Biopsy sample MHC-II expression kit for predicting curative effect of gastric cancer immunotherapy

Abstract

The invention discloses a gastric cancer immunotherapy curative effect prediction biopsy sample MHC-II expression kit, belongs to the field of kit preparation, and particularly relates to a preparation method of an antigen repair liquid. Citric acid, sodium citrate, an additive and pure water are mixed to prepare the antigen repair liquid; the additive comprises a glucosamido polyether derivative and a propyl gallate / calcium ion complex. The antigen repair liquid is adopted to form a gastric cancer immunotherapy curative effect prediction biopsy sample MHC-II expression kit; the kit is prepared from the following components in parts by mass: 20 to 30 parts of dewaxing liquid, 10 to 20 parts of antigen repair liquid, 2 to 10 parts of sheep serum, 10 to 20 parts of enhancement liquid, 15 to 25 parts of antibody, 5 to 15 parts of staining liquid and 10 to 20 parts of mounting medium. The invention provides the kit for predicting the MHC-II expression of the biopsy sample in the gastric cancer immunotherapy curative effect, which is high in sensitivity and high in specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of kit preparation, and in particular to a kit for predicting the efficacy of gastric cancer immunotherapy using biopsy samples containing MHC-II. Background Art

[0002] Gastric and gastroesophageal junction cancer ranks fifth among newly diagnosed malignancies worldwide and fourth among cancer-related deaths. Currently, a combination of perioperative therapy and D2 gastrectomy has become the standard treatment for locally advanced gastric and gastroesophageal junction cancer. Biomarkers associated with immunotherapy efficacy primarily include programmed cell death ligand 1 expression, microsatellite instability status, and tumor-infiltrating lymphocytes. Theoretically, cancer patients with high programmed cell death ligand 1 expression and microsatellite instability-high status tend to show enhanced responsiveness to immunotherapy. However, some patients may not fully respond to immunotherapy. Therefore, further exploration of precise biomarkers is crucial to identify the most suitable immunotherapy candidates and improve their efficacy.

[0003] Major histocompatibility complex (MHC) molecules, also known as human leukocyte antigen molecules, have been shown to allow the immune system to recognize tumor cells through major histocompatibility complexes (MHCs). MHCs are cell surface receptors that bind foreign peptides and present them to T lymphocytes. MHC class II molecules, encoded by polymorphic MHC genes, consist of non-covalent complexes of α and β chains. Helper T lymphocytes bind to antigenic peptides presented by MHC class II molecules. MHC class II molecules bind to antigenic peptides of 13-18 amino acids. Studies have shown that specific MHC class II expression in gastric cancer patients is closely associated with immunotherapy.

[0004] MHC-II expression can be detected through immunohistochemical staining technology, which is a technology that uses the principle of antigen-antibody reaction to perform in situ tissue or cytological detection of specific targets in various tissue samples. With the development of antibody and antibody coupling technology, immunohistochemical staining is currently widely used and can detect a large number of targets. As one of the convenient in situ detection technologies, it has become a platform technology for basic research, pathological diagnosis and molecular target determination in the clinical and basic medical fields. As an important target in the immune response of gastric malignant tumors, MHC-II is particularly important in clinical diagnosis and treatment for the early determination of MHC-II expression in gastric cancer patients through immunohistochemical staining. Therefore, it is particularly important to provide an MHC-II expression kit for biopsy samples to predict the efficacy of gastric cancer immunotherapy and obtain high-quality and accurate MHC-II expression detection results in a relatively short period of time. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing an antigen retrieval solution to improve the sensitivity and specificity of detecting MHC-II expression in biopsy samples for predicting the efficacy of gastric cancer immunotherapy.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A method for preparing an antigen repair solution comprises uniformly mixing at least three components of citric acid, sodium citrate, an additive, and pure water to obtain the antigen repair solution; the additive comprises at least a glucosamide polyether derivative and a propyl gallate / calcium ion complex, the glucosamide polyether derivative having a polyether bond and an amide group, and the propyl gallate / calcium ion complex having a phenolic hydroxyl group and a calcium ion coordination unit.

