfer buffer. Immerse the PVDF membrane in isopropanol for 1 min and then transfer it to the transfer buffer. Immerse the filter paper in the transfer buffer as well (both the PVDF membrane and the filter paper are cut into the same size as the gel). Rinse the graphite electrodes with the transfer buffer, lay three layers of filter paper, and drip a little transfer buffer. Lay the separating gel and drip a little transfer buffer. Lay the membrane and drip a little transfer buffer. Finally, lay three layers of filter paper, drip a little transfer buffer, and use a spreading rod to drive out the air bubbles. Cover the electrodes, adjust the voltage to the maximum, and transfer the membrane at 1.5 mA / cm² of gel volume for 1.5 h (the load voltage should not exceed 1 V / cm 2 );

[0081] d. Blocking: Take out the membrane and rinse it with PBST for 5 min (shaking on a horizontal shaker). Take out the membrane and immerse it in the blocking solution at 37 °C for 2 h or overnight at 4 °C (the blocking solution is 5% non-fat milk powder);

[0082] e. Incubate with primary antibody: Take out the membrane and wash it three times with PBST, 10 min each time (shaking on a horizontal shaker). Take out the membrane and immerse it in the primary antibody dilution solution diluted with 2.5% non-fat milk powder at 25 °C for 1 h;

[0083] f. Incubate with secondary antibody (anti-mouse IgG): Take out the membrane and wash it three times with PBST, 10 min each time (shaking on a horizontal shaker). Take out the membrane and immerse it in the secondary antibody dilution solution diluted with 5% non-fat milk powder (1:50000) at 25 °C for 1 h;

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

[0085] 1.5 Detection results:

[0086] The detection results of the serum titers of 6 mice after the third immunization showed that: in the case of a slight background in the pre-immune serum, the overall immune serum titers were relatively low, and the order of the serum titers of the mice was: No. 1 > No. 4 = No. 2 > No. 3 = No. 6 = No. 5.

[0087] For the fourth immunization of the mice, the immunization dose was increased. Among them, 4 mice (No. 1, 3, 4, 6) were adjusted to 75 μg / mouse, and 2 mice (No. 5, 2) were adjusted to 100 μg / mouse. According to the detection results of the serum titers after the fourth immunization, 2 mice (No. 3, 4) were abandoned, and 3 mice (No. 1, 6, 5) were given a quick-acting adjuvant of 50 μg for the fifth immunization. Among them, the titer of No. 1 mouse was better. The WB detection results of the 1 / 500 dilution of the serum of No. 1 mouse after the fourth immunization showed that there were obvious target bands for the HU, MO, and RA-PADI2 recombinant proteins; compared with HEK293T cells, there were no obvious target bands in HEK293T-PADI2 cells.

[0088] 1. The results of the fifth immunization titer detection of three mice numbered 1, 6, and 5 showed that: compared with the fourth immunization titer, the titers of all three mice were significantly increased. Among them, the titers of mice numbered 5 and 6 were increased more significantly. The order of mouse titers was: No. 1 > No. 6 > No. 5. The WB detection results of the 1 / 500 dilution of the fifth immunization sera of mice numbered 1, 6, and 5 showed that when there were obvious target bands for the HU-PADI2 recombinant protein, there were obvious target bands (about 75 kD) in the sera of mice numbered 1 and 5 on the HEK293T-PADI2 cell samples, and there were no obvious target bands on the HEK293T and MCF-7 samples; no obvious target bands were detected in the serum of mouse No. 6.

[0089] The results of the sixth immunization titer detection of three mice numbered 1, 6, and 5 showed that: compared with the fifth immunization titer, the titer of mouse No. 5 was increased, and the titers of mice numbered 1 and 6 were not significantly increased; there was a slight background in the detection of the his-tag unrelated protein. The order of mouse titers was: No. 5 > No. 1 = No. 6. According to the titer detection results, mice numbered 1 and 5 were selected for fusion.

[0090] Example 2 Screening of the spleen cell fusion of mice numbered 1 and 5

[0091] 2.1 Cell fusion

[0092] Take 50 μg of PADI2 protein, dilute it to 100 μL with PBS, mix it with the quick-acting adjuvant at a volume ratio of 1:1, and inject it intraperitoneally into mice numbered 1 and 5.

[0093] Take the shocked mice numbered 1 and 5, exsanguinate by removing the eyeballs and then completely decapitate them to death, collect the blood and separate it. The serum is used as the positive control serum for antibody detection. Sterilely take out the spleen and grind it into single cells (spleen cells), wash it twice with serum-free DMEM and then prepare for fusion.

