A high-affinity antibody pair for detecting PADI2 and its application

Abstract

The present invention relates to the field of antibodies. Specifically, the present invention provides a high-affinity antibody pair for detecting PADI2 and its use. The antibody pair comprises a first antibody and a second antibody. The antibody pair exhibits excellent specificity and high affinity for human PADI2, effectively detecting trace amounts of PADI2 protein in human serum. This improves the accuracy, reliability, and sensitivity of immunoassay results, providing a more precise and reliable antibody tool for detecting PADI2 protein levels.

Description

Technical Field

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

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

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

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

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

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

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

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

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

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

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

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

[0013] First, in a first aspect, the present invention provides an anti-PADI2 antibody pair, the antibody pair consisting 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 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 represented by SEQ ID NOs. 31, 47, and 63, and light chain CDR1, 2, 3 represented by SEQ ID NOs. 32, 48, and 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 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 shown in SEQ ID NO. 5 and 6, respectively; or

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

[0028] (5) The heavy chain and light chain of the antibody are 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 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 one embodiment, the first antibody is selected from any one of the following antibodies:

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

[0034] (2) The heavy chain and light chain of the antibody are shown in SEQ ID NOs. 7 and 8, respectively;

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

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

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

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

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

[0040] (5) The heavy chain and light chain of the antibody are shown in SEQ ID NOs. 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 through gene recombination expression technology or obtained from ascites.

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

[0045] In a second aspect, the present invention provides a reagent for detecting PADI2, comprising any one of the aforementioned anti-PADI2 antibody pairs.

[0046] The third aspect of the present invention provides a kit for detecting PADI2, wherein the kit comprises the aforementioned reagents.

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

[0048] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0049] Figure 1 The results of matching HU-PADI2 antigen with 10 antibody strains by chessboard method;

[0050] Figure 2 Results of the second mutual matching test for 9 antibodies;

[0051] Figure 3 These are the results of sandwich tests between 5 antibody pairs and PADIs;

[0052] Figure 4 Results of sandwich assays of 5 antibody pairs against human serum;

[0053] Figure 5 Comparative titer testing of 59H10D1 and 50H2D4 ascites purified antibodies and recombinantly expressed antibodies

[0054] Figure 6 To compare the titers of 59H10D1 and 50H2D4 ascites purified biotinylated antibodies and recombinantly expressed biotinylated antibodies;

[0055] Figure 7 This is the result of sandwich detection of PADI2 protein by 50H2D4 / 59H10D1 antibodies;

[0056] Figure 8 These are the test results for the 50H2D4 / 59H10D1 antibody pair and human serum. DETAILED DESCRIPTION

[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 used to illustrate and explain the present invention, and are not used to limit the present invention.

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

[0059] Reagents factory Item No. HumanPADI2 Cusabio CSB-MP896493HUd7 MousePADI2 Cusabio CSB-MP3642MO RATPADI2 Cusabio CSB-MP017377RA HumanPADI1 Cusabio CSB-MP891543HU HumanPADI3 Cusabio CSB-MP891552HU HumanPADI4 Cusabio CSB-MP890757HU HumanPADI6 Cusabio CSB-MP744226HU Goat anti-mouse secondary antibody-HRP Cusabio CSB-PA573747 Goat anti-human secondary antibody-HRP Cusabio CSB-PA160506 avidin-HRP Jackson 016-030-084 DMEM Hyclone SH30022.01 Dual antibody Hyclone SV30010 Fetal bovine serum ExCell FSS500 HT Sigma H0137-10VL HAT Sigma H0262-10VL DMSO Sigma D2650 Freund's adjuvant Sigma F5506, F5881 Rapid immune adjuvant Invivogen 10253-45-01 Subtype detection kit Frdbio FRD90100P8Rd Subtype detection kit Proteintech PK20002

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

[0061]

[0062]

[0063] *PADI2-positive / negative cell lines are obtained by chemically synthesizing the coding sequence (CDS) of the human PADI2 gene (Gene ID: 11240). The human PADI2 CDS sequence is cloned into the MSCV vector and packaged with the PCL-10A1 packaging plasmid to produce the MSCV virus containing the human PADI2 CDS sequence. PADI2-positive 293T cell lines are obtained by transducing the virus into 293T cells, and positive cell clones are isolated by flow cytometry using the GFP marker on the vector. Wild-type 293T cells serve as the negative control cell line.

