High-affinity antibody pair for detecting PADI2 and application thereof

Abstract

The invention relates to the field of antibodies, in particular to a high-affinity antibody pair for detecting PADI2 and application of the high-affinity antibody pair. The antibody pair consists of a first antibody and a second antibody. The antibody pair has good specificity and high affinity to human PADI2, can effectively detect trace PADI2 protein in human serum, improves the accuracy, reliability and sensitivity of an immunodetection result, and provides a more accurate and reliable antibody tool for detecting the PADI2 protein level.

Description

Technical Field

[0001] The present invention relates to the field of antibodies. Specifically, the present invention relates to a pair of high-affinity antibodies for detecting PADI2 and their applications. Background Art

[0002] PADI2 (Peptidyl Arginine Deiminase 2) is a calcium-dependent enzyme expressed in mammals, which is mainly responsible for deiminating arginine residues in proteins to citrulline. This process is called citrullination or deimination, and 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 by 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 inconsistent quality of existing PADI2 antibodies, with poor sensitivity, specificity, and stability, there is no mature product that can be applied to the clinical detection and treatment fields. 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 in the current market, and there is no mature product that can be applied to the field of clinical detection and treatment. Summary of the Invention

[0012] The applicant previously obtained 10 monoclonal antibodies with high affinity and high specificity for PADI2 through antibody pair screening experiments. On this basis, the applicant further provided a high - affinity antibody pair for detecting PADI2 and its application through antibody pairing screening. Specifically as follows:

[0013] First, in a first aspect, the present invention provides an antibody pair against PADI2, which is composed of a first antibody and a second antibody, wherein,

[0014] The first antibody and the second antibody are selected from any two of the following antibodies:

[0015] (1) The antibody contains 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

[0016] (2) The antibody contains 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

[0017] (3) The antibody contains 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

[0018] (4) The antibody contains 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

[0019] (5) The antibody comprises heavy chain CDR1, 2, 3 having sequences as shown in SEQ ID NO.25, 41, 57, and light chain CDR1, 2, 3 having sequences as shown in SEQ ID NO.26, 42, 58; or

[0020] (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

[0021] (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

[0022] (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.

[0023] In a preferred embodiment, the antibody pair consists of a first antibody and a second antibody, wherein the first antibody and the second antibody are selected from any two of the following antibodies:

[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 as 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 as 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 as shown in SEQ ID NO.15 and 16 respectively.

[0032] In one embodiment, the first antibody is selected from any one of the following antibodies:

[0033] (1) The heavy and light chains of the antibody are shown in SEQ ID NO.1 and 2 respectively; or

[0034] (2) The heavy and light chains of the antibody are shown in SEQ ID NO.7 and 8 respectively;

[0035] The second antibody is selected from any one of the following antibodies:

[0036] (1) The heavy and light chains of the antibody are shown in SEQ ID NO.1 and 2 respectively; or

[0037] (2) The heavy and light chains of the antibody are shown in SEQ ID NO.3 and 4 respectively; or

[0038] (3) The heavy and light chains of the antibody are shown in SEQ ID NO.5 and 6 respectively; or

[0039] (4) The heavy and light chains of the antibody are shown in SEQ ID NO.7 and 8 respectively; or

[0040] (5) The heavy and light chains of the antibody are shown in SEQ ID NO.7 and 8 respectively;

[0041] Wherein, the first antibody and the second antibody are different antibodies.

[0042] In one embodiment, the first antibody serves as a capture antibody and the second antibody serves as a detection antibody.

[0043] In one embodiment, the antibody pair is obtained by recombinant expression technology or by ascites.

[0044] In one embodiment, the second antibody is labeled with biotin.

[0045] In a second aspect of the present invention, there is provided a reagent for detecting PADI2, the reagent comprising the antibody pair against PADI2 as described above.

[0046] In a third aspect of the present invention, there is provided a kit for detecting PADI2, the kit comprising the aforementioned reagent.

