Detection reagent for detecting mhc-ii expression of tumor cells in gastric cancer tissue

Abstract

The reagent for detecting MHC-II expression of tumor cells in gastric cancer tissue comprises the following components in parts by mass: 10-30 parts of paraffin section preparation liquid, 2-10 parts of dewaxing liquid, 2-10 parts of sodium citrate antigen repair liquid, and 20-40 parts of immunohistochemical staining liquid; the dewaxing liquid comprises dimethylbenzene, and at least one of flavonol aminobenzoate derivative and sinapamide benzyl trimethylammonium benzoate salt; the flavonol aminobenzoate derivative has a nitrogen-containing group and a benzene ring, and the sinapamide benzyl trimethylammonium benzoate salt has a long-chain unsaturated alkane and a nitrogen-containing group. The reagent for detecting MHC-II expression of tumor cells in gastric cancer tissue can obtain high-quality immunohistochemical staining sections, thereby improving the sensitivity and specificity of MHC-II expression detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunohistochemical staining technology, and particularly relates to a detection reagent for detecting MHC-II expression of tumor cells in gastric cancer tissue. 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 candidates for immunotherapy 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 the amount of detection targets is large. 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 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. However, MHC-II expression has not yet become a routine detection indicator in clinical pathology, so there is no uniform standard and method for its preparation, staining, and interpretation, and its screening value still needs more clinical and basic verification. Therefore, it is particularly important to improve the staining efficiency of immunohistochemistry and obtain high-quality and accurate MHC-II immunohistochemical staining sections in a short period of time. SUMMARY

[0005] The present application aims to provide a detection reagent for detecting MHC-II expression of tumor cells in gastric cancer tissue, which can obtain high-quality immunohistochemical staining sections, thereby improving the sensitivity and specificity of detecting MHC-II expression.

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

[0007] The detection reagent for detecting MHC-II expression of tumor cells in gastric cancer tissue comprises the following components in mass fraction: 10-30 parts of paraffin section preparation liquid, 2-10 parts of dewaxing liquid, 2-10 parts of sodium citrate antigen repair liquid, and 20-40 parts of immunohistochemical staining liquid. The dewaxing liquid comprises xylene, and at least one of flavonol aminobenzoate derivative and sinapamide benzyl trimethylammonium benzoate salt. The flavonol aminobenzoate derivative has a nitrogen-containing group and a benzene ring, and the sinapamide benzyl trimethylammonium benzoate salt has a long-chain unsaturated alkane and a nitrogen-containing group. The flavonol aminobenzoate derivative and the sinapamide benzyl trimethylammonium benzoate salt have a large number of hydrophobic groups, which can reduce the oil-water interfacial tension and improve the dewaxing effect. At the same time, the sinapamide benzyl trimethylammonium benzoate salt has strong penetration, which can promote the penetration of organic solvents into the tissue, improve the solubility of lipophilic paraffin in organic solvents, restore the binding capacity of the tissue and the antigen, and thus enhance the immunohistochemical staining effect and improve the detection sensitivity and specificity of MHC-II expression.

[0008] Preferably, the flavonol aminobenzoate derivative is obtained by esterification of 3-hydroxyflavone and p-aminobenzoic acid under the catalysis of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine.

[0009] More preferably, the preparation method of the flavonol aminobenzoate derivative is as follows:

[0010] The 3-hydroxyflavone, p-aminobenzoic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in N,N-dimethylformamide, a magnetic stirrer is added for sufficient stirring until the solids are dissolved, and the mixture is stirred at room temperature for 6-24 h. Deionized water is added, the solid is retained by suction filtration, and the solid is washed with saturated sodium bicarbonate for 3-6 times and with saturated ammonium chloride for 3-6 times. After drying, the flavonol aminobenzoate derivative is obtained by column chromatography purification with a developing agent.

[0011] More preferably, the mass ratio of 3-hydroxyflavone to p-aminobenzoic acid is 1:1-2.

[0012] More preferably, the mass ratio of p-aminobenzoic acid and 1-ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride is 1:1-2.

[0013] More preferably, the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1-2.

