Preparation and application of specific monoclonal antibodies of different binding sites of tissue transglutaminase 2 (TGM2)

Abstract

The invention provides preparation and application of specific monoclonal antibodies of different binding sites of tissue transglutaminase 2 (TGM2). By combining protein informatics and immunology, polypeptides at different sites of tissue glutamine transferase 2 are designed and synthesized and coupled to KLH carrier protein to serve as immunogens, and mice are immunized to obtain the tissue glutamine transferase 2 immunogen. The monoclonal antibody provided by the invention can specifically recognize tissue transglutaminase 2, has no cross reaction with other connexins, has the advantages of high affinity, high sensitivity and high specificity when being used for detection, provides a method for accurately detecting the content of TGM2 protein in tissues, and has great significance for further deep research of TGM2.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody against TGM2 and application of the antibody. Background Art

[0002] Tissue transglutaminase 2 (TGM2) is the most widely expressed member of the transglutaminase family, with a molecular weight of 78 kDa. It exhibits multiple biological functions and is closely related to cell growth, differentiation and apoptosis. TGM2 is present in the extracellular matrix (ECM), plasma membrane, cytosol, mitochondria, circulating endosomes and nuclei, and its subcellular localization is one of the important determinants of its function. Intracellular TGM2 is believed to play an important role in cell apoptosis, and in the extracellular matrix, TGM2 is tightly bound to the ECM protein fibronectin. Extracellular TGM2 is associated with cell adhesion, cell tumorigenesis, ECM stability, wound healing, receptor signaling, cell proliferation and cell viability. TGM2 activity is affected by Ca 2+ , guanine nucleotides and redox signaling. TGM2 and Ca 2+ The interaction causes conformational changes, allowing TGM2 to exert transaminase activity or cell signal transduction function. In contrast, TGM2 binds to GTP or GDP to form a "closed" conformation, preventing its transamination activity. TGM2 exerts enzymatic activity without relying on Ca2+, that is, by hydrolyzing ATP and GTP, and interacts with a variety of proteins as an adhesion protein molecule. TGM2 can participate in the interaction of integrin-fibronectin, and the TGM2-FN complex promotes FN fibril deposition and arginyl-glycyl-aspartate (Arg-Gly-Asp, RGD) synthetic peptide-independent cell adhesion, and maintains the survival of osteoblasts, bone marrow-derived mesenchymal stem cells, and many tumor cells. TGM2 has protein disuloide isomerase (PDI) activity and is related to mitochondria-dependent apoptosis. TGM2 exerts unique serine / threonine kinase activity and phosphorylates multiple proteins such as insulin-like growth factor binding protein 3 and p53 tumor suppressor protein.

[0003] Existing studies have found that TGM2-related genes are involved and regulated in a variety of pathological changes such as endoplasmic reticulum stress, inflammation, viral infection, liver damage and apoptosis signals, and cancer, but its specific mechanism of action is still unclear and requires further research. The monoclonal antibody provided by the present invention can specifically recognize the connexin TGM2, has no cross-reaction with other connexins, has the advantages of high affinity, high sensitivity, and high specificity, and provides a method for accurately detecting the content of TGM2 protein in tissues, which is of great significance for further in-depth research on TGM2. Summary of the invention

[0004] The present invention provides three antibodies against TGM2 protein, wherein the antibodies are obtained by coupling TGM2 different binding site polypeptides (SEQ ID NO 7-9) to carrier proteins as immunogens and immunizing mice to prepare hybridoma cells. In a specific embodiment, the carrier protein is KLH, and the antibodies can specifically recognize TGM2 protein.

[0005] On the one hand, the present invention provides three anti-TGM2 protein antibodies, wherein the antibodies have a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID No.1-3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.4-6.

[0006] In one embodiment, the antibody is a recombinant antibody, a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

[0007] In one embodiment, the antibody is a murine antibody, a human antibody, or a chimeric antibody.

[0008] In a preferred embodiment, the antibody is a mouse antibody.

[0009] In one embodiment, the antibody further comprises a heavy chain constant region of an IgG selected from IgG1, IgG2, IgG3 or IgG4.

[0010] On the other hand, the present invention also provides a hybridoma cell producing the above-mentioned antibody.

[0011] On the other hand, the present invention also provides the use of the above antibody in the detection of TGM2 protein.

