Preparation and application of tissue glutamine transferase 2 (TGM2) different binding site specific monoclonal antibody

Abstract

This invention provides a method for preparing and applying monoclonal antibodies specific to different binding sites of tissue glutaminase 2 (TGM2). Combining protein informatics and immunology, peptides targeting different binding sites of TGM2 were designed and synthesized, coupled to a KLH carrier protein as immunogens, and obtained by immunizing mice. The monoclonal antibodies provided by this invention can specifically recognize tissue glutaminase 2, exhibiting no cross-reactivity with other linker proteins. They possess advantages of high affinity, high sensitivity, and high specificity for detection, and provide an accurate method for detecting TGM2 protein content in tissues, which is of great significance for further in-depth research on TGM2.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a monoclonal antibody against TGM2 and the application of the antibody. Background Technology

[0002] Tissue transglutaminase 2 (TGM2) is the most widely expressed member of the transglutaminase family, with a molecular weight of 78 kDa. It exhibits a variety of 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 nucleus; its subcellular localization is one of the important determinants of its function. Intracellular TGM2 is considered to play an important role in apoptosis, while in the ECM, TGM2 binds tightly to the ECM protein fibronectin. Extracellular TGM2 is involved in cell adhesion, tumorigenesis, ECM stability, wound healing, receptor signaling, cell proliferation, and cell viability. TGM2 activity is affected by calcium channel blockers (CCLs). 2+ Regulation of guanine nucleotides and redox signaling. TGM2 and Ca 2+ Interactions induce conformational changes, enabling TGM2 to exert its transaminase activity or cell signal transduction function. Conversely, TGM2 binds to GTP or GDP to form a "closed" conformation, preventing its transaminase activity. TGM2 exerts its enzymatic activity in a Ca2+-independent manner, i.e., by hydrolyzing ATP and GTP, and interacts with a variety of proteins as an adhesion protein molecule. TGM2 can participate in integrin-fibronectin interactions; the TGM2-FN complex promotes FN fibril deposition and arginyl-glycyl-aspartate (RGD) peptide-independent cell adhesion, and maintains the survival of osteoblasts, bone marrow-derived mesenchymal stem cells, and many tumor cells. TGM2 possesses protein disuloide isomerase (PDI) activity, which is associated with mitochondrial-dependent apoptosis. TGM2 exhibits unique serine / threonine kinase activity and phosphorylates various proteins, including insulin-like growth factor binding protein 3 and p53 tumor suppressor protein.

[0003] Existing research has found that TGM2-related genes are involved in and regulate various pathological changes, including endoplasmic reticulum stress, inflammation, viral infection, liver damage, apoptosis signaling, and cancer. However, their specific mechanisms of action remain unclear and require further investigation. The monoclonal antibody provided in this invention can specifically recognize the TGM2 linker protein, exhibits no cross-reactivity with other linker proteins, and possesses advantages such as high affinity, high sensitivity, and high specificity. It also provides an accurate method for detecting TGM2 protein levels in tissues, which is of great significance for further in-depth research on TGM2. Summary of the Invention

[0004] This invention provides three antibodies against the TGM2 protein. These antibodies are obtained by immunizing mice with TGM2-binding peptides (SEQ ID NO 7-9) conjugated to a carrier protein as immunogens to prepare hybridoma cells. In a specific embodiment, the carrier protein is KLH, and the antibodies specifically recognize the TGM2 protein.

[0005] On one hand, the present invention provides three antibodies against TGM2 protein, the antibodies having a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID No. 1-3, and the amino acid sequence of the heavy chain variable region being 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 includes a heavy chain constant region comprising IgG selected from IgG1, IgG2, IgG3, or IgG4.

[0010] On the other hand, the present invention also provides hybridoma cells that produce the above-mentioned antibodies.

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

[0012] On the other hand, the present invention also provides a method for quantitative detection of TGM2 protein using a double antibody sandwich method, the method comprising the step of detecting TGM2 protein using the aforementioned antibodies.

[0013] In one embodiment, the method is an indirect enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF).

[0014] On the other hand, the present invention also provides a reagent for detecting TGM2 protein, the reagent comprising the antibody described above.

[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 lysates from humans or other animals.