[0094] Take out a 9 ml fusion pool, soak it in an equal volume of 75% ethanol for 10 min, rinse it twice with sterile deionized water, and rinse it twice with BTX fusion buffer; take the myeloma cells (SP2 / 0) in the logarithmic growth phase and the prepared spleen cells, mix them at a ratio of 1:1, and then perform electrofusion. After completing the electrofusion procedure, let it stand for 2 - 3 min, then collect the cells in 40 mL of HAT screening medium, put it into a 37 °C 5% CO2 incubator, incubate for 1 h, supplement the HAT screening medium, plate the cells on a 96-well culture plate, culture it in a 37 °C 5% CO2 incubator, completely change the medium with HT complete medium on the 5th - 6th day, and detect the supernatant after culturing in a 37 °C 5% CO2 incubator for 24 - 48 h.

[0095] 2.2 ELISA screening of the fusion supernatant

[0096] Mice No.1 and No.5 were fused with 60 plates, and ELISA primary screening was carried out using PADI2 protein with his tag. According to the screening results, 378 original wells with OD>=2 were selected, and ELISA confirmation screening was carried out using HU-PADI2 protein coated at 2μg / ml, 0.5μg / ml, 0.2μg / ml together with a protein unrelated to the His tag. According to the detection results, 60 strains with a signal value of more than 1 for PADI2-2μg / ml coating, and a signal value of more than 0.25 for PADI2-0.5μg / ml and PADI2-0.2μg / ml coating were selected for subcloning.

[0097] Example 3 Screening of Subclones

[0098] Gently pipette the cells to be subcloned to make a suspension of single cells, add them to a counting plate for counting, and calculate the cell density; take 100μL of the cells to be subcloned and dilute them to 1×10 3 cells / mL; take 50-100μL of the cells and add them to 5-10mL of culture medium, mix well, and pipette 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 ELISA primary screening results of 60 subcloned cell strains showed that for each strain, the 5 monoclonal strains with the highest ELISA readings were selected for confirmation screening, and for cell strains with less than 5 monoclonal strains or no monoclonal strains, polyclones were supplemented for confirmation screening. (Among them, 5 strains had no positive monoclonal strains, and a total of 291 designated strain wells were selected for confirmation screening)

[0100] Among the detection results of the supernatant ELISA confirmation screening of 291 designated strain wells of 60 subcloned cell strains:

[0101] ① For 44 cell strains, the OD detected by HU-PADI2 coating at 0.2μg / ml in the supernatant was >1. Select Top 1 for designated strain preservation and expand the cell culture;

[0102] ② Discard 5 cell strains with no monoclonal strains;

[0103] ③ For 10 cell strains, the OD detected by HU-PADI2 coating at 0.2μg / ml in the supernatant was <1, and the cell strains were discarded;

[0104] ④ Discard 1 strain with background for the protein unrelated to the his tag.

[0105] Further, ELISA affinity ranking (supernatant gradient) was carried out on the supernatants of 44 sub-designated strain wells. HU-PADI2 was coated at 2μg / ml, and the cell supernatant was serially diluted 4-fold from 1 / 2 for 7 gradients. According to the detection results ( Figure 1) Select the 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) for protein gradient detection. The detection results show that ( Figure 2 ), when the antigen coating is diluted to 0.025 μg / ml (2.5 ng / well), the OD values of 11 strains are greater than 1. Select these 11 strains for cross-validation of Mouse PADI2, RAT PADI2, and PADIs series proteins.

[0106] Cross-recognition verification of the supernatant of 11 first-subcloned strains:

[0107] Detected proteins:

[0108] Name Catalog Number Batch Label HumanPADI2 CSB-MP896493HUd7 DA06085k1g0CX-1 HisTag PADI1 CSB-MP891543HU DD06009k1g0CX HisTag PADI3 CSB-MP891552HU DD06022k1g0CX HisTag PADI4 CSB-MP890757HU DD06020k1g0CX HisTag PADI6 CSB-MP744226HU DD06022k1g0CX HisTag RatPADI2 CSB-MP3642MO DD06042k1g0zn-2 HisTag MousePADI2 CSB-MP017377RA DD05767k1g0HP-3 HisTag

[0109] The ELISA detection results show that ( Figure 3 ), when the coating proteins of Mouse PADI2, RAT PADI2, and PADIs are 2 μg / ml, only 4A7D9, 50H2D4, and 59C3G2 do not recognize PADI1, PADI3, PADI4, and PADI6 proteins, and the other cell lines have strong signals. Re-cross-validate the supernatant of 44 subcloned strains with Mouse PADI2, RAT PADI2, and PADIs proteins, and it is found that 31 out of 44 subcloned strains do not recognize PADI1, PADI3, PADI4, and PADI6 proteins ( Figure 4 ). Select these 31 cell lines to replenish the protein gradient detection. In the detection of the supernatant of 31 cell lines that do not recognize PADIs ( Figure 5 ), when the antigen coating is 0.025 μg / ml, the OD values of 10 strains are >1; the OD values of 3 strains are between 1 and 0.9. (Among the 3 strains with OD>1, 3 do not recognize mice and rats; among the 3 strains with 1>OD>0.9, 1 recognizes mice and rats, and 2 only recognize mice). Select 9 strains with OD>1 (excluding 48C11E11 which does not recognize mice and rats) and 1 strain with 1>OD>0.9 (43C10H11 which recognizes mice and rats) for ascites production to prepare antibodies.