[0014] More preferably, the liquid-solid ratio of 4-dimethylaminopyridine and N,N- dimethylformamide is 1g:20-100mL.

[0015] More preferably, the water body temperature of deionized water is 2-10℃, and the mass ratio of N,N-dimethylformamide and deionized water is 1:2-10.

[0016] More preferably, the developing agent comprises ethyl acetate and petroleum ether, and the mass ratio of ethyl acetate and petroleum ether is 1:2-5.

[0017] Preferably, the erucyl amide benzoic acid benzyl trimethyl ammonium salt is obtained by first reacting erucic acid with oxalyl chloride and p-aminobenzoic acid, and then reacting with benzyl trimethyl ammonium hydroxide.

[0018] More preferably, the mass ratio of erucic acid and oxalyl chloride is 1:1-2.

[0019] More preferably, the preparation method of erucyl amide benzoic acid benzyl trimethyl ammonium salt is,

[0020] In mass parts, erucic acid is placed in a 40-60℃ water bath and stirred to melt, oxalyl chloride and N,N-dimethylformamide are slowly added dropwise, the reaction is carried out until no bubbles are generated, p-aminobenzoic acid and triethylamine are added, the reaction is carried out for 6-24h, then dilute hydrochloric acid is used to adjust the pH of the reaction solution to 2-3, benzyl trimethyl ammonium hydroxide is slowly added dropwise, and the reaction is carried out at 20-40℃ for 6-24h, the solvent is evaporated, and acetone is used for recrystallization at low temperature for 3-6 times to obtain erucyl amide benzoic acid benzyl trimethyl ammonium salt.

[0021] More preferably, the mass ratio of erucic acid and oxalyl chloride is 1:1-2.

[0022] More preferably, the mass ratio of erucic acid and p-aminobenzoic acid is 1:1-2.

[0023] More preferably, the mass ratio of p-aminobenzoic acid and benzyl trimethyl ammonium hydroxide is 1:1-2.

[0024] More preferably, the mass ratio of N,N-dimethylformamide and erucic acid is 1:2-10.

[0025] More preferably, the mass ratio of triethylamine and p-aminobenzoic acid is 1:2-3.

[0026] Preferably, the dewaxing solution comprises the following components in mass fraction: 10-30 parts of xylene, 5-20 parts of flavonol aminobenzoate derivative, and 5-20 parts of sinapamide benzyltrimethylammonium benzoate.

[0027] Preferably, the dewaxing solution comprises the following components in mass fraction: 10-30 parts of xylene, 5-20 parts of flavonol aminobenzoate derivative, 5-20 parts of sinapamide benzyltrimethylammonium benzoate, and 2-10 parts of sodium di-n-pentyl sulfosuccinate. The sodium di-n-pentyl sulfosuccinate has a solubilizing effect. The addition of the sodium di-n-pentyl sulfosuccinate in the dewaxing solution can further improve the dewaxing effect of the dewaxing solution, improve the detection sensitivity and specificity of MHC-II expression, and effectively reduce false negatives and the like.

[0028] More preferably, the sodium di-n-pentyl sulfosuccinate is obtained by reacting maleic anhydride and n-pentanol under the catalysis of p-toluenesulfonic acid.

[0029] More preferably, the preparation method of the sodium di-n-pentyl sulfosuccinate is as follows,

[0030] Maleic anhydride and n-pentanol are used as raw materials, p-toluenesulfonic acid is used as a catalyst, and the reaction is carried out for 1-4 h at a reaction temperature of 80-100°C. Then, the reaction is carried out under reduced pressure of 0.08-0.1 MPa for 2-5 h. A sodium carbonate solution is added dropwise to adjust the pH value of the reaction solution to neutral. Sodium bisulfite and deionized water are added, and the reaction is carried out at 120-160°C. After the reaction is completed, methanol is added to completely dissolve the reaction product. Filtration is performed, and rotary evaporation is carried out. The precipitate is dried at 80-120°C for 6-24 h. After drying, activated carbon is added, and stirring is carried out for 3-6 h. Filtration is performed, and rotary evaporation is carried out to obtain the sodium di-n-pentyl sulfosuccinate.