[0017] This invention uses peptides (SEQ ID NO 7-9) with different binding sites of TGM2 that can induce an immune response in the body, conjugated to KLH as immunogens to immunize mice. Hybridoma cell lines capable of continuously and stably secreting monoclonal antibodies against TGM2 protein were obtained through cell fusion and screening using hybridoma technology. Monoclonal antibodies were then secreted from these cell lines. The monoclonal antibodies prepared by conjugating three peptides with different TGM2 binding sites to a carrier protein as immunogens specifically recognize TGM2, with 9 positive clones in peptide group 1, 12 positive clones in peptide group 2, and 18 positive clones in peptide group 3. Further screening yielded 4 positive clones in peptide group 1, 3 positive clones in peptide group 2, and 6 positive clones in peptide group 3. Further screening yielded 1 positive clone in peptide group 1, 1 positive clone in peptide group 2, and 1 positive clone in peptide group 3. These monoclonal antibody lines were respectively named Clone1-2F8, Clone2-1E9, and Clone3-3A5. These monoclonal antibodies exhibit good affinity. Experiments show that when these monoclonal antibodies bind to TGM2 protein, the antibody titer can reach above 0.001 μg / mL, and there is no significant specific cross-contamination with other related proteins. Furthermore, the expression and distribution of TGM2 protein in vivo can be detected using Western blotting, immunofluorescence, and immunohistochemistry. In addition, since Clone 2-1E9 and Clone 3-3A5 recognize different antigenic sites of TGM2 protein, a double-antibody sandwich method can be used to detect TGM2 protein using two specifically recognized monoclonal antibodies. Therefore, this invention provides a method for detecting TGM2 protein, which can accurately detect the content of TGM2 protein in a sample. Attached Figure Description

[0018] Figure 1Indirect ELISA was used to screen and identify positive clones of TGM2. Among the 300 clones screened in the polypeptide group 1, 50 were positive clones. The 9 better positive clones were selected for further screening.

[0019] Figure 2 Indirect ELISA was used to screen positive clones of TGM2 protein. Among the 400 clones screened in the peptide group 2, 24 were positive clones. The 12 better positive clones were selected for further screening.

[0020] Figure 3 Indirect ELISA was used to screen positive clones of TGM2 protein. Among the 400 clones screened in the peptide group 3, 60 were positive clones. The 18 better positive clones were selected for further screening.

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

[0022] Figure 5 1-2F8, 2-1E9, and 3-3A5 were used to detect the expression and distribution of TGM2 protein in vivo using immunofluorescence methods, with NC serving as a negative control without the associated monoclonal antibody.

[0023] Figure 6 .1-2F8, 2-1E9, and 3-3A5 were used to detect the expression and distribution of TG M2 protein in vivo using immunohistochemistry, with NC serving as a negative control without the associated monoclonal antibody.

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

[0025] Figure 8 This is the fitted curve of the sensitivity assay for detecting TGM2 protein using an ELISA method. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0027] Example 1: Establishment of hybridoma cell lines

[0028] I. Experimental Materials

[0029] 1. Immunogen: Hybridoma cells were obtained by immunizing mice with peptides that bind to different binding sites of TGM2 coupled to a carrier protein.

[0030] 2. Culture media: DMEM medium was purchased from Hyclone; HAT and HT selective media and norphyrane were purchased from Sigma.

[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-Aldrich; PEG4000 was purchased from Fluka; HRP-goat anti-mouse IgG antibody was purchased from Jackson Immune; all other reagents were domestically produced analytical grade products.

[0033] II. Establishment of Hybridoma Cell Lines

[0034] 1. Animal immunization

[0035] 1) Basic immunization: Mix the antigen with Freund's complete adjuvant in equal volume and emulsify thoroughly. Administer the mixture subcutaneously at multiple sites. Each BALB / c mouse receives 100 μg per injection.

