High affinity antibody pair for detecting padi2 and use thereof

Abstract

The present application relates to the field of antibodies, and in particular, the present application provides a high-affinity antibody pair for detecting PADI2 and application thereof. The antibody pair consists of a first antibody and a second antibody. The antibody pair has good specificity for human PADI2, high affinity, and can effectively detect trace amounts of PADI2 protein in human serum, thereby improving the accuracy, reliability and sensitivity of the immune detection result, and providing a more accurate and reliable antibody tool for detecting PADI2 protein level.

Description

[0001] This application is a divisional application of the patent filed on April 14, 2025, with application number 202510457672.3, entitled "A High-Affinity Antibody Pair for Detecting PADI2 and Its Application". Technical Field

[0002] This invention relates to the field of antibodies, and more specifically, to a high-affinity antibody pair for detecting PADI2 and its application. 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] The applicant previously obtained 10 monoclonal antibodies with high affinity and high specificity for PADI2 through antibody pair screening experiments. Based on this, the applicant further developed a high-affinity antibody pair for detecting PADI2 and its application through antibody pairing screening. Details are as follows:

[0014] Firstly, in a first aspect, the present invention provides an antibody pair against PADI2, said antibody pair comprising a first antibody and a second antibody, wherein,

[0015] The first antibody and the second antibody are selected from any two of the following antibodies:

[0016] (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

[0017] (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

[0018] (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

[0019] (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

[0020] (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

[0021] (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

[0022] (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

[0023] (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.

[0024] In a preferred embodiment, the antibody pair consists of a first antibody and a second antibody, wherein,

[0025] The first antibody and the second antibody are selected from any two of the following antibodies:

[0026] (1) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.1 and 2, respectively; or

[0027] (2) The heavy chain and light chain of the antibody are shown in SEQ ID NO.3 and 4, respectively; or

[0028] (3) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 5 and 6, respectively; or

[0029] (4) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.7 and 8, respectively; or

[0030] (5) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 9 and 10, respectively; or

[0031] (6) The heavy chain and light chain of the antibody are shown in SEQ ID NO. 11 and 12, respectively; or

[0032] (7) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 13 and 14, respectively; or

[0033] (8) The heavy chain and light chain of the antibody are shown in SEQ ID NO.15 and 16, respectively.

[0034] In one embodiment, the first antibody is selected from any one of the following antibodies:

[0035] (1) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.1 and 2, respectively; or

[0036] (2) The heavy chain and light chain of the antibody are shown in SEQ ID NO.7 and 8, respectively;

[0037] The second antibody is selected from any one of 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

[0038] (2) The heavy chain and light chain of the antibody are shown in SEQ ID NO.3 and 4, respectively; or

[0039] (3) The heavy chain and light chain of the antibody are as shown in SEQ ID NO. 5 and 6, respectively; or

[0040] (4) The heavy chain and light chain of the antibody are as shown in SEQ ID NO.7 and 8, respectively; or

[0041] (5) The heavy chain and light chain of the antibody are shown in SEQ ID NO.7 and 8, respectively;

[0042] The first antibody and the second antibody are different antibodies.

[0043] In one embodiment, the first antibody is a capture antibody, and the second antibody is a detection antibody.

[0044] In one embodiment, the antibody pair is obtained through gene recombination expression technology or through ascites.

[0045] In one embodiment, the second antibody is biotin-labeled.

[0046] In a second aspect, the present invention provides a reagent for detecting PADI2, said reagent comprising any of the aforementioned anti-PADI2 antibody pairs.

[0047] A third aspect of the present invention provides a kit for detecting PADI2, the kit comprising the aforementioned reagents.

[0048] In one embodiment, the kit is a colloidal gold kit. Attached Figure Description

[0049] 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:

[0050] Figure 1 The results of pairing 10 antibodies against the HU-PADI2 antigen using the checkerboard method are shown.

[0051] Figure 2 The results of the second cross-matching test for the nine antibodies;

[0052] Figure 3 The results are from a sandwich assay of 5 antibody pairs and PADIs.