[0031] More preferably, the mass ratio of maleic anhydride to n-pentanol is 1:1.5-2.5.

[0032] More preferably, the mass ratio of maleic anhydride to p-toluenesulfonic acid is 1:0.2-1.0.

[0033] More preferably, the mass ratio of maleic anhydride to sodium bisulfite is 1:1-2.

[0034] More preferably, the liquid-solid ratio of sodium bisulfite to deionized water is 1 g:1.5-2.5 mL.

[0035] More preferably, the volume ratio of deionized water to methanol is 1:1.0-1.5.

[0036] More preferably, the liquid-solid ratio of activated carbon to methanol is 1 g:10-30 mL.

[0037] Preferably, the paraffin section preparation liquid comprises the following components in mass fraction: 10-50 parts of formalin solution, 10-50 parts of 80% ethanol solution, 10-50 parts of 90% ethanol solution, 10-50 parts of 100% ethanol solution, 20-40 parts of xylene, and 10-30 parts of paraffin.

[0038] More preferably, the mass fraction of the formalin solution is 2-10%.

[0039] Preferably, the sodium citrate antigen retrieval solution is prepared by mixing citric acid, sodium citrate, and pure water.

[0040] More preferably, in the sodium citrate antigen retrieval solution, the mass ratio of citric acid to sodium citrate is 1:5-10.

[0041] More preferably, in the sodium citrate antigen retrieval solution, the liquid-solid ratio of citric acid to pure water is 1g:2-3L.

[0042] Preferably, the immunohistochemical staining solution comprises the following components in mass fraction: 10-50 parts of 80% ethanol, 10-50 parts of 95% ethanol, 10-50 parts of 100% ethanol, 30-70 parts of phosphate buffered saline solution, 10-30 parts of hydrogen peroxide, 10-30 parts of sheep serum, 2-10 parts of enhancer solution, 1-5 parts of MHC Class II recombinant rabbit monoclonal antibody, 1-5 parts of enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody, 1-5 parts of diaminobenzidine developing solution, 1-5 parts of hematoxylin staining solution, and 1-5 parts of counterstaining solution.

[0043] More preferably, the mass fraction of the hydrogen peroxide is 2-10%.

[0044] More preferably, the MHC Class II recombinant rabbit monoclonal antibody comprises MHC Class II recombinant rabbit monoclonal antibody and MHC Class II recombinant rabbit monoclonal antibody diluent.

[0045] More preferably, the MHC Class II recombinant rabbit monoclonal antibody diluent comprises phosphate buffered saline solution and MHC Class II recombinant rabbit monoclonal antibody, and the volume ratio of MHC Class II recombinant rabbit monoclonal antibody to phosphate buffered saline solution is 1:50-150.

[0046] More preferably, the counterstaining solution is prepared by mixing sodium bicarbonate and pure water.

[0047] More preferably, in the counterstaining solution, the liquid-solid ratio of sodium bicarbonate to pure water is 1g:1-2L.

[0048] Preferably, the detection of MHC-II expression in gastric cancer tissue tumor cells, in particular,