[0047] In one embodiment, the kit is a colloidal gold kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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, but do not constitute a limitation to the present invention. In the drawings:

[0049] Figure 1 Results of pairing the HU-PADI2 antigen by the checkerboard method with 10 antibodies;

[0050] Figure 2 Results of the second cross-matching test with 9 antibodies;

[0051] Figure 3 Results of the sandwich assay with 5 pairs of antibodies and PADIs;

[0052] Figure 4 Results of the sandwich assay with 5 pairs of antibodies and human serum;

[0053] Figure 5 Comparison and detection of the titers of ascites-purified antibodies and recombinant expressed antibodies of 59H10D1 and 50H2D4

[0054] Figure 6 Comparison and detection of the titers of ascites-purified biotinylated antibodies and recombinant expressed biotinylated antibodies of 59H10D1 and 50H2D4;

[0055] Figure 7 Results of the sandwich assay of the 50H2D4 / 59H10D1 antibody pair with PADI2 protein;

[0056] Figure 8 Results of the detection of the 50H2D4 / 59H10D1 antibody pair with human serum. Specific embodiments

[0057] 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 for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention.

[0058] The reagents involved in the following examples are as follows:

[0059] Reagent Manufacturer Article 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

[0060] The biological materials involved in the following examples are as follows:

[0061]

[0062]

[0063] *The PADI2 positive / negative cell line refers to: the coding sequence (CDS) of the human PADI2 gene (Gene ID: 11240) was obtained by chemical synthesis method. The CDS sequence of human PADI2 was cloned into the MSCV vector, and the MSCV virus containing the CDS sequence of the human PADI2 gene was packaged using the PCL-10A1 packaging plasmid. The 293T cell line was transduced with the virus, and the GFP on the vector was used as a sorting marker. Positive cell clones were sorted by flow cytometry to obtain the PADI2 positive 293T cell line. Wild-type 293T was used as a negative control cell line.

[0064] Example 1 Antibody Pairing

[0065] In earlier studies, the applicant screened 8 monoclonal antibodies with good titers, high affinity, and the ability to specifically recognize the human PADI2 antigen through antibody pair screening experiments. The sequence information of the antibodies is shown in Table 1 below:

[0066] Table 1. Sequence Information of 8 Monoclonal Antibodies Specifically Recognizing Human PADI2

[0067]

[0068]

[0069] 1. Pair the HU-PADI2 antigen by antibody checkerboard method (two additional antibodies were selected for pairing in addition to the 8 antibodies shown in Table 1), and the process is as follows:

[0070] a. Coating: Dilute the capture antibody to 2 μg / mL with coating buffer CB, add 100 μL / well to the ELISA plate, and incubate overnight at 4°C;

[0071] 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 / well to the ELISA plate, incubate at 37°C for 2 h, and wash the plate 3 times with TBS;

[0072] c. Sample addition: Dilute the HU-PADI2 protein 7-fold in a 2-fold gradient starting from 1 μg / mL with 5% skim milk, use 5% skim milk as a negative control, add the sample at 100 μL / well, incubate at 37°C for 1 h, and wash the plate 3 times with TBS;

[0073] d. Detection antibody addition: Biotin-labeled antibody (diluted 1:1000 with enzyme dilution), 100 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times with TBS;

[0074] e. Add enzyme: dilute avidin-HRP enzyme 1:30000, 100 μL / well, incubate at 37℃ for 1 hour, wash the plate 5 times with TBS; f. Color development: add TMB substrate, 90 μL / well, incubate at 37℃ in dark for 15 minutes;

[0075] g. Stop: Add stop solution, 50 μL / well, and read with a microplate reader (wavelength 450 nm).