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

[0037] 2. Preparation of hybridoma cells

[0038] Mouse spleen cells were collected using standard methods and fused with SP2 / 0 cells at a ratio of 10:1 using 500 g / L PEG4000. Cells were selectively cultured in HAT medium. After 10–15 days post-fusion, the supernatant was collected, and hybridoma cell lines secreting anti-TGM2 recombinant protein were screened using an indirect ELISA method. Positive clones were subcloned using a limiting dilution method. The indirect ELISA procedure was as follows: 100 μL of TGM2 recombinant protein was used to coat the plate. Immune mouse serum (1:2000) was used as a positive control, and culture supernatant without clonal growth and normal mouse serum were used as negative controls. 100 μL of 1:2000 HRP-goat anti-mouse IgG was added to each well. Finally, the OD value at 450 nm was measured. Clones with an OD450 value greater than twice that of the negative control were preliminarily identified as positive clones. Group 1 had 9 positive clones, Group 2 had 12 positive clones, and Group 3 had 18 positive clones. Further screening revealed 4 positive clones in Group 1, 3 in Group 2, and 6 in Group 3. Figure 1-3 As 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 from peptide group 1, one from peptide group 2, and one from peptide group 3) were finally obtained that stably secrete monoclonal antibodies.

[0041] Table 1. Positive clones selected through rescreening (clones highlighted in red are retained clones)

[0042]

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

[0044] 1) Ascites titer determination in mice: The ascites titer prepared from Clone 1-2F8, 2-1E9, and 3-3A5 hybridoma cells was determined by indirect ELISA to be >1:20,000,000

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

[0046] 5. Passaging of hybridoma cell lines

[0047] The hybridoma cell lines were cultured and passaged in DMEM medium containing 10% fetal bovine serum. After 10 generations, the hybridoma cell lines were still able to grow well and be passaged stably, and the titer of the culture supernatant could still reach more than 1:10000.

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

[0049] After obtaining hybridoma cells capable of producing the desired monoclonal antibodies, a portion of these cells must be preserved. Otherwise, during continuous passage, mutations or chromosomal drift may occur, leading to the loss of their inherent characteristics or antibody-producing properties. Furthermore, contamination and eventual destruction are inevitable during long-term culture. Therefore, a portion must be refrigerated. 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 and is also destroyed by high pressure, so it cannot be filtered or sterilized by high pressure. It is sterile 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) Sterilized 2mL ampoules, etc.

[0055] 2. Operating Method

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

[0057] (3) Centrifuge at 1000 r / min for 10 min and discard the supernatant. Prepare a suspension of the cell pellet using 10% dimethyl sulfoxide (DMSO) to a concentration of 1.0 × 10⁻⁶. 7 Cells / mL.

[0058] (3) Take a sample, stain with trypan blue, and count the viable cells. The percentage 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 then seal the ampoules.

[0060] (5) Place at 4℃ for 2 hours.

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

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

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

[0064] I. Antibody Preparation

[0065] Adult BALB / c mice were selected and intraperitoneally injected with 0.5 mL of phytidine per mouse. 7–10 days later, each mouse was intraperitoneally injected with 16th generation 1-2F8, 2-1E9, and 3-3A5 hybridoma cells at a dose of 1 × 10⁻⁶ cells. 6 ~2×10 6 Five days later, when the abdomen is noticeably distended and the skin feels taut to the touch, ascites can be collected using a No. 9 needle.

[0066] The ascites fluid was centrifuged (13000 rpm for 30 minutes) to remove cellular components and other precipitates, and the supernatant was collected. Purification was performed using Protein G-Sepharose CL-4B, with 20 mmol / L PBS buffer as the loading buffer and 20 mmol / L glycine buffer as the elution buffer, yielding monoclonal antibodies against different sites of TGM2.

[0067] II. Identification of Antibodies

[0068] 1. Antibody purity identification:

[0069] SDS-PAGE electrophoresis identification showed a purity of over 95%. Figure 4 As shown.

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

[0071] The Ig subtypes of the antibodies produced by the hybridoma cells were identified using an indirect ELISA method with antibodies against various mouse Ig subtypes. The results are shown in Table 2. Clone 2-1E9 showed the strongest IgG2a signal, while Clone 1-2F8 and 3-3A5 showed the strongest IgG1 signal. Based on the subtype identification criteria, clone 2-1E9 was identified as IgG2b, and Clone 1-2F8 and 3-3A5 were identified as 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. Sequencing of the variable regions of clones 1-2F8, 2-1E9, and 3-3A5.