[0049] The gastric cancer tissue tumor cells are immersed in a 2-10% formaldehyde solution for fixation for 24-48 hours, and then sequentially immersed in 80% ethanol, 90% ethanol, and 100% ethanol for 1-4 hours, and then immersed in xylene until transparent, and then immersed in paraffin for 1-2 hours. After the wax block is solidified, a paraffin section is cut using a microtome. The paraffin section is baked at 60-80°C for 10-30 minutes, and then immersed in a deparaffinizing solution for 2-5 times, each time for 2-10 minutes. The section is sequentially immersed in 100% ethanol, 95% ethanol, 80% ethanol, and pure water, each time for 1-5 minutes. A sodium citrate antigen retrieval solution is added to a pressure cooker, and the section is immersed in the boiling sodium citrate antigen retrieval solution. After 60-120 seconds of timing from the time when the pressure valve blows, the power is turned off, and the cooker cover is opened after the pressure cooker is naturally cooled. The section is taken out and washed with water for 2-5 times, each time for 1-5 minutes, and then immersed in a phosphate buffered saline solution for 2-10 minutes. The section is dried and the size of the tissue is drawn on the section using an oil-based pen. The section is immersed in 2-4% hydrogen peroxide for 5-20 minutes. The section is washed with water for 2-5 times, each time for 1-5 minutes, and then immersed in a phosphate buffered saline solution for 2-10 minutes. The surface of the gastric cancer tissue tumor cells is completely covered with goat serum at room temperature, and blocked for 2-20 minutes. The section is dried and the surface of the gastric cancer tissue tumor cells is kept moist, and MHC Class II recombinant rabbit monoclonal antibody is added to completely cover the surface of the gastric cancer tissue tumor cells, and the section is kept at 2-6°C for 6-10 hours. The section is kept at room temperature for 20-40 minutes, washed with water for 2-5 times, each time for 1-5 minutes, and then immersed in a phosphate buffered saline solution for 2-10 minutes. The section is dried and the surface of the gastric cancer tissue tumor cells is kept moist, and a strengthening solution is added to completely cover the surface of the gastric cancer tissue tumor cells. The section is kept at room temperature for 10-30 minutes, washed with water for 2-5 times, each time for 1-5 minutes, and then immersed in a phosphate buffered saline solution for 2-10 minutes. The section is dried and the surface of the gastric cancer tissue tumor cells is kept moist, and a streptavidin-peroxidase polymer antibody is added to completely cover the surface of the gastric cancer tissue tumor cells, and the section is kept at room temperature for 20 minutes, washed with water for 2-5 times, each time for 1-5 minutes, and then immersed in a phosphate buffered saline solution for 2-10 minutes. The operation is carried out in the dark, 1-3 drops of diaminobenzidine chromogenic solution are added dropwise, the section is incubated at room temperature for 1-5 minutes, and the color development is terminated with water. The section is washed with water for 2-5 times, each time for 1-5 minutes, and then immersed in hematoxylin staining solution for 1-5 minutes. The section is washed with water for 1-5 minutes using a shaking table, dried, and then immersed in bluing solution for 1-5 minutes.The section is washed with a vibration bath for 1-5 min, and then is sequentially immersed in 80% ethanol for 10 s, 95% ethanol for 10 s, 100% ethanol for 1-5 min, 100% ethanol for 1-5 min, and 100% ethanol for 1-5 min. After being naturally dried, the section is sealed with a sealing agent, and then is observed under a low-power microscope to detect the expression of MHC-II.

[0050] The application further discloses a use of the dewaxing solution in preparation of a detection reagent.

[0051] The application has the following beneficial effects: the flavonol aminobenzoate derivative and the sinapamide benzyltrimethylammonium benzoate salt have a large number of hydrophobic groups, can reduce the oil-water interfacial tension, and improve the dewaxing effect; the sinapamide benzyltrimethylammonium benzoate salt has a strong penetration effect, can promote the penetration of the organic solvent into the tissue, improve the solubility of the lipophilic paraffin in the organic solvent, restore the binding capacity of the tissue and the antigen, and then enhance the immunohistochemical staining effect, and improve the sensitivity and specificity of the MHC-II expression detection; the sodium n-pentyl sulfonate has a solubilizing effect, further improves the dewaxing effect of the dewaxing solution, and obtains more reliable staining results, and further improves the sensitivity and specificity of the MHC-II expression detection. Therefore, the application provides the detection reagent which can improve the sensitivity and specificity of the MHC-II expression detection in the gastric cancer tissue tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The figure is an immunohistochemical staining result of the gastric cancer tissue tumor cells in Example 1. DETAILED DESCRIPTION

[0053] The application will be further described in detail in combination with the specific embodiments. The examples provided below are only for illustrating the application, and are not intended to limit the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0054] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the materials, reagents and the like are commercially available unless otherwise specified.