[0012] On the other hand, the present invention also provides a method for quantitatively detecting TGM2 protein by double antibody sandwich method, which comprises the step of detecting TGM2 protein by using the above-mentioned antibody.

[0013] In one embodiment, the method is indirect immunoenzyme-linked sorbent assay (ELISA), Western Blot (WB), immunohistochemistry (IHC) or immunofluorescence (IF).

[0014] On the other hand, the present invention also provides a reagent for detecting TGM2 protein, wherein the reagent comprises the above-mentioned antibody.

[0015] On the other hand, the present invention also provides a method for detecting TGM2 protein in a sample, the method comprising the step of detecting TGM2 protein using the above-mentioned antibody.

[0016] In one embodiment, the sample is selected from tissue cell lysate of human or other animals.

[0017] The present invention uses different binding site polypeptides (SEQ ID NO 7-9) of TGM2 that can induce immune response in the body to be coupled to KLH as immunogens to immunize mice, adopts hybridoma technology, and obtains hybridoma cell lines that can continuously and stably secrete anti-TGM2 protein monoclonal antibodies through cell fusion and screening, and obtains monoclonal antibodies secreted by the cell lines. The monoclonal antibodies prepared by coupling three different binding site polypeptides of TGM2 with carrier proteins as immunogens can specifically recognize TGM2, and there are 9 positive clones in polypeptide group 1, 12 positive clones in polypeptide group 2, and 18 positive clones in polypeptide group 3. Four positive clones in polypeptide group 1, 3 positive clones in polypeptide group 2, and 6 positive clones in polypeptide group 3 were screened out by repeated screening, and one clone in polypeptide group 1, one positive clone in polypeptide group 2, and one positive clone in polypeptide group 3 were further screened out, and these monoclonal antibodies were respectively called Clone1-2F8, Clone 2-1E9, and Clone 3-3A5. These monoclonal antibodies have good affinity. Experiments show that the antibody titer of these monoclonal antibodies can reach more than 0.001 μg / mL in the binding experiment of TGM2 protein, and there is no obvious specific cross with other related proteins; and protein immunoblotting, immunofluorescence, and immunohistochemistry can be used to detect the expression and distribution of TGM2 protein in vivo. In addition, since Clone 2-1E9 and Clone 3-3A5 recognize different antigenic sites of TGM2 protein, the double antibody sandwich method can be used to detect TGM2 protein using two specifically recognized monoclonal antibodies. Thus, the present invention provides a method for detecting TGM2 protein, which can accurately detect the content of TGM2 protein in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The indirect ELISA method was used to screen positive clones that recognized TGM2. Among them, 300 clones were screened in peptide group 1, 50 positive clones were found, and the 9 better positive clones were selected for the next step of rescreening.

[0019] Figure 2 The indirect ELISA method was used to screen positive clones that recognized TGM2 protein. 400 clones were screened in group 2 of peptides, 24 of which were positive clones. The 12 better positive clones were selected for the next step of rescreening.

[0020] Figure 3 The indirect ELISA method was used to screen positive clones that recognized TGM2 protein. 400 clones were screened in the three peptide groups, 60 of which were positive clones. The 18 better positive clones were selected for the next step of rescreening.

[0021] Figure 4 is the SDS-PAGE electrophoresis diagram of monoclonal antibody 1-2F8, 2-1E9, and 3-3A5, where M is the protein molecular weight standard (kDa);

[0022] Figure 5 The results are as follows: 1-2F8, 2-1E9, and 3-3A5 were used to detect the expression and distribution of TGM2 protein in vivo by immunofluorescence method, wherein NC was a negative control with no relevant monoclonal antibody.

[0023] Figure 6 The results are as follows: 1-2F8, 2-1E9, and 3-3A5 were used for immunohistochemistry to detect the expression and distribution of TG M2 protein in vivo, wherein NC was a negative control with no relevant monoclonal antibody.

[0024] Figure 7 This is the result of using 1-2F8, 2-1E9, and 3-3A5 to detect the expression of TGM2 protein in vivo using the Western blotting method.

[0025] Figure 8 This is the fitting curve of the ELISA sensitivity experiment for detecting TGM2 protein. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0027] Example 1. Establishment of hybridoma cell lines

[0028] 1. Experimental Materials

[0029] 1. Immunogen: The peptides with different binding sites of TGM2 are coupled to the carrier protein as immunogens, and hybridoma cells are prepared by immunizing mice.