[0007] The present invention prepares an antigen repair solution by mixing citric acid, sodium citrate, a glucosamide polyether derivative, a propyl gallate / calcium ion complex and pure water. The antigen repair solution can improve the sensitivity and specificity of a kit for predicting the MHC-II expression of biopsy samples for gastric cancer immunotherapy efficacy. The glucosamide polyether derivative and the propyl gallate / calcium ion complex have strong emulsifying properties and biological activity, can not only remove paraffin residual on the surface of tissue sections, but also can assist in antigen repair and antibody binding, thereby improving the sensitivity and specificity of the kit for predicting the MHC-II expression of biopsy samples for gastric cancer immunotherapy efficacy.

[0008] Preferably, in the preparation of the glucose amide polyether derivative, gluconolactone and diethylenetriamine are used as raw materials to carry out a ring-opening polymerization reaction, and then alkyl epoxy polyether and acyl chloride are added to react to obtain the glucose amide polyether derivative.

[0009] Preferably, the preparation of the glucose amide polyether derivative is specifically as follows: Dissolve gluconolactone in N,N-dimethylformamide, add diethylenetriamine, and react in a water bath at a constant temperature of 60-120°C for 2-10 hours. Then add alkyl epoxy polyether and react at a constant temperature of 60-120°C for 1-5 hours. Lower the temperature to 0-5°C, slowly add lauroyl chloride and triethylamine dropwise, and introduce chlorine gas to obtain a glucamide polyether derivative.

[0010] More preferably, the usage ratio of gluconolactone to N,N-dimethylformamide is 1 mmol:2-10 mL.

[0011] More preferably, the molar ratio of gluconolactone to diethylenetriamine is 1:0.5-2.

[0012] More preferably, the molar ratio of gluconolactone to the alkyl epoxy polyether is 1:1-2.

[0013] More preferably, the molar ratio of the alkyl epoxy polyether to the lauroyl chloride is 1:0.2-1.

[0014] More preferably, the usage ratio of gluconolactone to triethylamine is 1 mmol:0.5-2 mL.

[0015] Preferably, the propyl gallate / calcium ion complex is prepared by reacting propyl gallate and calcium chloride dihydrate.

[0016] Preferably, the preparation of propyl gallate / calcium ion complex is specifically as follows: Weigh propyl gallate, add deionized water, heat at 60-100°C for 1-3 hours, add calcium chloride dihydrate, heat and stir for 2-10 days, reduce the temperature to 40-60°C, wash with 40-60°C deionized water 2-4 times, and dry for 12-36 hours to obtain a propyl gallate / calcium ion complex.

[0017] More preferably, the usage ratio of propyl gallate to deionized water is 1 g:10-20 mL.

[0018] More preferably, the mass ratio of propyl gallate to calcium chloride dihydrate is 1:0.2-1.

[0019] Preferably, the mass ratio of citric acid to sodium citrate is 1:7-8.

[0020] Preferably, the mass ratio of citric acid to additives is 1:0.2-4.

[0021] Preferably, the usage ratio of citric acid to pure water is 1 g: 2-3 L.

[0022] Preferably, the additive includes a glucose amide polyether derivative and a propyl gallate / calcium ion complex.

[0023] Preferably, the additives include a glucosamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid. The use of coumarin-3-carboxylic acid can further enhance the antigen retrieval capability of the prepared antigen retrieval solution, thereby increasing the sensitivity and specificity of immunohistochemical detection of MHC-II expression.

[0024] Preferably, a method for preparing an antigen repair solution comprises: The citric acid, sodium citrate, glucose amide polyether derivative, propyl gallate / calcium ion complex, coumarin-3-carboxylic acid and pure water were uniformly mixed to obtain an antigen retrieval solution.

[0025] More preferably, the mass ratio of citric acid to sodium citrate is 1:7-8.

[0026] More preferably, the mass ratio of citric acid to the glucose amide polyether derivative is 1:0.5-1.5.

[0027] More preferably, the mass ratio of citric acid to propyl gallate / calcium ion complex is 1:0.5-1.5.

[0028] More preferably, the mass ratio of sodium citrate to coumarin-3-carboxylic acid is 1:0.2-1.