[0055] Example 1:

[0056] S1, the detection of the MHC-II expression in the gastric cancer tissue tumor cells, comprises,

[0057] The gastric cancer tissue tumor cells were immersed in a 10% formaldehyde solution for 24 hours, and then sequentially immersed in 80% ethanol, 90% ethanol, and 100% ethanol for 2 hours, and then immersed in xylene until transparent, and then immersed in paraffin for 1 hour. After the wax block was solidified, a paraffin section was cut using a microtome. The paraffin section was baked at 72°C for 20 minutes, and then immersed in a deparaffinizing solution three times, with a volume ratio of 20 parts of xylene, and each time for 5 minutes. The section was sequentially immersed in 100% ethanol, 95% ethanol, 80% ethanol, and pure water, each time for 3 minutes. Sodium citrate antigen retrieval solution was prepared by mixing 0.4 g of citric acid, 3 g of sodium citrate, and 1 L of pure water. The section was immersed in the boiling sodium citrate antigen retrieval solution, and then the power was turned off after 90 seconds of timing when the pressure valve blew. The section was taken out and washed with water three times, each time for 2 minutes, and then immersed in a phosphate buffered saline solution for 5 minutes. The phosphate buffered saline solution was purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd. After the section was dried, the size of the tissue was drawn on the section using an oil pen. The section was immersed in 3% hydrogen peroxide for 10 minutes. The section was washed with water three times, each time for 2 minutes, and then immersed in the phosphate buffered saline solution for 5 minutes. The surface of the gastric cancer tissue tumor cells was completely covered with goat serum at room temperature for 10 minutes. The goat serum was purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., with a product number of ZLI-9022. The section was dried and kept moist on the surface of the gastric cancer tissue tumor cells. MHC Class II recombinant rabbit monoclonal antibody was diluted with the phosphate buffered saline solution at a volume ratio of 100:1. The diluted MHC Class II recombinant rabbit monoclonal antibody was added to completely cover the surface of the gastric cancer tissue tumor cells, and then placed at 4°C for 8 hours. The MHC Class II recombinant rabbit monoclonal antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., with a product number of ET1704-13. The section was placed at room temperature for 30 minutes, washed with water three times, each time for 2 minutes, and then immersed in the phosphate buffered saline solution for 5 minutes. The section was dried and kept moist on the surface of the gastric cancer tissue tumor cells. Enhancing solution was added to completely cover the surface of the gastric cancer tissue tumor cells. The enhancing solution was purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd., with a product number of PV-9001. The section was placed at room temperature for 20 minutes, washed with water three times, each time for 2 minutes, and then immersed in the phosphate buffered saline solution for 5 minutes.The slice is spun dry and kept moist on the surface of the gastric cancer tissue tumor cells. Goat anti-rabbit IgG polymer enzyme-labeled antibody is added to completely cover the surface of the gastric cancer tissue tumor cells. The slice is placed at room temperature for 20 minutes, washed with water 3 times for 2 minutes each time, immersed in phosphate buffered saline solution for 5 minutes, and the goat anti-rabbit IgG polymer enzyme-labeled antibody is purchased from Beijing Zhongshanjinqiao Biotechnology Co., Ltd. with the product number PV-9001. Perform the operation in the dark. Add 2 drops of diaminobenzidine color developing solution, incubate at room temperature for 3 minutes, and terminate the color development with water. The diaminobenzidine color developing solution is purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. Wash the slice with water 3 times for 2 minutes each time, spin dry, and immerse in hematoxylin staining solution for 2 minutes. The hematoxylin staining solution is purchased from Ningbo Tongsheng Biotechnology Co., Ltd. Wash the slice with a shaking table for 3 minutes, spin dry, and immerse in counterstain solution for 2 minutes. The counterstain solution is prepared by mixing 2 g of sodium bicarbonate and 1 L of pure water. Wash the slice with a shaking table for 3 minutes, spin dry, and immerse in 80% ethanol for 10 seconds, 95% ethanol for 10 seconds, 100% ethanol for 3 minutes, 100% ethanol for 3 minutes, and 100% ethanol for 3 minutes. After natural air drying, use mounting medium for mounting. The mounting medium is purchased from Ningbo Tongsheng Biotechnology Co., Ltd. and the MHC-II expression detection is observed under a low-power microscope.