[0076] The results show that Figure 1 ): When 50H2D4 and 59H10D1 antibodies were used as the package antibodies (capture antibodies), the sandwich test results were good. When 12A4G6 antibody was used as the detection antibody (detection antibody), the results were basically negative and were discarded. In the next step, 50H2D4 and 59H10D1 antibodies were selected as the package antibodies, and 9 antibodies (12A4G6 was discarded) were adjusted for gradient retesting.

[0077] According to the above pairing process, the 9 antibodies were matched for the second time, and the test results showed that ( Figure 2 ), the detection effects of 5 antibody pairs 50H2D4 / 4A7D9-bio, 50H2D4 / 42A2G8-bio, 50H2D4 / 59H10D1-Bio, 59H10D1 / 50H2D4-bio, and 59H10D1 / 59C3G2-bio were the best, and these 5 antibody pairs were further used for antibody sandwich ELISA.

[0078] Example 2 Verification of the sandwich detection effect of five groups of antibody pairs on PADIs antigen

[0079] Table 2. Detected proteins

[0080]

[0081]

[0082] 1. Antibody pairing process:

[0083] a. Coating: Dilute the capture antibodies (50H2D4, 59H10D1) to 2 μg / mL with CB buffer, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight;

[0084] b. Blocking: Take out the ELISA plate and 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 3 times with TBS;

[0085] c. Sample addition: Human PADI2, PADI1, PADI3, PADI4, PADI6, Rat PADI2, and Mouse PADI2 were serially diluted 7-fold starting from 1 μg / ml with 5% defatted milk. 5% defatted milk was used as a negative control. Samples were added at 100 μL / well, incubated at 37 °C for 1 h, and the plates were washed 3 times with TBS;

[0086] d. Detection antibody addition: Biotin-labeled antibodies (4A7D9-bio, 42A2G8-bio, 59H10D1-Bio, 50H2D4-bio, 59C3G2-bio) were diluted 1:1000 with the enzyme, added at 100 μL / well, incubated at 37 °C for 1 h, and the plates were washed 5 times with TBS;

[0087] e. Enzyme addition: Avidin-HRP enzyme was diluted 1:30000, added at 100 μL / well, incubated at 37 °C for 1 h, and the plates were washed 5 times with TBS; f. Color development: TMB substrate was added at 90 μL / well, and incubated at 37 °C in the dark for 5 - 20 min;

[0088] g. Termination: Stop solution was added at 50 μL / well, and the absorbance was measured using a microplate reader (wavelength 450 nm).

[0089] 2. Sandwich assay detection results ( Figure 3 ):

[0090] ① All 5 antibody pairs could effectively recognize human PADI2; and none of them recognized PADI1, PADI3, PADI4, or PADI6;

[0091] ② The 50H2D4 / 4A7D9-bio and 50H2D4 / 42A2G8-bio antibody pairs had good recognition of Rat PADI2 and weak recognition of Mouse PADI2;

[0092] ③ The 50H2D4 / 59H10D1-bio antibody pair had good recognition of Mouse PADI2 and weak recognition of Rat PADI2;

[0093] ④ The 59H10D1 / 50H2D4-bio pair had weak recognition of Mouse PADI2 and hardly recognized Rat PADI2;

[0094] ⑤ The 59H10D1 / 59C3G2-bio antibody pair did not recognize Mouse PADI2 or Rat PADI2.

[0095] Example 3 Effect of 5 antibody pairs in detecting PADI2 protein in human serum samples

[0096] In this example, the normal human serum samples were from the sera of normal healthy volunteers enrolled in Peking University People's Hospital (who had signed informed consent forms).

