Gastric cancer immunotherapy efficacy prediction biopsy sample mhc-ii expression kit

Abstract

The application discloses a gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit and belongs to the field of kit preparation, and particularly relates to a preparation method of an antigen repair solution, which is prepared by mixing citric acid, sodium citrate, an additive and pure water, and the additive comprises a glucose amido polyether derivative and a propyl gallate / calcium ion complex. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit is composed of the antigen repair solution; the kit comprises the following components in parts by mass: 20-30 parts of a dewaxing solution, 10-20 parts of the antigen repair solution, 2-10 parts of sheep serum, 10-20 parts of an enhancing solution, 15-25 parts of an antibody, 5-15 parts of a staining solution and 10-20 parts of a mounting agent. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit provided by the application has high sensitivity and high specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of kit preparation, and particularly relates to a gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit. BACKGROUND

[0002] Gastric and gastroesophageal junction cancer ranks fifth in newly diagnosed malignancies worldwide and fourth in cancer-related deaths. At present, perioperative treatment combined with D2 gastrectomy has become the standard treatment for locally advanced gastric and gastroesophageal junction cancer. The main biomarkers related to the efficacy of immunotherapy include programmed cell death ligand 1 expression, microsatellite instability status, and tumor-infiltrating lymphocytes. In theory, cancer patients showing high programmed cell death ligand 1 expression and microsatellite instability-high status tend to show enhanced reactivity to immunotherapy. Nevertheless, some patients may not fully respond to immunotherapy. Therefore, it is crucial to further explore accurate biomarkers to determine the most suitable immunotherapy candidates and improve their efficacy.

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

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

[0005] The application aims to provide a preparation method of an antigen repair solution, and improve the sensitivity and specificity of MHC-II expression detection in a gastric cancer immunotherapy efficacy prediction biopsy sample.

[0006] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows:

[0007] The application discloses a preparation method of an antigen repair solution, which comprises the following steps: uniformly mixing at least three components of citric acid, sodium citrate, an additive and pure water to obtain the antigen repair solution; and the additive at least comprises a glucose amide polyether derivative and a propyl gallate / calcium ion complex, wherein the glucose amide 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.

[0008] The antigen repair solution is prepared by mixing citric acid, sodium citrate, the glucose amide polyether derivative, the propyl gallate / calcium ion complex and pure water, so that the sensitivity and specificity of a gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit can be improved, the glucose amide polyether derivative and the propyl gallate / calcium ion complex have strong emulsifying performance and biological activity, can remove residual paraffin on the surface of a tissue section, and can assist antigen repair and antibody combination, so that the sensitivity and specificity of the gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit can be improved.

[0009] Preferably, in the preparation of the glucose amide polyether derivative, a ring-opening polymerization reaction is performed on glucose lactone and diethylene triamine as raw materials, then alkyl epoxy polyether and acyl chloride are added to react, and the glucose amide polyether derivative is obtained.

[0010] Preferably, the preparation of the glucose amide polyether derivative is specifically as follows:

[0011] The glucose lactone is dissolved in N,N-dimethylformamide, diethylene triamine is added, and a constant-temperature reaction is performed in a 60-120 DEG C water bath for 2-10 hours; then alkyl epoxy polyether is added, and a constant-temperature reaction is performed at 60-120 DEG C for 1-5 hours; the temperature is reduced to 0-5 DEG C, lauryl chloride and triethylamine are slowly added dropwise, and chlorine is introduced, and the glucose amide polyether derivative is obtained.

[0012] More preferably, the amount ratio of the glucose lactone and the N,N-dimethylformamide is 1 mmol:2-10 mL.

[0013] More preferably, the molar ratio of the glucose lactone and the diethylene triamine is 1:0.5-2.

[0014] More preferably, the molar ratio of the glucose lactone and the alkyl epoxy polyether is 1:1-2.

[0015] More preferably, the molar ratio of the alkyl epoxy polyether and lauroyl chloride is 1:0.2-1.

[0016] More preferably, the ratio of the amount of use of the gluconolactone and triethylamine is 1 mmol:0.5-2 mL.

[0017] Preferably, the propyl gallate / calcium ion complex is prepared by reacting propyl gallate and calcium chloride dihydrate.

[0018] Preferably, the preparation of the propyl gallate / calcium ion complex is specifically,

[0019] The propyl gallate is weighed, deionized water is added, heated at 60-100°C for 1-3h, calcium chloride dihydrate is added, heated and stirred for 2-10d, the temperature is reduced to 40-60°C, washed with 40-60°C deionized water for 2-4 times, and dried for 12-36h to obtain the propyl gallate / calcium ion complex.