[0029] The purpose of the present invention is to provide a biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy, thereby improving the sensitivity and specificity of detecting MHC-II expression.

[0030] A gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit comprises the following components in parts by weight: 20-30 parts of dewaxing solution, 10-20 parts of antigen retrieval solution prepared by the above method, 2-10 parts of sheep serum, 10-20 parts of enhancement solution, 15-25 parts of antibody, 5-15 parts of staining solution, and 10-20 parts of mounting medium; the dewaxing solution comprises xylene.

[0031] Preferably, the antibodies include MHC Class II recombinant rabbit monoclonal antibody and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

[0032] More preferably, the mass ratio of the MHC Class II recombinant rabbit monoclonal antibody to the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is 1:2-10.

[0033] Preferably, the staining solution includes diaminobenzidine color developing solution, hematoxylin staining solution and anti-blue solution.

[0034] More preferably, the anti-blueing solution is prepared by mixing sodium bicarbonate and pure water, and the usage ratio of sodium bicarbonate to pure water is 1g:0.2-1L.

[0035] More preferably, the mass ratio of diaminobenzidine color developing solution to hematoxylin staining solution is 1:1-2.

[0036] More preferably, the mass ratio of diaminobenzidine color developing solution to anti-blueing solution is 1:1-2.

[0037] The present invention uses a glucosamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid to prepare an antigen retrieval solution. This antigen retrieval solution is then used to form a kit for detecting MHC-II expression in biopsy samples for predicting the efficacy of gastric cancer immunotherapy. This kit has the following beneficial effects: The kit has high sensitivity and specificity for detecting MHC-II expression, with a sensitivity of 94-99% and a specificity of 87-92%. Therefore, the present invention provides a kit for detecting MHC-II expression in biopsy samples with high sensitivity and specificity for predicting the efficacy of gastric cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1This is a diagram showing the immunostaining results of detecting MCH-II expression using the kit obtained in Example 1. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0041] Example 1: S1. Preparation of antigen retrieval solution, including: Combine citric acid, sodium citrate, and purified water and stir thoroughly to obtain an antigen retrieval solution. The mass ratio of citric acid to sodium citrate is 1:7.5; the amount ratio of citric acid to purified water is 1g:2.5L.

[0042] S2. A kit for predicting the efficacy of gastric cancer immunotherapy using biopsy samples containing MHC-II expression, comprising the following components in parts by mass: 27 parts dewaxing solution, 14 parts antigen retrieval solution obtained in step S1, 5 parts sheep serum, 17 parts antibody, 14 parts enhancing solution, 9 parts staining solution, and 14 parts mounting medium. The dewaxing solution is xylene. The antibodies include an MHC Class II recombinant rabbit monoclonal antibody and an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody. The mass ratio of the MHC Class II recombinant rabbit monoclonal antibody to the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is 1:5. The staining solution includes a diaminobenzidine colorimetric solution, a hematoxylin staining solution, and an anti-blueing solution. The anti-blueing solution is prepared by mixing 2 g of sodium bicarbonate and 1 L of pure water. The mass ratio of the diaminobenzidine colorimetric solution to the hematoxylin staining solution is 1:1, and the mass ratio of the diaminobenzidine colorimetric solution to the anti-blueing solution is 1:1. In this example, goat serum was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., MHC Class II recombinant rabbit monoclonal antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., enhancement solution was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., diaminobenzidine colorimetric solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd., hematoxylin staining solution was purchased from Ningbo Tongsheng Biotechnology Co., Ltd., and mounting medium was purchased from Ningbo Tongsheng Biotechnology Co., Ltd.

[0043] Example 2: S1. Preparation of glucose amide polyether derivatives, comprising: Gluconolactone was dissolved in N,N-dimethylformamide, diethylenetriamine was added, and the reaction was maintained at 90°C in a water bath for 5 hours. Alkyl epoxy polyether was then added and the reaction was maintained at 90°C for 3 hours. The temperature was then lowered to 0°C, and lauroyl chloride and triethylamine were slowly added dropwise while chlorine gas was introduced to obtain a gluconic acid amide polyether derivative. The molar ratio of gluconic acid amide to N,N-dimethylformamide was 1 mmol:5 mL; the molar ratios of gluconic acid amide to diethylenetriamine were 1:1, the molar ratios of gluconic acid amide to alkyl epoxy polyether were 1:1.5, and the molar ratios of alkyl epoxy polyether to lauroyl chloride were 1:0.6. The molar ratio of gluconic acid amide to triethylamine was 1 mmol:1.5 mL.