[0030] 2. Culture medium: DMEM culture medium was purchased from Hyclone Company; HAT, HT selection culture medium and pristane were purchased from Sigma Company.

[0031] 3. Experimental animals: BALB / c mice, 8-12 weeks old, female, SPF grade animal culture.

[0032] 4. Other materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; PEG4000 was purchased from Fluka; HRP-goat anti-mouse IgG antibody was purchased from Jackson Immune; the remaining reagents were all domestic analytical grade products.

[0033] 2. Establishment of hybridoma cell lines

[0034] 1. Animal immunization

[0035] 1) Basic immunization: Mix equal volumes of antigen and Freund's complete adjuvant and fully emulsify, then inject subcutaneously at several points. Each injection volume is 100 μg per BALB / c mouse.

[0036] 2) Booster immunization: Booster immunization uses an emulsion of antigen and Freund's incomplete adjuvant. Three days before cell fusion, 150 μg of antigen in physiological saline solution is injected intraperitoneally.

[0037] 2. Preparation of hybridoma cells

[0038] The spleen cells of mice were collected according to the conventional method and fused with SP2 / 0 cells at a ratio of 10:1 with 500g / L PEG4000. The cells were selected and cultured with HAT medium. 10-15 days after fusion, the supernatant was taken and the hybridoma cell lines secreting anti-TGM2 recombinant protein were screened by indirect ELISA. The positive clones were subcloned by limiting dilution. The operation steps of the indirect ELISA method are as follows: 100μL of TGM2 recombinant protein was coated on the plate, 1:2000 of immune mouse serum was used as a positive control, the culture supernatant without clone growth and normal mouse serum were used as negative controls, 100μL of 1:2000 HRP-goat anti-mouse IgG was added to each well, and the OD value at 450nm was measured. Any clone with an OD450 value more than twice that of the negative control can be preliminarily determined as a positive clone. There are 9 positive clones in polypeptide group 1, 12 positive clones in polypeptide group 2, and 18 positive clones in polypeptide group 3. The rescreening screened out 4 positive clones in polypeptide group 1, 3 positive clones in polypeptide group 2, and 6 positive clones in polypeptide group 3. Figure 1-3 shown.

[0039] 3. Establishment of hybridoma cell lines

[0040] After four rounds of subcloning and indirect ELISA or cell ELISA screening, three hybridoma cell lines (one in polypeptide group 1, one in polypeptide group 2, and one in polypeptide group 3) that stably secreted monoclonal antibodies were finally obtained.

[0041] Table 1. Rescreening of positive clones (retained clones marked in red)

[0042]

[0043] 4. Potency detection of monoclonal antibodies obtained using the above hybridoma cell lines

[0044] 1) Mouse ascites titer determination: The ascites titer prepared by the above Clone 1-2F8, 2-1E9, and 3-3A5 hybridoma cells detected by indirect ELISA is: >1:20000000

[0045] 2) Determination of purified antibody titer: The titer of the purified antibody prepared by the above hybridoma cells was detected by indirect ELISA method: >0.001 μg / mL.

[0046] 5. Subculture of hybridoma cell lines

[0047] The hybridoma cell line was further cultured and passaged in a DMEM medium containing 10% fetal bovine serum. After the 10th passage, the hybridoma cell line was still able to grow well and be stably passaged, and the titer of the culture supernatant could still reach above 1:10,000.

[0048] The above results indicate that the obtained hybridoma cell line can be stably propagated and can continuously and stably secrete anti-TGM2 monoclonal antibodies.

[0049] After obtaining hybridoma cells that produce the desired monoclonal antibodies, a portion of the hybridoma cells must be preserved, otherwise during the continuous passage process, mutations or chromosome drift may occur, resulting in loss of inherent characteristics or loss of antibody production characteristics. In addition, during the long-term culture process, contamination and even destruction are inevitable. Therefore, a portion must be refrigerated for preservation. The preservation method is as follows:

[0050] 1. Materials

[0051] (1) Cells: Take cells in the logarithmic growth phase.