[0058] Example 2:

[0059] S1, preparation of flavonol aminobenzoic acid ester derivatives, comprising,

[0060] S1, preparation of flavonol aminobenzoic acid ester derivatives, comprising,

[0061] S2, preparation of sinapamide benzyl trimethylammonium benzoate salt, comprising,

[0062] In mass parts, 1 part of erucic acid was placed in a 50℃ water bath and stirred to melt, 1.05 parts of oxalyl chloride was slowly added dropwise, and 0.2 parts of N,N-dimethylformamide was added dropwise, the reaction was carried out until no bubbles were generated, 1.1 parts of p-aminobenzoic acid and 0.5 parts of triethylamine were added, the reaction was carried out for 12 hours, then the pH of the reaction solution was adjusted to 2 using dilute hydrochloric acid, 1.1 parts of benzyltrimethylammonium hydroxide was slowly added dropwise, the reaction was carried out at 30℃ for 12 hours, the solvent was evaporated, and the product was recrystallized 4 times at low temperature using acetone to obtain erucyl amide p-aminobenzoic acid benzyltrimethylammonium salt.

[0063] S3, detection of MHC-II expression in gastric cancer tissue tumor cells, except that the mass components of the deparaffinizing solution were changed to 20 parts of xylene, 10 parts of flavonol aminobenzoate derivative and 10 parts of erucyl amide p-aminobenzoic acid benzyltrimethylammonium salt, and the rest of the conditions were the same as in Example 1.

[0064] Example 3:

[0065] Except that in the detection of MHC-II expression in gastric cancer tissue tumor cells, the mass components of the deparaffinizing solution were changed to 20 parts of xylene, 5 parts of flavonol aminobenzoate derivative and 10 parts of erucyl amide p-aminobenzoic acid benzyltrimethylammonium salt, and the rest of the conditions were the same as in Example 1.

[0066] Example 4:

[0067] Except that in the detection of MHC-II expression in gastric cancer tissue tumor cells, the mass components of the deparaffinizing solution were changed to 20 parts of xylene, 10 parts of flavonol aminobenzoate derivative and 5 parts of erucyl amide p-aminobenzoic acid benzyltrimethylammonium salt, and the rest of the conditions were the same as in Example 1.

[0068] Example 5:

[0069] S1, preparation of flavonol aminobenzoate derivative, same as in Example 2.

[0070] S2, preparation of erucyl amide p-aminobenzoic acid benzyltrimethylammonium salt, same as in Example 2.

[0071] S3, preparation of sodium di-n-pentyl succinate sulfonate, including,

[0072] In mass parts, 1 part of maleic anhydride and 1.8 parts of n-pentanol were used as raw materials, 0.17 parts of p-toluenesulfonic acid was used as a catalyst, the reaction was carried out for 2 hours at a reaction temperature of 90℃, then the pressure was reduced to 0.1 MPa for 3 hours, sodium carbonate solution was added dropwise to adjust the pH of the reaction solution to neutral, 1.1 parts of sodium bisulfite and 1.8 parts of deionized water were added, and the reaction was carried out at 140℃, after the reaction was completed, 1.8 parts of methanol was added to completely dissolve the reaction product, filtration was carried out, rotary evaporation was carried out, the precipitate was dried at 100℃ for 12 hours, after drying, 0.11 parts of activated carbon was added, stirring was carried out for 4 hours, then the precipitate was filtered, rotary evaporation was carried out, and sodium di-n-pentyl succinate sulfonate was obtained.

[0073] S4, detection of MHC-II expression in gastric cancer tissue tumor cells, except that the mass components of the deparaffinizing solution were changed to 20 parts of xylene, 10 parts of flavonol aminobenzoate derivative, 10 parts of sinapamide benzoic acid benzyl trimethyl ammonium salt and 5 parts of sodium di-n-pentyl sulfonate succinate, and other conditions were the same as in Example 1.

[0074] Example 6:

[0075] Except that in the detection of MHC-II expression in gastric cancer tissue tumor cells, the mass components of the deparaffinizing solution were changed to 20 parts of xylene, 10 parts of flavonol aminobenzoate derivative, 10 parts of sinapamide benzoic acid benzyl trimethyl ammonium salt and 2 parts of sodium di-n-pentyl sulfonate succinate, and other conditions were the same as in Example 5.