[0064] Example 1 Antibody pairing

[0065] In earlier studies, the applicant obtained 8 monoclonal antibodies with good titer, high affinity and specific recognition of human PADI2 antigen through antibody 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]

[0069] 1. Antibody checkerboard method was used to pair HU-PADI2 antigens (in addition to the 8 antibodies shown in Table 1, two additional antibodies were selected for pairing). The process is as follows:

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

[0071] b. Blocking: Remove the ELISA plate and pat dry the liquid in the wells. Block with 5% skim milk (dissolved in PBS) and add 200 μL / well of the ELISA plate. Incubate at 37°C for 2 h. Wash the plate three times with TBS.

[0072] c. Sample loading: HU-PADI2 protein was diluted 2-fold in 5% skim milk starting from 1 μg / mL for seven dilutions. 5% skim milk was used as a negative control. 100 μL of sample was added to each well. Incubate at 37°C for 1 hour. Wash the plate three times with TBS.

[0073] d. Add detection antibody: biotin-labeled antibody (1:1000 enzyme dilution), 100 μL / well, incubate at 37°C for 1 hour, and wash the plate five times with TBS;

[0074] e. Add enzyme: dilute avidin-HRP enzyme 1:30,000, 100 μL / well, incubate at 37°C for 1 hour, and wash the plate five times with TBS. f. Color development: add TMB substrate, 90 μL / well, incubate at 37°C in the dark for 15 minutes.

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

[0076] The results show that ( Figure 1 ) Sandwich assays yielded good results using 50H2D4 and 59H10D1 as the capture antibodies. However, using 12A4G6 as the detection antibody yielded essentially negative results and was discarded. Next, 50H2D4 and 59H10D1 were selected as the capture antibodies, and a gradient retest was performed using nine antibodies (12A4G6 was discarded).

[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 the five antibody pairs 50H2D4 / 4A7D9-bio, 50H2D4 / 42A2G8-bio, 50H2D4 / 59H10D1-Bio, 59H10D1 / 50H2D4-bio, and 59H10D1 / 59C3G2-bio were the best, and these five antibody pairs were further used for antibody sandwich ELISA.

[0078] Example 2 Verification of the sandwich detection effect of five 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 and 59H10D1) to 2 μg / mL in CB buffer, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight.

[0084] b. Blocking: Remove the ELISA plate and pat dry the liquid in the wells. Block with 5% skim milk (dissolved in PBS) and add 200 μL / well of the ELISA plate. Incubate at 37°C for 2 h. Wash the plate three times with TBS.

[0085] c. Sample loading: Human PADI2, PADI1, PADI3, PADI4, PADI6, Rat PADI2, and Mouse PADI2 were diluted 2-fold in 5% skim milk starting at 1 μg / ml in seven steps. 5% skim milk was used as a negative control. 100 μL of sample was added to each well. Incubate at 37°C for 1 hour. Wash the plate three times with TBS.

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

[0087] e. Add enzyme: dilute avidin-HRP enzyme 1:30,000, 100 μL / well, incubate at 37°C for 1 hour, and wash the plate five times with TBS. f. Develop: add TMB substrate, 90 μL / well, incubate at 37°C in the dark for 5-20 minutes.

[0088] g. Stop: Add stop solution, 50 μL / well, and read on a microplate reader (wavelength 450 nm).

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

[0090] ① All five antibody pairs can effectively recognize human PADI2; none of them recognize PADI1, PADI3, PADI4, and PADI6;

[0091] ②The 50H2D4 / 4A7D9-bio and 50H2D4 / 42A2G8-bio antibody pairs recognized Rat PADI2 well but weakly recognized Mouse PADI2;

[0092] ③The 50H2D4 / 59H10D1-bio antibody pair has good recognition for Mouse PADI2 but weak recognition for Rat PADI2;

[0093] ④59H10D1 / 50H2D4-bio weakly recognized Mouse PADI2 and almost did not recognize Rat PADI2;

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

[0095] Example 3 Effects of five groups of antibodies on the detection of PADI2 protein in human serum samples

[0096] In this example, normal human serum samples were obtained from serum of normal healthy volunteers recruited from Peking University People's Hospital (with signed informed consent).

[0097] 1. Antibody Pairing Process

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

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

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

[0101] c. Sample loading: Normal human serum was diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, and 128-fold with 5% skim milk. 5% skim milk was used as a negative control. 100 μL of sample was added to each well. Incubate at 37°C for 1 hour. Wash the plate three times with TBS.