[0052] (2) 10% dimethyl sulfoxide protective solution (dimethyl sulfoxide can damage the filter, but is also destroyed by high pressure, so it cannot be filtered or sterilized by high pressure. It is sterile in itself): contains 10% dimethyl sulfoxide, 20% inactivated fetal bovine serum, and 70% RPMI-1640 solution.

[0053] (3) 20% FCS-1640 culture medium: containing 100 U / mL penicillin and 100 μg / mL streptomycin.

[0054] (4) Sterile 2mL ampoules, etc.

[0055] 2. Operation method

[0056] (1) Remove the old culture medium in the cell culture bottle and add 10% FCS-1640 solution to suspend the cells.

[0057] (3) Centrifuge at 1000 r / min for 10 min and remove the supernatant. The cell pellet was suspended in 10% dimethyl sulfoxide protective solution to make a 1.0×10 7 cells / mL.

[0058] (3) Take samples, stain with trypan blue, and count the viable cells, which should be above 95%.

[0059] (4) Use a syringe to dispense the cells into ampoules, 0.5 mL to 1.0 mL per ampoule, and seal the ampoule.

[0060] (5) Incubate at 4°C for 2 hours.

[0061] (6) Release the gaseous part of the liquid nitrogen tank (-70℃) for 15 hours.

[0062] (7) Transfer to liquid nitrogen part.

[0063] Example 2: Preparation of monoclonal antibodies against different sites of TGM2 protein

[0064] 1. Antibody Preparation

[0065] Adult BALB / c mice were selected and intraperitoneally inoculated with pristane (0.5 mL per mouse). 7 to 10 days later, 16th generation 1-2F8, 2-1E9, 3-3A5 hybridoma cells were intraperitoneally inoculated (1×10 per mouse). 6 ~2×10 6 After an interval of 5 days, when the abdomen is obviously swollen and the skin feels tense when touched, the ascites can be collected with a No. 9 needle.

[0066] The ascites was centrifuged (13000r / min for 30 minutes) to remove the cell components and other precipitates, and the supernatant was collected. It was purified with Protein G-Sepharose CL-4B, the column liquid was 20mmol / L PBS buffer, and the column chromatography eluent was: pH 2.7, 20mmol / L glycine buffer, to obtain monoclonal antibodies against different sites of TGM2.

[0067] 2. Identification of Antibodies

[0068] 1. Antibody purity identification:

[0069] SDS-PAGE electrophoresis identification, the purity is above 95%, such as Figure 4 shown.

[0070] 2. Identification of antibody classes and subclasses:

[0071] The indirect ELISA method was used to identify the Ig subtype of the antibodies produced by the above hybridoma cells using antibodies against various mouse Ig subtypes. The results are shown in Table 2. Clone 2-1E9 showed the strongest IgG2a signal, and Clone1-2F8 and 3-3A5 showed the strongest IgG1 signal. According to the criteria for subtype identification results, clone 2-1E9 was IgG2b subtype, and Clone 1-2F8 and 3-3A5 were IgG1.

[0072] Table 2. Antibody subtype identification results

[0073] 1-2F8 2-1E9 3-3A5 IgG1 0.115 IgG2a 0.012 0.011 IgG2b 0.008 0.008 0.008 IgG3 0.002 0.001 0.001 IgA 0.006 0.007 0.007 IgM 0.017 0.020 0.005

[0074] 3. Sequence determination of variable regions of clones 1-2F8, 2-1E9, and 3-3A5

[0075] The cell mRNA of the three clones was extracted and reverse transcribed into cDNA. High-fidelity PCR amplification was performed using universal primers for the variable region. The PCR product fragments were inserted into the T vector for DNA sequence determination, and the obtained sequences were translated into the amino acid sequence of the protein. The amino acid sequence of the variable region of Clone1-1-2F8 antibody: the amino acid sequence of the light chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.4. The amino acid sequence of the variable region of Clone2-1E9 antibody: the amino acid sequence of the light chain variable region is shown in SEQ ID No.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.5. The amino acid sequence of the variable region of Clone3-3A5 antibody: the amino acid sequence of the light chain variable region is shown in SEQ ID No.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.6. After comparing the sequences, no identical sequences were shown, indicating that the obtained sequences were clone-specific sequences, which were marked as 1-2F8, 2-1E9, and 3-3A5 sequences respectively.