[0044] S2. Preparation of propyl gallate / calcium ion complex, comprising: Weigh propyl gallate, add deionized water, and heat at 85°C for 2 hours. Add calcium chloride dihydrate and heat with stirring for 5 days. Lower the temperature to 50°C, wash three times with 50°C deionized water, and dry for 24 hours to obtain a propyl gallate / calcium ion complex. The ratio of propyl gallate to deionized water is 1 g:10 mL, and the mass ratio of propyl gallate to calcium chloride dihydrate is 1:0.7.

[0045] S3. Preparation of antigen retrieval solution, including: Mix citric acid, sodium citrate, a glucosamide polyether derivative, propyl gallate / calcium ion complex, and pure water and stir thoroughly to obtain an antigen retrieval solution. The mass ratio of citric acid to sodium citrate is 1:7.5, the mass ratio of citric acid to glucosamide polyether derivative is 1:1, and the mass ratio of citric acid to propyl gallate / calcium ion complex is 1:1. The ratio of citric acid to pure water is 1g:2.5L.

[0046] S4. A biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy is prepared with the same conditions as in Example 1, except that the antigen retrieval solution is replaced with the antigen retrieval solution obtained in step S3 of this example.

[0047] Example 3: Except that the mass ratio of citric acid to glucosamide polyether derivative was changed to 1:0.5 in the preparation of antigen retrieval solution, other conditions were the same as those in Example 2.

[0048] Example 4: Except that the mass ratio of citric acid and propyl gallate / calcium ion complex was changed to 1:0.5 in the preparation of antigen retrieval solution, other conditions were the same as those in Example 2.

[0049] Example 5: S1, glucose amide polyether derivative, same as in Example 2.

[0050] S2. Preparation of propyl gallate / calcium ion complex, same as in Example 2.

[0051] S3. Preparation of antigen retrieval solution, including: Mix citric acid, sodium citrate, a glucosamide polyether derivative, propyl gallate / calcium ion complex, coumarin-3-carboxylic acid, and purified water and stir thoroughly to obtain an antigen retrieval solution. The mass ratio of citric acid to sodium citrate is 1:7.5, the mass ratio of citric acid to glucosamide polyether derivative is 1:1, the mass ratio of citric acid to propyl gallate / calcium ion complex is 1:1, and the mass ratio of sodium citrate to coumarin-3-carboxylic acid is 1:0.5. The amount of citric acid to purified water is 1 g:2.5 L.

[0052] S4. A biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy is prepared with the same conditions as in Example 1, except that the antigen retrieval solution is replaced with the antigen retrieval solution obtained in step S3 of this example.

[0053] Example 6: Except that the mass ratio of citric acid to coumarin-3-carboxylic acid was changed to 1:0.1 in the preparation of the antigen retrieval solution, other conditions were the same as those in Example 5.

[0054] Comparative Example 1: S1. Preparation of glucose amide polyether derivatives, same as in Example 2.

[0055] S2. Preparation of antigen retrieval solution, including: Combine citric acid, sodium citrate, a glucosamide polyether derivative, and purified water and stir thoroughly to obtain an antigen retrieval solution. The mass ratio of citric acid to sodium citrate is 1:7.5, the mass ratio of citric acid to glucosamide polyether derivative is 1:1, and the amount of citric acid to purified water is 1g:2.5L.

[0056] S3. A biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy is used. Except that the antigen retrieval solution is replaced by the antigen retrieval solution obtained in step S2 of this comparative example, other conditions are the same as those in Example 1.

[0057] Comparative Example 2: S1. Preparation of propyl gallate / calcium ion complex, same as in Example 2.