[0102] d. Add detection antibody: dilute the biotinylated antibody 1:1000 with enzyme diluent, add 100 μL / well, incubate at 37°C for 1 hour, and wash the plate 5 times with TBS.

[0103] e. Add enzyme: dilute avidin-HRP enzyme 1:30,000, 100 μL / well, incubate at 37°C for 1 hour, and wash the plate five times with TBS.

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

[0105] g. Stop: Add stop solution, 50 μL / well, and read on 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 five groups of 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 expression vector:

[0110] a. Primer synthesis and enzyme digestion were performed using linearized plasmids containing the light and heavy chain sequences of 59H10D1 and 50H2D4 antibodies, respectively;

[0111] b. Primer synthesis and enzyme digestion of 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 spliced ​​product to competent cells and incubate on ice for 30 minutes; heat shock at 42°C for 90 seconds (the time here must be accurate); incubate on ice for 1 minute; add 800 μL of preheated LB medium and shake at 37°C at 158 ​​rpm for 120 minutes. Remove 800 μL of supernatant in a clean bench, mix the remaining bacterial solution, and spread it on a plate containing the corresponding antibiotic; invert the plate and incubate in a 37°C constant temperature incubator. Colonies should appear after 12-16 hours, and they can be detected by spot picking.

[0114] e. Transfer the correctly sequenced bacterial suspension to 200 mL of LB (containing the corresponding antibiotics). After overnight culture, centrifuge at 8000 rpm for 3 minutes at 4°C. Discard the supernatant and retain the precipitate. Use a plasmid extraction kit to extract the plasmid.

[0115] 2. Recombinant antibody expression and purification:

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

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

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

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

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

[0121] f. Elution was performed with 0.1 M glycine (pH 3.0), and the eluate was dialyzed into PBS (pH 7.4) overnight. After ultrafiltration, the concentration and purity were determined; SDS-PAGE was performed and the product was sent for quality control.

[0122] The purification of 50H2D4 and 59H10D1 recombinant antibodies was completed, and 500 μg of each antibody was labeled with biotin. The antibodies and labeled antibodies were tested by ELISA.

[0123] 3. Efficacy test of 50H2D4 and 59H10D1 recombinant antibodies

[0124] Detection protein:

[0125]

[0126] The titer of the antibody pair was detected by ELISA in the same manner as in the previous example, and the recombinant antibody pair was tested against PADI2 protein and human serum samples.

[0127] The ELISA test results showed that the titer comparison of 59H10D1, 50H2D4 ascites purified antibodies and recombinant expressed antibodies showed that the titer of recombinant expressed antibodies was better than that of ascites purified antibodies ( Figure 5 The titer comparison test of ascites purified biotinylated antibody and recombinant expressed biotinylated antibody showed that the titer difference between the several groups of antibodies was small ( Figure 6 ).

[0128] The results of sandwich detection of Human PADI2 protein by 50H2D4 / 59H10D1 antibody pair showed that ( Figure 7 ), 59H10D1 antibody was used as the coating antibody, HU-PADI2 protein was diluted 4-fold in a gradient, and 50H2D4-Bio was used as the detection antibody for detection. The results showed that there was almost no difference in the detection results between ascites antibodies and recombinantly expressed antibodies; 50H2D4 antibody was used as the coating antibody, HU-PADI2 protein was diluted 4-fold in a gradient, and 59H10D1-Bio was used as the detection antibody for detection. 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 test was performed on normal human serum samples using 50H2D4 / 59H10D1 antibodies. Normal human serum was diluted 2-fold. The experimental results showed that ( Figure 8 ), the results of ascites antibodies and recombinant antibodies were slightly different, 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 may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An anti-PADI2 antibody pair, 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 as shown in SEQ ID NO. 17, 33, 49, and light chain CDR1, 2, 3 as shown in SEQ ID NO. 18, 34, 50; The second 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.

2. An anti-PADI2 antibody pair, 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 shown in SEQ ID NO. 1 and 2, respectively; The heavy chain and light chain of the second antibody are shown in SEQ ID NO. 3 and 4, respectively.

3. The anti-PADI2 antibody pair according to claim 1 or 2, characterized in that: The first antibody serves as a capture antibody, and the second antibody serves as a detection antibody.

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

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

6. A reagent for detecting PADI2, characterized in that The invention comprises an anti-PADI2 antibody pair according to any one of claims 1 to 5.

7. A kit for detecting PADI2, characterized in that: The kit comprises the reagent according to claim 6.

8. The kit according to claim 7, characterized in that The kit is a colloidal gold kit.

Images