[0076] Example 3: Affinity and specificity verification using purified antibodies

[0077] The affinity of monoclonal antibodies 1-2F8, 2-1E9, and 3-3A5 was tested by ELISA to determine their binding titers to TGM2 recombinant protein.

[0078] Detection method: On the first day, prepare the plate for TGM2 recombinant protein (KLH protein and Cx43 recombinant protein as control protein), and plate 100μL per well in 96-wells overnight. On the second day, after removing the supernatant, wash 3 times with PBS, and add 200μL / well 3% BSA for blocking for 1h. After washing three times with PBS, add gradient dilutions of Clone1-2F8, 2-1E9, and 3-3A5, incubate at 37°C for 1h, wash three times with PBS, add HRP (horseradish peroxidase) labeled mouse secondary antibody (1:2000), incubate for 1h, wash 5 times with PBS (5min for the first three times, 10min for the last 2 times), add colorimetric agent for 15min, and detect OD on the machine. 450 .

[0079] The formula of color developer solution A is as follows: 1 g of urea peroxide, 10.3 g of citric acid, 35.8 g of Na2HPO4·12H2O, and 100 μL of Tween-20 are added to every 1000 mL of water, and the pH is adjusted to 5; the formula of solution B is as follows: 700 mg of tetramethylbenzidine (TMB) (dissolved in 40 mL of DMSO) and 10.3 g of citric acid are added to every 1000 mL of distilled water, and the pH is adjusted to 2.4.

[0080] Table 3. Antibody affinity and specificity ELISA test

[0081]

[0082] The results are shown in Table 3. The titers of antibodies 1-2F8, 2-1E9, and 3-3A5 all reached above 0.001 μg / mL, showing high affinity, and no obvious specific cross-linking with other related proteins such as KLH and Cx, showing the characteristics of high affinity, high specificity, and high sensitivity.

[0083] Example 4: In vivo application of monoclonal antibodies 1-2F8, 2-1E9, 3-3A5

[0084] In this embodiment, the in vivo application of monoclonal antibodies 1-2F8, 2-1E9, and 3-3A5 was tested.

[0085] (1) The immunofluorescence experimental method is used for testing, and the steps of the method are as follows:

[0086] 1. Cell preparation: For monolayer growing cells, when subculturing, inoculate the cells into a culture dish with a treated coverslip in advance. When the cells are close to growing into a monolayer, remove the coverslip and wash twice with PBS. For suspended growing cells, take logarithmically growing cells, wash them twice by centrifugation with PBS (1000r / min, 5min), prepare cell slices with a cell centrifuge or directly prepare cell smears.

[0087] 2. Fixation and permeabilization: Wash the coverslips with cells in the culture plate with 1×PBS 3 times, 3 minutes each time. Fix the coverslips with 4% paraformaldehyde for 15 minutes, and wash the coverslips with 1×PBS 3 times, 3 minutes each time. Permeabilize the cells with 0.5% Triton X-100 (prepared with 1×PBS) at room temperature for 15 minutes (omit this step for antigens expressed on the cell membrane), and wash the coverslips with 1×PBS 3 times, 3 minutes each time.

[0088] 3. Blocking: Dry 1× PBS with absorbent paper, add 5% normal serum (similar to or consistent with the species of the secondary antibody) on the slide, and block at room temperature for 1 hour.

[0089] 4. Antibody incubation: Use absorbent paper to absorb the blocking solution. Do not wash. Add a sufficient amount of diluted antibody 1-1E2 to each slide and place it in a wet box. Incubate overnight at 4°C.

[0090] 5. Add fluorescent secondary antibody: Wash the slides 3 times with PBST, 3 min each time. After absorbing the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody. Incubate in a wet box at 37°C for 1 h. Wash the slides 3 times with PBST, 3 min each time.

[0091] 6. Fixed photo taking

[0092] 7. Re-staining the nucleus: Add DAPI and incubate in the dark for 5 minutes to stain the nucleus of the specimen, and wash away the excess DAPI with PBST for 5 minutes × 4 times.

[0093] 8. Use absorbent paper to absorb the liquid on the slide, seal the slide with sealing solution containing anti-fluorescence quenching agent, and observe and collect images under a fluorescence microscope.