[0075] mRNA was extracted from cells of three clones, reverse transcribed into cDNA, and amplified using high-fidelity PCR with universal primers for the variable region. The PCR product fragments were inserted into a T vector for DNA sequencing, and the obtained sequences were translated into the amino acid sequences of the proteins. The amino acid sequences of the variable region of Clone1-1-2F8 antibody are as follows: light chain variable region amino acid sequence as shown in SEQ ID No. 1, heavy chain variable region amino acid sequence as shown in SEQ ID No. 4. The amino acid sequences of the variable region of Clone2-1E9 antibody are as follows: light chain variable region amino acid sequence as shown in SEQ ID No. 2, heavy chain variable region amino acid sequence as shown in SEQ ID No. 5. The amino acid sequences of the variable region of Clone3-3A5 antibody are as follows: light chain variable region amino acid sequence as shown in SEQ ID No. 3, heavy chain variable region amino acid sequence as shown in SEQ ID No. 6. No identical sequences were found after sequence alignment, indicating that the obtained sequences are clone-specific and were labeled as 1-2F8, 2-1E9, and 3-3A5 sequences, respectively.

[0076] Example 3: Verification of the affinity and specificity of purified antibodies

[0077] The affinity assays of monoclonal antibodies 1-2F8, 2-1E9, and 3-3A5 with TGM2 recombinant protein were performed using ELISA to determine their binding titers.

[0078] Detection Method: On day 1, TGM2 recombinant protein (KLH and Cx43 recombinant proteins were used as controls) was prepared by plate-planting 100 μL per well into 96 wells and incubating overnight. On day 2, after removing the supernatant, the plates were washed three times with PBS, and then blocked with 200 μL / well 3% BSA for 1 h. After washing three times with PBS, serially diluted Clone 1-2F8, 2-1E9, and 3-3A5 were added and incubated at 37°C for 1 h. After washing three times with PBS, HRP (horseradish peroxidase)-labeled mouse secondary antibody (1:2000) was added and incubated for 1 h. After washing five times with PBS (5 min for the first three times and 10 min for the last two times), chromogenic reagent was added and developed for 15 min before OD detection. 450 .

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

[0080] Table 3. ELISA detection of antibody affinity and specificity

[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. They also showed no obvious specific cross-linking with other related proteins such as KLH and Cx, exhibiting the characteristics of high affinity, high specificity, and high sensitivity.

[0083] Example 4: In vivo application of monoclonal antibodies 1-2F8, 2-1E9, and 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 assay was performed, and the steps of the assay are as follows:

[0086] 1. Cell preparation: For monolayer cells, during passage culture, seed the cells into a culture dish with pre-treated coverslips. Once the cells have almost grown into a monolayer, remove the coverslips and wash twice with PBS. For suspension cells, take logarithmic cells and wash twice with PBS (1000 r / min, 5 min). Prepare cell slides using a cell centrifuge or directly prepare cell smears.

[0087] 2. Fixation and permeabilization: In the culture plate, wash the coverslips with the cells that have already spread to the cell culture with 1×PBS three times, 3 min each time. Fix the coverslips with 4% paraformaldehyde for 15 min, then wash the coverslips with 1×PBS three times, 3 min each time. Permeabilize the cells with 0.5% Triton X-100 (prepared with 1×PBS) at room temperature for 15 min (omit this step if the antigen is expressed on the cell membrane), then wash the coverslips with 1×PBS three times, 3 min each time.

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

[0089] 4. Antibody incubation: Absorb the blocking solution with absorbent paper, do not wash, add a sufficient amount of diluted antibody 1-1E2 to each slide and place it in a humidified chamber, incubate overnight at 4°C.

[0090] 5. Add fluorescent secondary antibody: Wash the slides with PBST 3 times for 3 minutes each time. After blotting off the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody. Incubate in a humidified chamber at 37°C for 1 hour. Wash the slides with PBST 3 times for 3 minutes each time.

[0091] 6. Fixed photo taking

[0092] 7. Counterstaining the nucleus: Add DAPI and incubate in the dark for 5 min to stain the nucleus. Wash away excess DAPI by PBST for 5 min × 4 times.

[0093] 8. Blot the liquid off the slide with absorbent paper, seal the slide with mounting solution containing anti-fluorescence quencher, and observe the acquired image 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 methods.

[0095] (2) The immunohistochemical assay was performed, and the steps of the assay are as follows:

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

[0097] 2. Sectioning: Remove the embedded tissue from the mold, place it on a paraffin microtome and section it. Place the slide containing the intact tissue in 40°C warm water and then remove it. Dry it in a 37°C oven.