[0020] More preferably, the ratio of the amount of use of the propyl gallate and deionized water is 1g:10-20mL.

[0021] More preferably, the mass ratio of the propyl gallate and calcium chloride dihydrate is 1:0.2-1.

[0022] Preferably, the mass ratio of the citric acid and sodium citrate is 1:7-8.

[0023] Preferably, the mass ratio of the citric acid and the additive is 1:0.2-4.

[0024] Preferably, the ratio of the amount of use of the citric acid and pure water is 1g:2-3L.

[0025] Preferably, the additive includes a gluconamido polyether derivative and a propyl gallate / calcium ion complex.

[0026] Preferably, the additive includes a gluconamido polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid. The use of coumarin-3-carboxylic acid can further improve the antigen repair capacity of the prepared antigen repair solution, thereby improving the sensitivity and specificity of immunohistochemical detection of MHC-II expression.

[0027] Preferably, a preparation method of an antigen repair solution is specifically,

[0028] The citric acid, sodium citrate, gluconamido polyether derivative, propyl gallate / calcium ion complex, coumarin-3-carboxylic acid, and pure water are mixed uniformly to obtain the antigen repair solution.

[0029] More preferably, the mass ratio of the citric acid and sodium citrate is 1:7-8.

[0030] More preferably, the mass ratio of the citric acid and the glucose amide-based polyether derivative is 1:0.5-1.5.

[0031] More preferably, the mass ratio of the citric acid and the propyl gallate / calcium ion complex is 1:0.5-1.5.

[0032] More preferably, the mass ratio of the sodium citrate and the coumarin-3-carboxylic acid is 1:0.2-1.

[0033] The purpose of the present application is to provide a gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit to improve the sensitivity and specificity of MHC-II expression detection.

[0034] The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit comprises the following components in mass parts: 20-30 parts of a deparaffinizing solution, 10-20 parts of an antigen repairing solution prepared by the above method, 2-10 parts of sheep serum, 10-20 parts of an enhancer, 15-25 parts of an antibody, 5-15 parts of a staining solution, and 10-20 parts of a mounting agent; the deparaffinizing solution comprises xylene.

[0035] Preferably, the antibody comprises an MHC Class II recombinant rabbit monoclonal antibody and an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

[0036] More preferably, the mass ratio of the MHC Class II recombinant rabbit monoclonal antibody and the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is 1:2-10.

[0037] Preferably, the staining solution comprises a diaminobenzidine developing solution, a hematoxylin staining solution, and an unbluing solution.

[0038] More preferably, the unbluing solution is prepared by mixing sodium bicarbonate and pure water, and the mass ratio of the sodium bicarbonate and the pure water is 1g:0.2-1L.

[0039] More preferably, the mass ratio of the diaminobenzidine developing solution and the hematoxylin staining solution is 1:1-2.

[0040] More preferably, the mass ratio of the diaminobenzidine developing solution and the unbluing solution is 1:1-2.

[0041] The antigen repair solution is prepared by using a glucose amide-based polyether derivative, propyl gallate / calcium ion complex and coumarin-3-carboxylic acid, and then the antigen repair solution is used to constitute a gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, so that the kit has the following beneficial effects: the sensitivity and specificity of the kit for detecting MHC-II expression are high, the sensitivity of the kit for detecting MHC-II expression is 94-99%, and the specificity is 87-92%. Therefore, the present application provides a gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit with high sensitivity and high specificity. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 An immunostaining result diagram for detecting MCH-II expression by using the kit obtained in Example 1. DETAILED DESCRIPTION

[0043] The present application will be further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0044] In the following examples, the experimental methods are conventional methods unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0045] Example 1:

[0046] S1, preparation of the antigen repair solution, comprising,

[0047] Mix citric acid, sodium citrate and pure water, and stir until uniform to obtain the antigen repair solution. The mass ratio of citric acid to sodium citrate is 1:7.5; the dosage ratio of citric acid to pure water is 1g:2.5L.