[0094] The results are as follows Figure 5 As shown, 1-2F8, 2-1E9, and 3-3A5 can be used to detect the distribution and expression of TGM2 protein in vivo using immunofluorescence method.

[0095] (2) The immunohistochemical method is used for testing, and the steps of the method are as follows:

[0096] 1. Embedding tissue: Add liquid paraffin to the iron mold, cool it slightly, place the tissue to be embedded in the paraffin, arrange it neatly, cover the plastic mold box, add a little liquid paraffin, freeze it, and make the paraffin become solid.

[0097] 2. Sectioning: Remove the embedded tissue from the mold, place it on a paraffin slicer for sectioning, place the slide containing the intact tissue in 40℃ warm water, then take it out and dry it in a 37℃ oven.

[0098] 3. Dewaxing: Place the slides in xylene-xylene-100% alcohol-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol in sequence for 10 minutes in each reagent.

[0099] 4. Antigen repair: Rinse in clean water after dewaxing, soak in 3% H2O2 for 10 minutes, wash twice with clean water, add citric acid buffer, cook in a microwave for 3 minutes (medium heat) until boiling, cool to room temperature, cook again, and cool to room temperature.

[0100] 5. Serum blocking: After cooling to room temperature, pour out the citric acid buffer, wash twice with water, place the slide in PBS for 5 minutes, wash twice, wipe the PBS solution around the tissue, and immediately add serum blocking, incubate at 37℃ for 30 minutes. Dilute the serum 10 times (900μL PBS: 100μL serum blocking solution).

[0101] 6. Add primary antibody: Take the slide out of the incubator, absorb the serum with absorbent paper, add primary antibody, and store in a 4°C refrigerator overnight.

[0102] 7. Add secondary antibody: Take the slide out of the refrigerator, wash it with PBS three times, 5 minutes each time, wipe off the PBS, add secondary antibody, and place it in a 37℃ incubator for 30 minutes.

[0103] 8. Add SABC: Take the slide out of the incubator, wash it with PBS three times, 5 minutes each time, wipe off the PBS and add SABC, incubate it at 37℃ for 30 minutes. Dilute SABC 100 times (990μL PBS: 10μL SABC).

[0104] 9. Add color developer: Take the slide out of the incubator, wash it with PBS for 3 times, 5 minutes each time, wipe off the PBS and add color developer. (Color developer configuration: add 1 drop of color developer A to 1 mL of water, shake well, then add 1 drop of color developer B, shake well, then add 1 drop of color developer C, shake well) A: DAB; B: H2O2; C: phosphate buffer.

[0105] 10. Re-staining: Rinse the colored slide with clean water for a period of time, and then soak it in hematoxylin for staining for 30 seconds.

[0106] 11. Dehydration: After washing the restained slides in water, place the slides in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-100% alcohol-xylene-xylene in turn. Place them in each reagent for 2 minutes, and finally soak them in xylene and move them to the fume hood.

[0107] 12. Sealing: Drop neutral gum next to the tissue, cover with a coverslip, and place in a fume hood to dry.

[0108] The results are as follows Figure 6 As shown, antibodies 1-2F8, 2-1E9, and 3-3A5 can be used to detect the expression and distribution of TGM2 protein in vivo by immunohistochemistry, wherein the brown area indicates a positive reaction.

[0109] (3) Western blot (WB) was used for testing, and the steps of the method were as follows:

[0110] 1. SDS-PAGE electrophoresis: After cleaning the glass plate, put it into the clamp and clamp it tightly, then clamp it vertically on the rack to prepare for gel filling. Prepare 10% SDS separation gel, calculate the volume of the solution containing 50ng protein as the loading amount. Take out the loading sample into a 0.5mL centrifuge tube, add 5×SDS loading buffer to a final concentration of 1×, and perform electrophoresis after loading. Stop the electrophoresis until bromophenol blue just runs out, and transfer to the membrane.