[0097] 1. Antibody pairing process

[0098] a. Coating: Dilute the capture antibodies to 2 μg / mL with CB buffer, add 100 μL / well to the ELISA plate, and incubate overnight at 4°C;

[0099] b. Blocking: Take out the ELISA plate, pat dry the liquid in the wells, block with 5% skim milk (dissolved in PBS), 200 μL / well

[0100] Add to the ELISA plate, incubate at 37°C for 2 h, and wash the plate 3 times with TBS;

[0101] c. Sample addition: Dilute normal human serum 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, and 128-fold with 5% skim milk. Use 5% skim milk as the negative control. Add samples at 100 μL / well, incubate at 37°C for 1 h, and wash the plate 3 times with TBS;

[0102] d. Add detection antibody: Dilute the biotin-labeled antibody 1:1000 with enzyme, 100 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times with TBS;

[0103] e. Add enzyme: Dilute avidin-HRP enzyme 1:30000, 100 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times with TBS;

[0104] f. Color development: Add TMB substrate, 90 μL / well, incubate at 37°C in the dark for 5 - 20 min;

[0105] g. Termination: Add termination solution, 50 μL / well, and read with an ELISA reader (wavelength 450 nm).

[0106] 2. Detection results( Figure 4 ):

[0107] Although the content of PADI2 protein in normal human serum is extremely low (the concentration of PADI2 protein in serum is between 0 - 5 ng / ml), the detection results show that all 5 groups of antibody pairs can effectively detect PADI2 protein in human serum.

[0108] Example 4 Recombinant expression and verification of antibody pairs (taking 59H10D1 / 50H2D4 as an example)

[0109] 1. Construction of expression vector:

[0110] a. Use the linearized plasmids containing the light and heavy chain sequences of 59H10D1 and 50H2D4 antibodies as templates for primer synthesis and enzyme digestion respectively;

[0111] b. Synthesize primers and perform restriction digestion on the expression vectors pSecTag2A-M-IgG2b and pSecTag2A-M-KP;

[0112] c. Ligate the digested products of the light and heavy chains of the 59H10D1 and 50H2D4 antibodies with the digested vector, and the ligation products;

[0113] d. Add the ligation products to competent cells, incubate on ice for 30 min; heat shock at 42 °C for 90 s (the time here must be accurate); incubate on ice for 1 min; add 800 μL of pre-warmed LB medium, place in a shaker at 37 °C at 158 rpm for 120 min, aspirate 800 μL of the supernatant in a laminar flow hood, mix the remaining bacterial solution, spread it on a plate containing the corresponding antibiotic; invert the plate and culture it in a 37 °C constant temperature incubator. Colonies can appear after 12 - 16 hours, and pick colonies for detection;

[0114] e. Transfer the bacterial solution with correct sequencing results to 200 mL of LB (containing the corresponding antibiotic), culture overnight, centrifuge at 8000 rpm for 3 min at 4 °C, discard the supernatant medium, retain the precipitate, and use a plasmid extraction kit for large-scale plasmid extraction.

[0115] 2. Recombinant antibody expression and purification:

[0116] a. Take 100 μg of the mixed plasmid with VH:VL = 1:2, dilute it to 5 mL and mix it with PET in equal volume, and let it stand for 10 min to prepare the plasmid-vector complex;

[0117] b. Prepare HEK293F cells in the logarithmic growth phase and dilute the cell density to 2×10 6 cells / mL;

[0118] c. Take 100 mL of the diluted cells, add the prepared plasmid-vector complex while shaking, 100 μL / well, incubate in a 5% CO2, 37 °C constant temperature shaker for 72 h;

[0119] d. Collect the cell suspension, centrifuge to obtain the supernatant, filter it through a 0.45 μm filter and prepare for purification;

[0120] e. Use the protein A column from Zhongke Senhui, equilibrate it with 20 mM PB (pH 7.0) and load the cell supernatant onto the column twice;

[0121] f. Elute with 0.1 M glycine (pH 3.0), dialyze the eluate overnight into PBS (pH 7.4), ultrafilter and detect the concentration and purity; perform SDS-PAGE detection and send it for quality control.

[0122] The purification of the 50H2D4 and 59H10D1 recombinant antibodies is completed. Take 500 μg of each antibody to label with biotin, and perform ELISA detection on the antibody and the labeled antibody.