[0053] Figure 4 The results of sandwich assay of 5 pairs of antibodies against human serum;

[0054] Figure 5 Comparative detection of the titers of purified antibodies and recombinant expressed antibodies from ascites fluid containing 59H10D1 and 50H2D4.

[0055] Figure 6 Comparative detection of the titers of purified biotinylated antibodies and recombinant expressed biotinylated antibodies from ascites fluid of 59H10D1 and 50H2D4;

[0056] Figure 7 The results of the 50H2D4 / 59H10D1 antibody sandwich assay for PADI2 protein are shown.

[0057] Figure 8 The results are for the detection of the 50H2D4 / 59H10D1 antibody pair against human serum. Detailed Implementation

[0058] 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.

[0059] The reagents involved in the following examples are as follows:

[0060]

[0061] The biomaterials involved in the following embodiments are as follows:

[0062]

[0063] 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.

[0064] Example 1 Antibody Pairing

[0065] In earlier studies, the applicant screened eight monoclonal antibodies with good titers, high affinity, and the ability to specifically recognize 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 that specifically recognize human PADI2

[0067]

[0068] 1. Antibody checkerboard method for pairing HU-PADI2 antigen (in addition to the 8 antibodies shown in Table 1, two more antibodies were selected for pairing), the procedure is as follows:

[0069] a. Coating: Dilute the coating antibody to 2 μg / mL with coating buffer CB, add 100 μL / well to the microplate, and incubate overnight at 4°C;

[0070] 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.

[0071] c. Sample loading: HU-PADI2 protein was serially diluted 2-fold starting from 1 μg / mL with 5% skim milk for 7 gradients, with 5% skim milk as a negative control. The sample was loaded at 100 μL / well, incubated at 37℃ for 1 h, and washed 3 times with TBS.

[0072] d. Add detection antibody: Biotin-labeled antibody (1:1000 enzyme dilution), 100 μL / well, incubate at 37℃ for 1 h, wash the plate 5 times with TBS;

[0073] e. Add enzyme: dilute avidin-HRP enzyme 1:30000, 100 μL / well, incubate at 37℃ for 1 h, wash plate 5 times with TBS;

[0074] f. Color development: Add TMB substrate, 90 μL / well, incubate at 37°C in the dark for 15 min;

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

[0076] The results show that ( Figure 1 When using 50H2D4 and 59H10D1 antibodies as coating antibodies (capture antibodies), the sandwich detection results were good. When using 12A4G6 antibody as the detection antibody (detection antibody), the results were mostly negative and were discarded. The next step is to select 50H2D4 and 59H10D1 antibodies as coating antibodies, and perform gradient retesting with 9 antibody strains (12A4G6 was discarded).

[0077] Following the above pairing procedure, a second pairing was performed on the nine antibodies, and the test results showed ( Figure 2 The antibody pairs 50H2D4 / 4A7D9-bio, 50H2D4 / 42A2G8-bio, 50H2D4 / 59H10D1-Bio, 59H10D1 / 50H2D4-bio, and 59H10D1 / 59C3G2-bio showed the best detection performance. These five antibody pairs were then used to perform a sandwich ELISA.

[0078] Example 25: Verification of the sandwich detection efficacy of the antibody group against PADI antigens.

[0079] Table 2. Protein Detection

[0080]

[0081] 1. Antibody pairing process:

[0082] a. Coating: Dilute the capture antibodies (50H2D4, 59H10D1) to 2 μg / mL with CB buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C;

[0083] 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.

[0084] c. Add samples: Human PADI2, PADI1, PADI3, PADI4, PADI6, Rat PADI2,

[0085] Mouse PADI2 was prepared by 2-fold dilution of 1 μg / ml in 5% skim milk for 7 gradients, with 5% skim milk as a negative control. The sample was added at 100 μL / well and incubated at 37°C for 1 h. The plate was washed 3 times with TBS.