[0058] S2. Preparation of antigen retrieval solution, including: Combine citric acid, sodium citrate, propyl gallate / calcium ion complex, and purified water and stir thoroughly to obtain an antigen retrieval solution. The mass ratio of citric acid to sodium citrate is 1:7.5, the mass ratio of citric acid to propyl gallate / calcium ion complex is 1:1, and the amount of citric acid to purified water is 1g:2.5L.

[0059] S3. A biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy is used. Except that the antigen retrieval solution is replaced by the antigen retrieval solution obtained in step S2 of this comparative example, other conditions are the same as those in Example 1.

[0060] Comparative Example 3: S1. Preparation of antigen retrieval solution, including: Combine citric acid, sodium citrate, coumarin-3-carboxylic acid, and purified water and stir thoroughly to obtain an antigen retrieval solution. The mass ratio of citric acid to sodium citrate is 1:7.5, the mass ratio of sodium citrate to coumarin-3-carboxylic acid is 1:0.5, and the amount of citric acid to purified water is 1g:2.5L.

[0061] S2. A biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy is used. The conditions are the same as those in Example 1, except that the antigen retrieval solution is replaced by the antigen retrieval solution obtained in step S1 of this comparative example.

[0062] Experimental example: The kits obtained in Examples 1-6 and Comparative Examples 1-3 were used to detect MHC-II expression by immunohistochemical staining. The specific method is as follows: After baking paraffin sections at 72°C for 20 minutes, immediately immerse them in deparaffinization solution three times for 5 minutes each time to obtain dewaxed tissue sections. Immerse tissue sections in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 80% ethanol, and purified water, sequentially for 3 minutes each time. Add antigen retrieval solution to a pressure cooker, bring to a boil, and place tissue sections. After the pressure valve sounds and a 90-second timer begins, turn off the power. Allow the pressure cooker to cool naturally before opening the lid. Remove the tissue sections and rinse them three times with water for 2 minutes each time. Then, immerse them in phosphate-buffered saline (PBS) for 5 minutes. After drying the tissue sections, use a marker to mark the tissue size on the sections. Immerse the tissue sections completely in 3% hydrogen peroxide for 10 minutes. Rinse the tissue sections three times with water for 2 minutes each time, and then immerse them in PBS for 5 minutes. Cover the sections completely with goat serum at room temperature and block for 10 minutes. Shake dry the sections while keeping the surface moist. Dilute MHC Class II recombinant rabbit monoclonal antibody in phosphate-buffered saline to a final concentration of 1%. Add the diluted MHC Class II recombinant rabbit monoclonal antibody to completely cover the surface of the sections and incubate at 4°C for 8 hours. Incubate the sections at room temperature for 30 minutes, rinse with water three times for 2 minutes each, and immerse in phosphate-buffered saline for 5 minutes. Shake dry the sections while keeping the surface moist. Add enhancement solution to completely cover the surface of the sections. Incubate the sections at room temperature for 20 minutes, rinse with water three times for 2 minutes each, and immerse in phosphate-buffered saline for 5 minutes. Shake dry the sections while keeping the surface moist. Add enhancement solution labeled with enzyme-linked goat anti-rabbit IgG polymer antibody to completely cover the surface of the sections. Incubate at room temperature for 20 minutes, rinse with water three times for 2 minutes each, and immerse in phosphate-buffered saline for 5 minutes. Protect from light, add two drops of diaminobenzidine colorimetric solution, incubate at room temperature for 3 minutes, and terminate color development with water. Rinse the sections with water three times for 2 minutes each, shake dry, and immerse in hematoxylin staining solution for 2 minutes. Wash sections in a vibrating trough for 3 minutes, spin dry, and immerse in anti-blue solution for 2 minutes. Rinse sections in a vibrating trough for 3 minutes, spin dry, and immerse in 80% ethanol for 10 seconds, 95% ethanol for 10 seconds, 100% ethanol for 3 minutes, anhydrous ethanol for 3 minutes, and anhydrous ethanol for 3 minutes. After air drying, mount the sections with mounting media and observe under a low-power microscope for MHC-II expression.

[0063] 1. Immunohistochemical staining According to the above method, the expression of MHC-II was detected using the kit obtained in Example 1, and the proportion of cells with stained cell membranes and the staining intensity were observed under a low-power microscope.