[0123] 3. Potency Detection of 50H2D4 and 59H10D1 Recombinant Antibodies

[0124] Protein to be detected:

[0125]

[0126] In the same manner as in the foregoing embodiments, the titers of the antibody pairs were detected by ELISA, and the recombinant antibody pairs were detected with PADI2 protein and human serum samples.

[0127] The ELISA detection results showed that the comparison of the titers of the ascites-purified antibodies and recombinant expressed antibodies of 59H10D1 and 50H2D4 showed that the titer of the recombinant expressed antibody was better than that of the ascites-purified antibody ( Figure 5 ). The comparison of the titers of the ascites-purified biotinylated antibodies and recombinant expressed biotinylated antibodies showed that the differences in the titers of several groups of antibodies were small ( Figure 6 ).

[0128] The sandwich detection results of the 50H2D4 / 59H10D1 antibody pair against Human PADI2 protein showed ( Figure 7 ), with 59H10D1 antibody as the coating antibody, HU-PADI2 protein serially diluted 4-fold, and 50H2D4-Bio as the detection antibody for detection. The results showed that there was almost no difference between the ascites antibody and the recombinant expressed antibody; with 50H2D4 antibody as the coating antibody, HU-PADI2 protein serially diluted 4-fold, and 59H10D1-Bio as the detection antibody for detection. The results showed that the recombinant antibody was better than the ascites antibody, and the 50H2D4 / 59H10D1-Bio recombinant antibody pair was better than the 59H10D1 / 50H2D4-Bio recombinant antibody pair.

[0129] Using the 50H2D4 / 59H10D1 antibody pair for sandwich detection of normal human serum samples, normal human serum was serially diluted 2-fold. The experimental results showed ( Figure 8 ) that the difference between the ascites antibody and the recombinant antibody was small, and the 50H2D4 / 59H10D1-Bio recombinant antibody pair was better than the 59H10D1 / 50H2D4-Bio recombinant antibody pair.

[0130] 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. An anti-PADI2 antibody pair, characterized in that, The antibody pair consists of a first antibody and a second antibody, wherein the first antibody and the second antibody are any two selected from the following antibodies: (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. An anti-PADI2 antibody pair, characterized in that, The antibody pair consists of a first antibody and a second antibody, wherein the first antibody and the second antibody are any two selected from the following antibodies: (1) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.1 and 2 respectively; or (2) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.3 and 4 respectively; or (3) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.5 and 6 respectively; or (4) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.7 and 8 respectively; or (5) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.9 and 10 respectively; or (6) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.11 and 12 respectively; or (7) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.13 and 14 respectively; or (8) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.15 and 16 respectively.

3. The anti-PADI2 antibody pair according to claim 1 or 2, characterized in that the first antibody is selected from any one of the following antibodies: (1) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.1 and 2; or (2) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.7 and 8; the second antibody is selected from any one of the following antibodies: (1) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.1 and 2; or (2) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.3 and 4; or (3) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.5 and 6; or (4) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.7 and 8; or (5) the heavy chain and light chain of the antibody are respectively shown in SEQ ID NO.7 and 8; wherein, the first antibody and the second antibody are different antibodies.

4. The anti-PADI2 antibody pair according to any one of claims 1-3, characterized in that, The first antibody is used as a capture antibody, and the second antibody is a detection antibody.

5. The antibody pair according to any one of claims 1-3, characterized in that, The antibody pair is obtained by recombinant expression technology or by ascites.

6. The antibody pair according to any one of claims 1-3, characterized in that The second antibody is labeled with biotin.

7. A reagent for detecting PADI2, characterized in that, Comprising the anti-PADI2 antibody pair according to any one of claims 1-6.

8. A kit for detecting PADI2, characterized in that, The kit contains the reagent according to claim 7.

9. The kit according to claim 8, characterized in that, The kit is a colloidal gold kit.

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