[0048] S2, the gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, includes the following quality parts of components: 27 parts of the deparaffinizing fluid, 14 parts of the antigen repairing fluid of step S1, 5 parts of the goat serum, 17 parts of the antibody, 14 parts of the enhancer, 9 parts of the staining fluid and 14 parts of the mounting medium, the deparaffinizing fluid is dimethylbenzene;The antibody includes MHC Class II recombinant rabbit monoclonal antibody and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody, and the mass ratio of MHC Class II recombinant rabbit monoclonal antibody and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is 1:5;The staining fluid includes diaminobenzidine color developing fluid, hematoxylin staining fluid and reverse blue liquid, the reverse blue liquid is prepared by mixing 2g of sodium bicarbonate and 1L of pure water, the mass ratio of diaminobenzidine color developing fluid and hematoxylin staining fluid is 1:1, and the mass ratio of diaminobenzidine color developing fluid and reverse blue liquid is 1:1.In this embodiment, the goat serum is purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., the MHC Class II recombinant rabbit monoclonal antibody is purchased from Hangzhou Huaan Biotechnology Co., Ltd., the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., the enhancer is purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., the diaminobenzidine color developing fluid is purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., the hematoxylin staining fluid is purchased from Ningbo Tongsheng Biotechnology Co., Ltd., and the mounting medium is purchased from Ningbo Tongsheng Biotechnology Co., Ltd.

[0049] Example 2:

[0050] S1, preparation of glucose amide polyether derivative, comprising,

[0051] Glucolactone is dissolved in N,N-dimethylformamide, diethylene triamine is added, and constant temperature reaction is carried out in a 90℃ water bath for 5h, then alkyl epoxy polyether is added, and constant temperature reaction is carried out at 90℃ for 3h, the temperature is reduced to 0℃, lauryl chloride and triethylamine are slowly added dropwise, and chlorine gas is introduced, to obtain a glucose amide polyether derivative.The dosage ratio of glucolactone and N,N-dimethylformamide is 1mmol:5mL;The molar ratio of glucolactone and diethylene triamine is 1:1, the molar ratio of glucolactone and alkyl epoxy polyether is 1:1.5, the molar ratio of alkyl epoxy polyether and lauryl chloride is 1:0.6, and the dosage ratio of glucolactone and triethylamine is 1mmol:1.5mL.

[0052] S2, preparation of propyl gallate / calcium ion complex, comprising,

[0053] Propyl gallate was weighed, deionized water was added, heated at 85℃ for 2h, calcium chloride dihydrate was added, heated and stirred for 5d, the temperature was reduced to 50℃, washed with 50℃ deionized water for 3 times, and dried for 24h to obtain propyl gallate / calcium ion complex. The mass ratio of propyl gallate to deionized water was 1g:10mL, and the mass ratio of propyl gallate to calcium chloride dihydrate was 1:0.7.

[0054] S3, preparation of antigen repair solution, comprising,

[0055] Citric acid, sodium citrate, glucose amido polyether derivative, propyl gallate / calcium ion complex and pure water were mixed and stirred uniformly to obtain an antigen repair solution. The mass ratio of citric acid to sodium citrate was 1:7.5, the mass ratio of citric acid to glucose amido polyether derivative was 1:1, the mass ratio of citric acid to propyl gallate / calcium ion complex was 1:1, and the mass ratio of citric acid to pure water was 1g:2.5L.

[0056] S4, gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, except that the antigen repair solution is replaced by the antigen repair solution obtained in step S3 of this embodiment, other conditions are the same as in example 1.

[0057] Example 3:

[0058] Except that in the preparation of the antigen repair solution, the mass ratio of citric acid to glucose amido polyether derivative is changed to 1:0.5, other conditions are the same as in example 2.

[0059] Example 4:

[0060] Except that in the preparation of the antigen repair solution, the mass ratio of citric acid to propyl gallate / calcium ion complex is changed to 1:0.5, other conditions are the same as in example 2.

[0061] Example 5:

[0062] S1, glucose amido polyether derivative, same as example 2.

[0063] S2, preparation of propyl gallate / calcium ion complex, same as example 2.

[0064] S3, preparation of antigen repair solution, comprising,

[0065] Mixing citric acid, sodium citrate, glucose amido polyether derivative, propyl gallate / calcium ion complex, coumarin-3-carboxylic acid and pure water, stirring uniformly, obtaining antigen repair solution. The mass ratio of citric acid and sodium citrate is 1:7.5, the mass ratio of citric acid and glucose amido polyether derivative is 1:1, the mass ratio of citric acid and propyl gallate / calcium ion complex is 1:1, the mass ratio of sodium citrate and coumarin-3-carboxylic acid is 1:0.5; the dosage ratio of citric acid and pure water is 1g:2.5L.

[0066] S4, gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, except that the antigen repair solution is replaced by the antigen repair solution obtained in step S3 of the embodiment, the other conditions are the same as in example 1.