[0086] d. Add detection antibodies: Biotin-labeled antibodies (4A7D9-bio, 42A2G8-bio, 59H10D1-Bio, 50H2D4-bio, 59C3G2-bio) were diluted 1:1000 with enzyme, 100 μL / well, incubated at 37℃ for 1 h, and washed 5 times with TBS.

[0087] e. Add enzyme: dilute avidin-HRP enzyme 1:30000, 100 μL / well, incubate at 37℃ for 1 h, wash plate 5 times with TBS;

[0088] f. Color development: Add TMB substrate, 90 μL / well, incubate at 37°C in the dark for 5-20 min;

[0089] g. Termination: Add stop solution, 50 μL / well, and read the value using a microplate reader (wavelength 450 nm).

[0090] 2. Sandwich method test results ( Figure 3 ):

[0091] ① All five antibody pairs can effectively recognize human PADI2; and none of them recognize PADI1, PADI3, PADI4, or PADI6.

[0092] ② The 50H2D4 / 4A7D9-bio and 50H2D4 / 42A2G8-bio antibodies showed good recognition of Rat PADI2 but weak recognition of Mouse PADI2.

[0093] ③ The 50H2D4 / 59H10D1-bio antibody shows good recognition of Mouse PADI2 but weak recognition of Rat PADI2;

[0094] ④ 59H10D1 / 50H2D4-bio has weak recognition of Mouse PADI2 and almost no recognition of Rat PADI2;

[0095] ⑤ The 59H10D1 / 59C3G2-bio antibody does not recognize Mouse PADI2 or Rat PADI2.

[0096] Example 35: Effect of antibody group on detection of PADI2 protein in human serum samples

[0097] In this embodiment, the normal human serum sample was obtained from the serum of normal healthy volunteers enrolled at Peking University People's Hospital (informed consent forms have been signed).

[0098] 1. Antibody pairing process

[0099] a. Coating: Dilute the capture antibody to 2 μg / mL with CB buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C;

[0100] 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.

[0101] c. Sample addition: Normal human serum was diluted with 5% skim milk at 2, 4, 8, 16, 32, 64 and 128 times. 5% skim milk was used as a negative control. 100 μL of sample was added to each well and incubated at 37°C for 1 h. The plate was washed 3 times with TBS.

[0102] d. Add detection antibody: Dilute biotin-labeled antibody 1:1000 with enzyme solution, 100 μL / well, incubate at 37℃ 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℃ for 1 h, wash 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 stop solution, 50 μL / well, and read the value using a microplate reader (wavelength 450 nm).

[0106] 2. Test 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 test results showed that all 5 antibody pairs could 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 the expression carrier:

[0110] a. Primers were synthesized and enzyme digested using linearized plasmids containing the light and heavy chain sequences of antibodies 59H10D1 and 50H2D4, respectively, as templates;

[0111] b. Primer synthesis and enzyme digestion were performed on the expression vectors pSecTag2A-M-IgG2b and pSecTag2A-M-KP;

[0112] c. The light and heavy chain digestion products of 59H10D1 and 50H2D4 antibodies were spliced ​​with the digested vector to obtain the spliced ​​product;

[0113] d. Add the splicing product to competent cells and incubate on ice for 30 min; heat shock at 42℃ for 90 s (the timing must be accurate); incubate on ice for 1 min; add 800 μL of preheated LB medium and incubate at 37℃ for 120 min at 158 ​​rpm in a shaker; remove 800 μL of supernatant in a clean bench, mix the remaining bacterial culture, and spread it onto agar plates containing the appropriate antibiotic; invert the plates and incubate at 37℃; colonies will appear after 12–16 hours, and the colonies can be detected by spot picking.

[0114] e. Transfer the correctly sequenced bacterial culture to 200 mL LB (containing the corresponding antibiotic), incubate overnight, centrifuge at 8000 rpm for 3 min at 4℃, discard the supernatant culture medium, retain the precipitate, and perform large-scale plasmid extraction using a plasmid extraction kit.