[0110] Example 4 Preparation of Ascites and Antibody Detection

[0111] Use kits (Frdbio kit / Proteintech kit) to detect the subtypes of the antibodies produced by 10 ascites-producing cell lines. The results show that (Figure 6 ) Among them, the subtypes of 6 cell lines (4A7D9, 42A2G8, 50H2D4, 51E10D11, 59H10D1, 59C3G2) are IgG2b, Igκ; the subtypes of 2 cell lines (12A4G6, 57B4B2) are IgG1, Igκ; the subtype of 1 cell line (50G9E6) is IgG2b, Igλ; the subtype of 1 cell line (43C10H11) is IgG2C, Igκ.

[0112] 4.1 Preparation of ascites:

[0113] a. Inject incomplete adjuvant into the peritoneal cavity of mice one week in advance, 0.5 mL / mouse;

[0114] b. Centrifuge to collect the subcultured cells, resuspend them with 1 mL of DMEM, with a concentration of 1×10 6 / mL. Aspirate the cells with a sterile syringe and inject them into the peritoneal cavity of mice;

[0115] c. After 6 days, observe the production of ascites in mice every day. If the abdomen is significantly enlarged and feels tense when touched by hand, the ascites can be collected with a sterile syringe and ascites needle.

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

[0117] 4.2 Purification and labeling of antibodies:

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

[0119] b. Take ascites, centrifuge at 12,000 rpm for 10 min at 4°C, collect the supernatant, filter it, and mix it with sodium acetate buffer;

[0120] c. Load the Protein G column at a speed of 0.5 ml / min, collect the penetration. After the loading is completed, continue to rinse with sodium acetate buffer until the G250 detection is colorless;

[0121] d. Rinse the column bed with ice 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 for 10 column bed volumes;

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

[0123] f. Ultrafiltrate and concentrate the elution peak to the same volume as the serum, and load it into a dialysis bag for overnight dialysis;

[0124] g. Change the solution once after 12 hours (5 L of PBS);

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

[0126] i. Purify the qualified antibodies, and take 500 μg each to label with biotin. Perform ELISA detection on the antibodies and biotin-labeled antibodies.

[0127] The results of antibody titer detection showed that among the 10 antibodies, except for the relatively low titer of 43C10H11, the titers of other antibodies were relatively high ( Figure 7 ). Among the 10 biotin-labeled antibodies, the titer of 21E10G7 was relatively low, and the titers of other antibodies were relatively high, and the titers were generally better than those of unbiotinylated antibodies.

[0128] Example 5 Monoclonal Antibody Sequencing

[0129] Select 8 cell lines with the best antibody effects therefrom and entrust Wuhan Jinkairui Biotechnology Co., Ltd. to perform sequencing. Analyze the antibody sequences of the obtained sequencing results. The sequence information is as follows:

[0130]

[0131]

[0132] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A PADI2 antibody with high affinity, characterized in that, The antibody is selected from any of the following: (1) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.17, 33, 49, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.18, 34, 50; or (2) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.19, 35, 51, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.20, 36, 52; or (3) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.21, 37, 53, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.22, 38, 54; or (4) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.23, 39, 55, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.24, 40, 56; or (5) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.25, 41, 57, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.26, 42, 58; or (6) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.27, 43, 59, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.28, 44, 60; or (7) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.29, 45, 61, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.30, 46, 62; or (8) The antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.31, 47, 63, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO.32, 48, 64.

2. The PADI2 antibody according to claim 1, wherein The antibody is selected from any of the following: (1) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; or (2) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; or (3) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively; or (4) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.7 and SEQ ID NO.8 respectively; or (5) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.9 and SEQ ID NO.10 respectively; or (6) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.11 and SEQ ID NO.12 respectively; or (7) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.13 and SEQ ID NO.14 respectively; or (8) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.15 and SEQ ID NO.16 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 according to claim 3.

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

6. An engineered bacterium, characterized in that, Comprising 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, Comprising culturing the recombinant cell according to claim 5, or the engineered bacterium according to claim 6, to produce an antibody.

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

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

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