[0067] Example 6:

[0068] Except that in the preparation of the antigen repair solution, the mass ratio of citric acid and coumarin-3-carboxylic acid is changed to 1:0.1, the other conditions are the same as in example 5.

[0069] Comparative Example 1:

[0070] S1, preparation of glucose amido polyether derivative, same as example 2.

[0071] S2, preparation of antigen repair solution, including,

[0072] Mixing citric acid, sodium citrate, glucose amido polyether derivative and pure water, stirring uniformly, obtaining antigen repair solution. The mass ratio of citric acid and sodium citrate is 1:7.5, the mass ratio of citric acid and glucose amido polyether derivative is 1:1, the dosage ratio of citric acid and pure water is 1g:2.5L.

[0073] S3, gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, except that the antigen repair solution is replaced by the antigen repair solution obtained in step S2 of the comparative example, the other conditions are the same as in example 1.

[0074] Comparative Example 2:

[0075] S1, preparation of propyl gallate / calcium ion complex, same as example 2.

[0076] S2, preparation of antigen repair solution, including,

[0077] Mixing citric acid, sodium citrate, propyl gallate / calcium ion complex and pure water, stirring uniformly, obtaining antigen repair solution. The mass ratio of citric acid and sodium citrate is 1:7.5, the mass ratio of citric acid and propyl gallate / calcium ion complex is 1:1, the dosage ratio of citric acid and pure water is 1g:2.5L.

[0078] S3, the gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, except that the antigen repair solution is replaced by the antigen repair solution obtained in step S2 of the present comparative example, and other conditions are the same as in example 1.

[0079] Comparative Example 3:

[0080] S1, preparation of the antigen repair solution, comprising,

[0081] Citric acid, sodium citrate, coumarin-3-carboxylic acid and pure water were mixed and stirred uniformly to obtain the antigen repair solution. The mass ratio of citric acid to sodium citrate was 1:7.5, the mass ratio of sodium citrate to coumarin-3-carboxylic acid was 1:0.5, and the dosage ratio of citric acid to pure water was 1g:2.5L.

[0082] S2, the gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, except that the antigen repair solution is replaced by the antigen repair solution obtained in step S1 of the present comparative example, and other conditions are the same as in example 1.

[0083] Experimental Example:

[0084] The kits obtained in examples 1-6 and comparative examples 1-3 were used to detect MHC-II expression by immunohistochemical staining, and the specific method was as follows:

[0085] The paraffin section of the sample is baked at 72°C for 20 min and then immersed in the deparaffinizing solution for 3 times, each time for 5 min, to obtain the deparaffinized tissue section. The tissue section is immersed in anhydrous ethanol, anhydrous ethanol, 95% ethanol, 80% ethanol and pure water, each time for 3 min. The antigen repairing solution is added into the pressure cooker, and after boiling, the tissue section is put into the pressure cooker. After 90 s of timing from the blowing of the pressure valve, the power is turned off, and the cover of the pressure cooker is opened after the pressure cooker is naturally cooled. The tissue section is taken out and washed with water for 3 times, each time for 2 min, and then immersed in the phosphate buffered saline solution for 5 min. After the tissue section is dried, the size of the tissue section is drawn on the tissue section with an oil pen. The tissue section is completely immersed in the 3% hydrogen peroxide solution for 10 min. The tissue section is washed with water for 3 times, each time for 2 min, and then immersed in the phosphate buffered saline solution for 5 min. The surface of the tissue section is completely covered with the sheep serum at room temperature for 10 min. The section is dried and the surface of the tissue section is kept wet, and the MHC Class II recombinant rabbit monoclonal antibody is diluted with the phosphate buffered saline solution to a final concentration of 1%. The MHC Class II recombinant rabbit monoclonal antibody diluent is added to completely cover the surface of the tissue section, and the section is placed at 4°C for 8 h. The section is placed at room temperature for 30 min, washed with water for 3 times, each time for 2 min, and immersed in the phosphate buffered saline solution for 5 min. The section is dried and the surface of the tissue section is kept wet, and the enhancer is added to completely cover the surface of the tissue section. The section is placed at room temperature for 20 min, washed with water for 3 times, each time for 2 min, and immersed in the phosphate buffered saline solution for 5 min. The section is dried and the surface of the tissue section is kept wet, and the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is added to completely cover the surface of the tissue section. The section is placed at room temperature for 20 min, washed with water for 3 times, each time for 2 min, and immersed in the phosphate buffered saline solution for 5 min. The operation is carried out in the dark, 2 drops of diaminobenzidine color developing solution are added dropwise, and the color development is terminated with water after incubation at room temperature for 3 min. The section is washed with water for 3 times, each time for 2 min, and then immersed in the hematoxylin staining solution for 2 min. The section is washed with the shaking water for 3 min, and then immersed in the destaining solution for 2 min. The section is washed with the shaking water for 3 min, and then immersed in 80% ethanol for 10 s, 95% ethanol for 10 s, 100% ethanol for 3 min, anhydrous ethanol for 3 min and anhydrous ethanol for 3 min. After natural air drying, the section is mounted with the mounting medium, and the detection of the expression of MHC-II is observed under a low-power microscope.