[0111] 2. Transfer: Soak the cut NC membrane or PVDF membrane in water for 2 hours. Use tweezers to pinch one side of the membrane and gently place it in a flat dish with ultrapure water. To make the membrane float on the water, put the transfer clip, two sponge pads, a glass rod, filter paper and the soaked membrane in the enamel dish with transfer solution. Open the clip to keep the black side horizontal. Put a sponge pad on it and roll it back and forth with a glass rod several times to roll out the bubbles inside. Put three layers of filter paper on the pad, fix the filter paper with one hand and use the glass rod with the other hand to roll out the bubbles. Carefully peel off the separation glue and cover it on the filter paper, adjust it with your hands to align it with the filter paper, and gently roll out the bubbles with the glass rod. Cover the membrane on the glue and remove the bubbles. Cover the membrane with 3 pieces of filter paper and remove the bubbles. Finally, cover it with another sponge pad and roll it a few times to close the clip. The whole operation is carried out in the transfer solution, and bubbles must be rolled out continuously. Put the clip into the transfer tank and transfer at 80V for 1 hour. After transfer, stain the membrane with 1× Ponceau red dye for 5 minutes (shake on a decolorizing shaker). Then rinse off the unstained dye with water to see the protein on the membrane. Let the membrane dry for later use.

[0112] 3. Immunoreaction: After soaking the membrane with TBS from bottom to top, move it to a plate containing blocking solution, shake it on a decolorizing shaker at room temperature for 1 hour, dilute the antibodies 2-1G11 and 3-2G12 to appropriate concentrations with TBST (in a 1.5mL centrifuge tube); incubate at room temperature for 1 to 2 hours, wash it twice with TBST on a decolorizing shaker at room temperature for 10 minutes each time; then wash it once with TB S for 10 minutes. Prepare the secondary antibody dilution solution in the same way as above and contact it with the membrane for chemiluminescence reaction.

[0113] 4. Chemiluminescence: Mix equal volumes of reagents A and B on a plastic wrap; After 1 minute, place the membrane protein side down in full contact with the mixture; After 1 minute, move the membrane to another plastic wrap, remove all residual liquid, wrap it, and put it in an X-ray film holder. In a dark room, pour 1× developer and fixer into plastic trays respectively; take out the X-ray film under a red light and cut it to an appropriate size with a paper cutter (1 cm larger than the length and width of the film); open the X-ray film holder and place the X-ray film on the film. Once placed, it cannot be moved. Close the X-ray film holder. After the exposure is completed, open the X-ray film holder, take out the X-ray film, and quickly immerse it in the developer for development. When obvious bands appear, stop the development immediately.

[0114] 5. Gel image analysis: Scan or photograph the film, and use the gel image processing system to analyze the molecular weight and net optical density value of the target band.

[0115] The results are as follows Figure 7 As shown, antibodies 1-2F8, 2-1E9, and 3-3A5 can be used to detect the expression of TGM2 protein in vivo by Western blotting.

[0116] Example 5: Establishment of a TGM2 protein sandwich ELISA quantitative detection method using purified antibodies

[0117] Clone3-3A5 and Clone 2-1E9 HRP labeled antibodies (using modified sodium periodate method to label antibodies) were used for pairing experiments. Clone3-3A5 was used as the coating antibody and HRP labeled Clone 2-1E9 was used as the detection antibody. The sandwich ELISA quantitative detection method was determined, and the sensitivity could reach 1ng / ml (Table 4, Figure 8 ).

[0118] Table 4 Quantitative detection of TGM2 protein by sandwich ELISA

[0119]

[0120] Detection method: The coated antibody Clone3-3A5 was diluted to 10μg / mL with a carbonate buffer solution of pH 9.60.05mol / L, and 100μL was added to each well of the ELISA plate. The plate was coated overnight at 4°C, the coating solution was removed, the plate was washed 3 times with PBST, and patted dry. Then 200μL of 1% gelatin was added to each well, and the plate was placed in a 37°C incubator for 2 hours, and then washed 3 times with PBST, patted dry, and stored. The antibody of Clone 2-1E was labeled with horseradish peroxidase to obtain 2-1E9-HRP and stored. Add 100 μL / well of TGM2 recombinant protein gradient dilution to the ELISA plate, incubate at 37°C for 1 hour, then add 100 μL / well of 2-1E9-HRP (1:4000 dilution), incubate at 37°C for 1 hour, add 50 μL / well of color developer A and B for color development, incubate at 37°C for 10 min, add 50 μL / well of stop solution, and read the results.