[0115] 2. Recombinant antibody expression and purification:

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

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

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

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

[0120] e. Using Zhongke Senhui's Protein A column, after equilibration with 20 mM PB (pH 7.0), load the cell supernatant onto the column twice;

[0121] f. Elute with 0.1 M glycine (pH 3.0), dialyze the eluent overnight to make PBS (pH 7.4), and then detect the concentration and purity after ultrafiltration; analyze by SDS-PAGE and send to quality control.

[0122] The recombinant antibodies 50H2D4 and 59H10D1 were purified. 500 μg of biotin was added to each antibody, and the antibodies and labeled antibodies were subjected to ELISA detection.

[0123] 3. Potential assay of recombinant antibodies against 50H2D4 and 59H10D1

[0124] Protein detection:

[0125]

[0126] The titer of the antibody pair was detected by ELISA using the same method as in the previous embodiments, and the recombinant antibody pair was tested in conjunction with PADI2 protein and human serum samples.

[0127] ELISA results showed that the titer of the purified ascites fluid antibody and the recombinant expressed antibody was superior to that of the purified ascites fluid antibody (59H10D1, 50H2D4). Figure 5 A comparative analysis of the titers of purified biotinylated antibodies and recombinant expressed biotinylated antibodies from ascites fluid showed that the differences in antibody titers among the groups were small. Figure 6 ).

[0128] The results of the 50H2D4 / 59H10D1 antibody sandwich assay against Human PADI2 protein showed that ( Figure 7Using 59H10D1 antibody as the coating antibody, HU-PADI2 protein was serially diluted 4-fold, and 50H2D4-Bio was used as the detection antibody. The results showed that there was almost no difference in the detection results between the ascites antibody and the recombinant expression antibody. Using 50H2D4 antibody as the coating antibody, HU-PADI2 protein was serially diluted 4-fold, and 59H10D1-Bio was used as the detection antibody. The results showed that the recombinant antibody was superior to the ascites antibody, and the 50H2D4 / 59H10D1-Bio recombinant antibody pair was superior to the 59H10D1 / 50H2D4-Bio recombinant antibody pair.

[0129] Sandwich assays were performed on normal human serum samples using 50H2D4 / 59H10D1 antibodies. The normal human serum was serially diluted twofold. The experimental results showed that... Figure 8 The results of ascites antibody and recombinant antibody were relatively similar, and the 50H2D4 / 59H10D1-Bio recombinant antibody pair was superior to the 59H10D1 / 50H2D4-Bio recombinant antibody pair.

[0130] 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. An antibody pair against PADI2, characterized in that, The antibody pair consists of a first antibody and a second antibody, wherein The first antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO. 17, 33, 49 respectively, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO. 18, 34, 50 respectively; The second antibody comprises heavy chain CDR1, 2, 3 with sequences as shown in SEQ ID NO. 23, 39, 55 respectively, and light chain CDR1, 2, 3 with sequences as shown in SEQ ID NO. 24, 40, 56 respectively.

2. An antibody pair against PADI2, characterized in that, The antibody pair consists of a first antibody and a second antibody, wherein The heavy chain and light chain of the first antibody are as shown in SEQ ID NO. 1, 2 respectively; The heavy chain and light chain of the second antibody are as shown in SEQ ID NO. 7, 8 respectively.

3. The pair of antibodies against PADI2 according to claim 1 or 2, characterized in that, The first antibody is a capture antibody, and the second antibody is a detection antibody.

4. The antibody pair according to claim 1 or 2, characterized in that, The antibody pair is obtained by genetic recombination expression technology or by ascites.

5. The antibody pair according to claim 1 or 2, characterized in that, The second antibody is labeled by biotin.

6. A reagent for detecting PADI2, characterized by, The kit comprises an antibody pair against PADI2 as claimed in any one of claims 1-5.

7. A kit for detecting PADI2, characterized by, The kit comprises a reagent as claimed in claim 6.

8. The kit of claim 7, wherein The kit is a colloidal gold kit.

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