[0086] 1. Immunohistochemical staining

[0087] According to the above method, the expression of MHC-II is detected by using the kit obtained in Example 1, and the proportion of the cell membrane colored cells and the color intensity are observed under a low-power microscope.

[0088] Figure 1The immunostaining results of detecting MHC-II expression using the kit of Example 1 are shown in the figure. The negative results show incomplete and weak cell membrane staining, and the positive results show strong and complete cell membrane staining.

[0089] 2. Determination of kit sensitivity

[0090] According to the above method, the sensitivity of detecting MHC-II expression of each group was determined by detecting MHC-II expression using the kits of Examples 1-6 and Comparative Examples 1-3, and the sensitivity (%) = the number of MHC-II positive samples / total sample number x 100%. The results are shown in Table 1. Table 1 shows the sensitivity (%) of detecting MHC-II expression.

[0091] Table 1 Sensitivity (%) of detecting MHC-II expression

[0092]

[0093] As shown in Table 1, the sensitivity of detecting MHC-II expression of Example 2 is higher than that of Example 1, which is due to the additional addition of glucose amide polyether derivative and propyl gallate / calcium ion complex in the preparation of the antigen repair solution in Example 2. This shows that the method of Example 2 can effectively improve the sensitivity of detecting MHC-II expression. The sensitivity of detecting MHC-II of Example 2 is higher than that of Comparative Examples 1-2, which is due to the additional addition of glucose amide polyether derivative and propyl gallate / calcium ion complex in the preparation of the antigen repair solution in Example 2. Comparative Example 1 only adds glucose amide polyether derivative, and Comparative Example 2 adds propyl gallate / calcium ion complex. The sensitivity of detecting MHC-II of Example 2 is higher than that of Examples 3-4, which is due to the different amounts of glucose amide polyether derivative and propyl gallate / calcium ion complex used in the preparation of the antigen repair solution. This shows that the synergistic use of glucose amide polyether derivative and propyl gallate / calcium ion complex in the preparation of the antigen repair solution makes the sensitivity of detecting MHC-II expression better.

[0094] The sensitivity of Example 5 for detecting MCH-II is higher than that of Example 2, because in the preparation of the antigen repair solution, Example 5 additionally adds glucose amide polyether derivative, propyl gallate / calcium ion complex and coumarin-3-carboxylic acid, and Example 2 additionally adds glucose amide polyether derivative and propyl gallate / calcium ion complex; the sensitivity of Example 5 for detecting MCH-II is higher than that of Comparative Example 3, because in the preparation of the antigen repair solution, Comparative Example 3 additionally adds coumarin-3-carboxylic acid, and Example 5 additionally adds glucose amide polyether derivative, propyl gallate / calcium ion complex and coumarin-3-carboxylic acid; the sensitivity of Example 5 for detecting MCH-II is higher than that of Example 6, because the use amount of coumarin-3-carboxylic acid in the antigen repair solution is different. This shows that adding coumarin-3-carboxylic acid in the preparation of the antigen repair solution can further improve the sensitivity to MCH-II expression.

[0095] 3. Determination of specificity of the kit

[0096] According to the above method, the kits obtained from Examples 1-6 and Comparative Examples 1-3 are used to detect the expression of MHC-II in samples, the specificity of each group for detecting MCH-II expression is determined, the specificity (%) = MCH-II negative sample number / total sample number x 100%, and the determination results are shown in Table 2. Table 2 is the specificity (%) of detecting MCH-II expression.