[0098] 3. Dewaxing: Place the slide in xylene-xylene-100% ethanol-100% ethanol-95% ethanol-90% ethanol-80% ethanol-70% ethanol in sequence, and leave it in each reagent for 10 minutes.

[0099] 4. Antigen retrieval: After dewaxing, rinse with clean water, soak in 3% H2O2 for 10 minutes, rinse twice with clean water, add citrate buffer, microwave for 3 minutes (medium heat) until boiling, cool to room temperature, boil again, and cool to room temperature.

[0100] 5. Serum blocking: After cooling to room temperature, discard the citrate buffer, wash twice with water, place the slide in PBS for 5 min, wash twice, wipe the PBS around the tissue dry, immediately add serum for blocking, and incubate at 37°C for 30 min. The serum is diluted 10 times (900 μL PBS: 100 μL serum blocking solution).

[0101] 6. Add primary antibody: Remove the slide from the incubator, blot the serum dry with absorbent paper, add primary antibody, and store in a 4°C refrigerator overnight.

[0102] 7. Add secondary antibody: Remove the slide from the refrigerator, wash it 3 times with PBS for 5 minutes each time, wipe the PBS dry, add the secondary antibody, and incubate at 37°C for 30 minutes.

[0103] 8. Add SABC: Remove the slides from the incubator, wash three times with PBS for 5 minutes each time, wipe dry the PBS, add SABC, and incubate at 37°C for 30 minutes. Dilute SABC 100 times (990 μL PBS: 10 μL SABC).

[0104] 9. Add chromogenic reagent: Remove the slides from the incubator, wash three times with PBS for 5 minutes each time, wipe the PBS dry, and then add the chromogenic reagent. (Preparation of chromogenic reagent: Add 1 drop of chromogenic reagent A to 1 mL of water, shake well, then add 1 drop of chromogenic reagent B, shake well, then add 1 drop of chromogenic reagent C, shake well) A: DAB; B: H2O2; C: phosphate buffer.

[0105] 10. Counterstaining: After rinsing the developed slides with water for a period of time, soak them in hematoxylin for 30 seconds.

[0106] 11. Dehydration: After rinsing the counterstained slides in water, place the slides in the following order: 70% ethanol - 80% ethanol - 90% ethanol - 95% ethanol - 100% ethanol - 100% ethanol - xylene - xylene. Let each reagent soak for 2 minutes, then finally immerse in xylene and move to a fume hood.

[0107] 12. Mounting: Apply a drop of neutral resin 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 using immunohistochemistry, with the brown areas indicating positive reactions.

[0109] (3) Western blot (WB) was used for testing. The steps of the method are as follows:

[0110] 1. SDS-PAGE electrophoresis: After cleaning the glass plate, place it in the clamp and secure it, then vertically clamp it onto the rack in preparation for gel loading. Prepare a 10% SDS separating gel; the volume of solution containing 50 ng of protein is the loading volume. Transfer the sample to a 0.5 mL centrifuge tube, add 5× SDS loading buffer to a final concentration of 1×, load the sample, and perform electrophoresis. Stop electrophoresis when bromophenol blue just appears, then proceed with membrane transfer.

[0111] 2. Transfer: Immerse the cut NC or PVDF membrane in water for 2 hours. Gently place the membrane, holding one side with tweezers, into a petri dish containing ultrapure water, ensuring it floats. Place the transfer clamp, two sponge pads, a glass rod, filter paper, and the soaked membrane in an enamel dish containing transfer buffer. Open the clamp, keeping the black side horizontal. Place a sponge pad on top and use the glass rod to gently roll it back and forth several times to remove air bubbles. Place three layers of filter paper on the pad, holding the filter paper in place with one hand while using the glass rod to remove air bubbles. Carefully peel off the separating gel and place it on the filter paper, adjusting it to align with the filter paper. Gently roll it with the glass rod to remove air bubbles. Place the membrane on the gel and remove air bubbles. Cover the membrane with three layers of filter paper and remove air bubbles. Finally, cover with another sponge pad, roll it a few times, and then close the clamp. The entire operation is performed in the transfer buffer, continuously removing air bubbles. Place the clamp in the transfer tank and transfer at 80V for 1 hour. After transfer, stain the membrane with 1× Ponceau S for 5 minutes (shaking on a destaining shaker). Then rinse off any unstained stain with water to reveal the protein on the membrane. Allow the membrane to air dry for later use.