[0064] Figure 1This is a diagram showing the immunostaining results of detecting MCH-II expression using the kit obtained in Example 1. Negative results show incomplete and weak cell membrane staining, while positive results show strong and complete cell membrane staining.

[0065] 2. Determination of kit sensitivity According to the above method, the kits obtained in Examples 1-6 and Comparative Examples 1-3 were used to detect MHC-II expression, and the sensitivity of each group in detecting MCH-II expression was determined. Sensitivity (%) = number of MCH-II positive samples / total number of samples × 100%. The results are shown in Table 1. Table 1 shows the sensitivity (%) of detecting MCH-II expression.

[0066] Table 1 Sensitivity of detecting MCH-II expression (%)

[0067] As shown in Table 1, compared with Example 1, Example 2 had a higher sensitivity for detecting MCH-II expression than Example 1. This is because Example 2 additionally added a glucosamido polyether derivative and a propyl gallate / calcium ion complex in the preparation of the antigen retrieval solution. This indicates that the method of Example 2 can effectively improve the sensitivity of detecting MCH-II expression. Compared with Comparative Examples 1-2, Example 2 had a higher sensitivity for detecting MCH-II than Comparative Examples 1-2. This is because Example 2 additionally added a glucosamido polyether derivative and a propyl gallate / calcium ion complex in the preparation of the antigen retrieval solution, while Comparative Example 1 only added a glucosamido polyether derivative and Comparative Example 2 additionally added a propyl gallate / calcium ion complex. Compared with Examples 3-4, Example 2 had a higher sensitivity for detecting MCH-II than Examples 3-4. This is because different amounts of the glucosamido polyether derivative and the propyl gallate / calcium ion complex were used in the preparation of the antigen retrieval solution. This indicates that in the preparation of antigen retrieval solution, the synergistic use of glucosamide polyether derivatives and propyl gallate / calcium ion complexes makes the sensitivity of detecting MCH-II expression better.

[0068] Compared with Example 2, Example 5 showed a higher sensitivity for detecting MCH-II than Example 2. This is because, in the preparation of the antigen retrieval solution, Example 5 additionally added a glucosamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid, while Example 2 additionally added a glucosamido polyether derivative and a propyl gallate / calcium ion complex. Compared with Comparative Example 3, Example 5 showed a higher sensitivity for detecting MCH-II than Comparative Example 3. This is because, in the preparation of the antigen retrieval solution, Comparative Example 3 additionally added coumarin-3-carboxylic acid, while Example 5 additionally added a glucosamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid. Compared with Example 6, Example 5 showed a higher sensitivity for detecting MCH-II than Example 6. This is because the amount of coumarin-3-carboxylic acid used in the antigen retrieval solution was different. This suggests that adding coumarin-3-carboxylic acid to the antigen retrieval solution can further improve the sensitivity of detecting MCH-II expression.

[0069] 3. Determination of kit specificity According to the above method, the kits obtained in Examples 1-6 and Comparative Examples 1-3 were used to detect the expression of MHC-II in the samples, and the specificity of each group for detecting MCH-II expression was determined. Specificity (%) = number of MCH-II negative samples / total number of samples × 100%. The results are shown in Table 2. Table 2 shows the specificity (%) for detecting MCH-II expression.

[0070] Table 2 Specificity of detecting MCH-II expression (%)

[0071] As shown in Table 2, compared with Example 1, Example 2 had a higher specificity for detecting MCH-II expression than Example 1. This is because Example 2 additionally added a glucosamido polyether derivative and a propyl gallate / calcium ion complex during the preparation of the antigen retrieval solution. This demonstrates that the method of Example 2 can effectively improve the specificity for detecting MCH-II expression. Compared with Comparative Examples 1-2, Example 2 had a higher specificity for detecting MCH-II than Comparative Examples 1-2. This is because Example 2 additionally added a glucosamido polyether derivative and a propyl gallate / calcium ion complex during the preparation of the antigen retrieval solution, while Comparative Example 1 only added a glucosamido polyether derivative, while Comparative Example 2 additionally added a propyl gallate / calcium ion complex. Compared with Examples 3-4, Example 2 had a higher specificity for detecting MCH-II than Examples 3-4. This is because different amounts of the glucosamido polyether derivative and the propyl gallate / calcium ion complex were used during the preparation of the antigen retrieval solution. This indicates that in the preparation of antigen retrieval solution, the synergistic use of glucosamide polyether derivatives and propyl gallate / calcium ion complexes can improve the specificity of detecting MCH-II expression.