[0097] Table 2 Specificity (%) of detecting MCH-II expression

[0098]

[0099] As shown in Table 2, the specificity of detecting MCH-II expression of Example 2 is higher than that of Example 1, which is because that the glucose amide polyether derivative and propyl gallate / calcium ion complex are additionally added in the preparation of the antigen repair solution in Example 2, which indicates that the method of Example 2 can effectively improve the specificity of detecting MCH-II expression. The specificity of detecting MCH-II of Example 2 is higher than that of Comparative Examples 1-2, which is because that the glucose amide polyether derivative and propyl gallate / calcium ion complex are additionally added in the preparation of the antigen repair solution in Example 2, the glucose amide polyether derivative is additionally added in Comparative Example 1, and the propyl gallate / calcium ion complex is additionally added in Comparative Example 2; the specificity of detecting MCH-II of Example 2 is higher than that of Examples 3-4, which is because that the usage amount of the glucose amide polyether derivative and the propyl gallate / calcium ion complex is different in the preparation of the antigen repair solution. This indicates that the synergistic use of the glucose amide polyether derivative and the propyl gallate / calcium ion complex in the preparation of the antigen repair solution makes the specificity of detecting MCH-II expression better.

[0100] The specificity of detecting MCH-II of Example 5 is higher than that of Example 2, which is because that the glucose amide polyether derivative, the propyl gallate / calcium ion complex and the coumarin-3-carboxylic acid are additionally added in the preparation of the antigen repair solution in Example 5, and the glucose amide polyether derivative and the propyl gallate / calcium ion complex are additionally added in Example 2; the specificity of detecting MCH-II of Example 5 is higher than that of Comparative Example 3, which is because that the coumarin-3-carboxylic acid is additionally added in the preparation of the antigen repair solution in Comparative Example 3, and the glucose amide polyether derivative, the propyl gallate / calcium ion complex and the coumarin-3-carboxylic acid are additionally added in Example 5; the specificity of detecting MCH-II of Example 5 is higher than that of Example 6, which is because that the usage amount of the coumarin-3-carboxylic acid is different in the antigen repair solution. This indicates that the addition of the coumarin-3-carboxylic acid in the preparation of the antigen repair solution can further improve the specificity of detecting MCH-II expression.

[0101] The routine operations in the operation steps of the present application are well known to those skilled in the art, and will not be described here.

[0102] The above-described examples have described the technical solutions of the present application in detail, and it should be understood that the above-described only is the specific embodiments of the present application, and is not used to limit the present application, and any changes or modifications made within the principle range of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing an antigen retrieval solution, comprising mixing citric acid, sodium citrate, an additive, and pure water uniformly to obtain the antigen retrieval solution; the additive comprises a glucose amide-based polyether derivative, a propyl gallate / calcium ion complex, and coumarin-3-carboxylic acid, the glucose amide-based 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 of claim 1, wherein the antigen retrieval solution is prepared by mixing the components in the following order: 1) the citrate buffer, 2) the urea, 3) the surfactant, and 4) the pH adjuster. In the preparation of the glucose amide-based polyether derivative, a ring-opening polymerization reaction is performed on glucose lactone and diethylenetriamine as raw materials, and then alkyl epoxy polyether and acyl chloride are added for reaction to obtain the glucose amide-based polyether derivative.

3. The method of claim 1, wherein the antigen retrieval solution is prepared by mixing the primary antibody with the diluent. The propyl gallate / calcium ion complex is prepared by reacting propyl gallate and calcium chloride dihydrate. 4.A gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit, comprising the following components in mass parts: 20-30 parts of a deparaffinizing solution, 10-20 parts of the antigen retrieval solution of claim 1, 2-10 parts of sheep serum, 10-20 parts of an enhancer solution, 15-25 parts of an antibody, 5-15 parts of a staining solution, and 10-20 parts of a mounting medium; the deparaffinizing solution is xylene.

5. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit according to claim 4, characterized in that, The antibody comprises an MHC Class II recombinant rabbit monoclonal antibody and an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

6. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit of claim 5, wherein, The mass ratio of the MHC Class II recombinant rabbit monoclonal antibody and the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody is 1:2-10.

7. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit of claim 4, wherein, The staining solution comprises a diaminobenzidine developing solution, a hematoxylin staining solution, and an anti-blue solution.

8. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit of claim 7, wherein, The anti-blue solution is obtained by mixing sodium bicarbonate and pure water, and the mass ratio of sodium bicarbonate to pure water is 1g:0.2-1L.

9. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit of claim 7, wherein, The mass ratio of the diaminobenzidine developing solution to the hematoxylin staining solution is 1:1-2.

10. The gastric cancer immunotherapy efficacy prediction biopsy sample MHC-II expression kit of claim 7, wherein, The mass ratio of the diaminobenzidine developing solution to the anti-blue solution is 1:1-2.

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