[0121] The formula of color developer solution A is as follows: 1 g of urea peroxide, 10.3 g of citric acid, 35.8 g of Na2HPO4·12H2O, and 100 μL of Tween-20 are added to every 1000 mL of water, and the pH is adjusted to 5; the formula of solution B is as follows: 700 mg of tetramethylbenzidine (TMB) (dissolved in 40 mL of DMSO) and 10.3 g of citric acid are added to every 1000 mL of distilled water, and the pH is adjusted to 2.4.

[0122] The results are as follows Figure 8 As shown in Figure 2, the sensitivity of the TGM2 protein quantitative detection method is about 1 ng / mL, R 2 The linear range is 1.95-125 ng / mL.

Claims

1. Three antibodies or antigen-binding fragments thereof that recognize different sites of anti-tissue transglutaminase 2, wherein the antibodies have a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID No.1-3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.4-6.

2. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody, a polyclonal antibody or a recombinant antibody.

3. The antibody according to claim 1 or 2, characterized in that The antibody is a murine antibody, a human antibody or a chimeric antibody.

4. The antibody according to claim 1, characterized in that The antibody further comprises a heavy chain constant region of an IgG selected from IgG1, IgG2, IgG3 or IgG4.

5. An anti-TGM2 antibody or antigen-binding fragment thereof, wherein when bound to TGM2, it binds to an epitope located within the amino acid sequence of REKLVVRRGQPFWLTLH (SEQ ID NO: 7).

6. An anti-TGM2 antibody or antigen-binding fragment thereof, wherein when bound to TGM2, it binds to an epitope located within the amino acid sequence of SRRSSPVYVGRVVSGMVN (SEQ ID NO: 8).

7. An anti-TGM2 antibody or antigen-binding fragment thereof, wherein when bound to TGM2, it binds to an epitope located within the amino acid sequence of EVKVRMDLLPLHMGLHKLVVNF (SEQ ID NO: 9).

8. The antibody or fragment thereof according to claim 5, wherein the epitope may comprise one or more amino acid sequences selected from: REKLVVRRGQPFWLTLH of SEQ ID NO:

7.

9. The antibody or fragment thereof of claim 5, wherein the epitope consists of REKLVVRRGQPFWLTLH of SEQ ID NO:

7.

10. The antibody or fragment thereof of claim 5, wherein the epitope comprises all seventeen amino acids of SEQ ID NO:

7.

11. The antibody or fragment thereof according to claim 6, wherein the epitope comprises one or more amino acid sequences selected from the group consisting of SRRSSPVYVGRVVSGMVN of SEQ ID NO:

8.

12. The antibody or fragment thereof of claim 6, wherein the epitope consists of SRRSSPVYVGRVVSGMVN of SEQ ID NO:

8.

13. The antibody or fragment thereof of claim 6, wherein the epitope comprises all eighteen amino acids of SEQ ID NO:

8.

14. The antibody or fragment thereof according to claim 7, wherein the epitope may comprise one or more amino acid sequences selected from: EVKVRMDLLPLHMGLHKLVVNF of SEQ ID NO:

9.

15. The antibody or fragment thereof of claim 7, wherein the epitope consists of EVKVRMDLLPLHMGLHKLVVNF of SEQ ID NO:

9.

16. The antibody or fragment thereof according to claim 7, wherein the epitope comprises all twenty-two amino acids of SEQ ID NO:

9.

17. Use of the antibody according to any one of claims 1 to 4 in detecting TGM2.

18. A method for detecting TGM2, comprising the step of detecting TGM2 using the antibody according to any one of claims 1 to 4.

19. The antibody according to claim 18, characterized in that The method is indirect immunoenzyme-linked immunosorbent assay (ELISA), Western Blot (WB), immunohistochemistry (Immunohistochemistry, IHC) or immunofluorescence (Immunofluorescence, IF).

20. A method for quantitatively detecting TGM2, the method being a double antibody sandwich method, wherein one monoclonal antibody according to claims 1 to 4 is used as a coating antibody, and another monoclonal antibody labeled with an enzyme is used as a detection antibody.

21. The method according to claim 20 comprises the step of detecting TGM2 using the antibody according to any one of claims 1 to 4.

22. A reagent for detecting TGM2, comprising the antibody according to any one of claims 1 to 4.

23. A method for detecting TGM2 in a sample, the method comprising the step of detecting TGM2 using the antibody according to any one of claims 1 to 4.

24. The method according to claim 20, characterized in that The sample is selected from tissues of human body or other animals.

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