[0112] 3. Immunoreaction: After wetting the membrane with TBS from bottom to top, transfer it to a petri dish containing blocking solution and block on a decolorizing shaker at room temperature for 1 hour. Dilute antibodies 2-1G11 and 3-2G12 to appropriate concentrations with TBST (in 1.5 mL centrifuge tubes); incubate at room temperature for 1–2 hours, then wash twice with TBST on a decolorizing shaker at room temperature for 10 minutes each time; wash once with TBS for 10 minutes. Prepare secondary antibody dilution solution using the same method and contact it with the membrane for chemiluminescent reaction.

[0113] 4. Chemiluminescence: Mix equal volumes of reagents A and B on plastic wrap; after 1 minute, place the membrane protein side down in the mixture for full contact; after 1 minute, transfer the membrane to another piece of plastic wrap, remove any residual liquid, wrap it up, and place it in an X-ray film holder. In a darkroom, pour 1× developer and fixer into separate plastic trays; under a red light, remove the X-ray film and cut it to an appropriate size (1 cm larger than the length and width of the membrane); open the X-ray film holder, place the X-ray film on the membrane, and once placed, do not move it. Close the X-ray film holder. After exposure, open the X-ray film holder, remove the X-ray film, and quickly immerse it in the developer. Once clear bands appear, immediately stop development.

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

[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 using Western blotting.

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

[0117] Pairing experiments were performed using HRP-labeled antibodies of Clone 3-3A5 and Clone 2-1E9 (labeled using a modified sodium periodate method). Using Clone 3-3A5 as the coating antibody and HRP-labeled Clone 2-1E9 as the detection antibody, a sandwich ELISA quantitative detection method was established, achieving a sensitivity of 1 ng / ml (Table 4). Figure 8 ).

[0118] Table 4. Quantitative detection of TGM2 protein using sandwich ELISA method.

[0119]

[0120] Detection method: The coating antibody Clone3-3A5 was diluted to 10 μg / mL with 0.05 mol / L carbonate buffer at pH 9.6. 100 μL was added to each well of the ELISA plate, and the plate was incubated overnight at 4°C. The coating solution was discarded, and the plate was washed three times with PBST, blotted dry, and then 200 μL of 1% gelatin was added to each well. The plate was then incubated at 37°C for 2 hours, washed three times with PBST, blotted dry, and stored in an airtight container. The antibody against Clone 2-1E was labeled with horseradish peroxidase to obtain 2-1E9-HRP, which was then stored. Add 100 μL of TGM2 recombinant protein gradient dilution to each well of the microplate and incubate at 37°C for 1 hour. Then add 100 μL of 2-1E9-HRP (1:4000 dilution) and incubate at 37°C for 1 hour. Add 50 μL each of chromogenic reagent A and B to each well for color development and incubate at 37°C for 10 minutes. Add 50 μL of stop solution to each well and take the reading.

[0121] The colorimetric reagent A solution is formulated as follows: add 1g of urea peroxide, 10.3g of citric acid, 35.8g of Na2HPO4·12H2O, and 100μL of Tween-20 per 1000mL of water, and adjust the pH to 5. The colorimetric reagent B solution is formulated as follows: add 700mg of tetramethylbenzidine (TMB) (dissolved in 40mL of DMSO) and 10.3g of citric acid per 1000mL of distilled water, and adjust the pH to 2.4.

[0122] The results are as follows Figure 8 As shown, the sensitivity of the TGM2 protein quantification method is approximately 1 ng / mL, R 2 The value was 0.9945, and the linear range was 1.95-125 ng / mL.

Claims

1. An antibody that recognizes anti-tissue glutaminase 2, said antibody having a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region being shown in SEQ ID No. 1, and the amino acid sequence of the heavy chain variable region being shown in SEQ ID No.

4.

2. The antibody of claim 1, wherein 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 of claim 1, wherein The antibody also includes a heavy chain constant region of IgG selected from IgG1, IgG2, IgG3 or IgG4.

5. The use of the antibody according to any one of claims 1-4 in the preparation of a reagent for detecting TGM2.

6. A method for detecting TGM2, the method comprising the step of detecting TGM2 using the antibody according to any one of claims 1-4, the method being a method for non-disease diagnosis and treatment purposes.

7. The method according to claim 6, characterized in that, The method is indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, or immunofluorescence.

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

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