[0072] Compared with Example 2, Example 5 showed a higher specificity for detecting MCH-II than Example 2. This is because Example 5 additionally added a glucosamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid during the preparation of the antigen retrieval solution, while Example 2 additionally added a glucosamido polyether derivative and a propyl gallate / calcium ion complex. Compared with Comparative Example 3, Example 5 showed a higher specificity for detecting MCH-II than Comparative Example 3. This is because Comparative Example 3 additionally added coumarin-3-carboxylic acid during the preparation of the antigen retrieval solution, while Example 5 additionally added a glucosamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid. Compared with Example 6, Example 5 showed a higher specificity for detecting MCH-II than Example 6 due to the different amounts of coumarin-3-carboxylic acid used in the antigen retrieval solution. This suggests that adding coumarin-3-carboxylic acid to the antigen retrieval solution can further improve the specificity for detecting MCH-II expression.

[0073] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0074] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any changes or modifications made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an antigen repair solution, comprising uniformly mixing at least three components: citric acid, sodium citrate, an additive, and pure water to obtain an antigen repair solution; the additive comprises at least a glucosamide polyether derivative and a propyl gallate / calcium ion complex, wherein the glucosamide polyether derivative has a polyether bond and an amide group, and the propyl gallate / calcium ion complex has a phenolic hydroxyl group and a calcium ion coordination unit.

2. The method for preparing an antigen repair solution according to claim 1, wherein: In the preparation of the glucose amide polyether derivative, gluconolactone and diethylenetriamine are used as raw materials to carry out a ring-opening polymerization reaction, and then alkyl epoxy polyether and acyl chloride are added to react to obtain the glucose amide polyether derivative.

3. The method for preparing an antigen repair solution according to claim 1, wherein: The propyl gallate / calcium ion complex is prepared by reacting propyl gallate and calcium chloride dihydrate.

4. A biopsy sample MHC-II expression kit for predicting the efficacy of gastric cancer immunotherapy, comprising the following components in parts by mass: 20-30 parts of dewaxing solution, 10-20 parts of the antigen retrieval solution of claim 1, 2-10 parts of sheep serum, 10-20 parts of enhancement solution, 15-25 parts of antibody, 5-15 parts of staining solution, and 10-20 parts of mounting medium; the dewaxing solution is xylene.

5. The MHC-II expression kit for biopsy samples for predicting the efficacy of gastric cancer immunotherapy according to claim 4, characterized in that: The antibodies include MHC Class II recombinant rabbit monoclonal antibodies and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibodies.

6. The MHC-II expression kit for biopsy samples for predicting the efficacy of gastric cancer immunotherapy according to claim 5, characterized in that: The mass ratio of the MHC Class II recombinant rabbit monoclonal antibody to the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is 1:2-10.

7. The MHC-II expression kit for biopsy samples for predicting the efficacy of gastric cancer immunotherapy according to claim 4, characterized in that: The staining solution comprises diaminobenzidine color developing solution, hematoxylin staining solution and anti-blue solution.

8. The MHC-II expression kit for biopsy samples for predicting the efficacy of gastric cancer immunotherapy according to claim 7, characterized in that: The anti-blue solution is obtained by mixing sodium bicarbonate and pure water, and the usage ratio of sodium bicarbonate to pure water is 1g:0.2-1L.

9. The MHC-II expression kit for biopsy samples for predicting the efficacy of gastric cancer immunotherapy according to claim 7, characterized in that: The mass ratio of the diaminobenzidine color developing solution to the hematoxylin staining solution is 1:1-2.

10. The MHC-II expression kit for biopsy samples for predicting the efficacy of gastric cancer immunotherapy according to claim 7, characterized in that: The mass ratio of the diaminobenzidine color developing solution to the anti-blueing solution is 1:1-2.

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