[0076] Comparative Example 1:

[0077] Except that in the detection of MHC-II expression in gastric cancer tissue tumor cells, the mass components of the deparaffinizing solution were changed to 20 parts of xylene and 10 parts of flavonol aminobenzoate derivative, and other conditions were the same as in Example 2.

[0078] Comparative Example 2:

[0079] Except that in the detection of MHC-II expression in gastric cancer tissue tumor cells, the mass components of the deparaffinizing solution were changed to 20 parts of xylene and 10 parts of sinapamide benzoic acid benzyl trimethyl ammonium salt, and other conditions were the same as in Example 2.

[0080] Comparative Example 3:

[0081] Except that in the detection of MHC-II expression in gastric cancer tissue tumor cells, the mass components of the deparaffinizing solution were changed to 20 parts of xylene and 5 parts of sodium di-n-pentyl sulfonate succinate, and other conditions were the same as in Example 5.

[0082] Experimental Example:

[0083] 1. Staining effect of gastric cancer tissue tumor cells

[0084] The gastric cancer tissue tumor cells were immunohistochemically stained according to the method of Example 1, and the proportion of the gastric cancer tissue tumor cells with cell membrane coloring and the coloring intensity were observed under a low power lens.

[0085] Figure 1 The immunohistochemical staining result chart of the gastric cancer tissue tumor cell slice obtained in Example 1 is shown in FIG. 1. The gastric cancer tissue tumor cells showed strong, complete and uniform cell membrane staining, which indicated that the method could obtain an immunohistochemically stained slice of the gastric cancer tissue tumor cells.

[0086] 2. Sensitivity of detecting MHC-II expression in gastric cancer tissue tumor cells

[0087] The expression of MHC-II in tumor cells of gastric cancer tissue was detected according to the methods of Examples 1-6 and Comparative Examples 1-3, respectively, the sensitivity of each group in detecting MCH-II expression was determined, the sensitivity (%) = the number of MCH-II positive samples / total sample number x 100%, and the determination results are shown in Table 1. Table 1 is the sensitivity (%) of detecting MCH-II expression.

[0088] Table 1 Sensitivity (%) of detecting MCH-II expression

[0089]

[0090] As can be seen from Table 1, the sensitivity of detecting MCH-II expression of Example 2 is higher than that of Example 1, which is because in the dewaxing liquid, Example 1 uses xylene, and Example 2 uses xylene, flavonol aminobenzoate derivative and sinapamide benzyl trimethylammonium benzoate salt, which shows that the method of Example 2 can effectively improve the sensitivity of detecting MCH-II expression. The sensitivity of detecting MCH-II of Example 2 is higher than that of Comparative Example 1-2, which is because in the dewaxing liquid, Example 2 uses xylene, flavonol aminobenzoate derivative and sinapamide benzyl trimethylammonium benzoate salt, Comparative Example 1 uses xylene and flavonol aminobenzoate derivative, and Comparative Example 2 uses xylene and sinapamide benzyl trimethylammonium benzoate salt; the sensitivity of detecting MCH-II of Example 2 is higher than that of Examples 3-4, which is because the amount of flavonol aminobenzoate derivative and sinapamide benzyl trimethylammonium benzoate salt used in the dewaxing liquid is different. This shows that in the dewaxing liquid, the synergistic use of xylene, flavonol aminobenzoate derivative and sinapamide benzyl trimethylammonium benzoate salt makes the sensitivity of detecting MCH-II expression better.

[0091] Compared with Example 2, the sensitivity of Example 5 for detecting MCH-II is higher than that of Example 2, because in the dewaxing solution, Example 5 uses xylene, flavonol aminobenzoate derivative, erucamide benzoic acid benzyl trimethyl ammonium salt and sodium di-n-pentyl sulfosuccinate, while Example 2 uses xylene, flavonol aminobenzoate derivative and erucamide benzoic acid benzyl trimethyl ammonium salt; compared with Comparative Example 3, the sensitivity of Example 5 for detecting MCH-II is higher than that of Comparative Example 3, because in the dewaxing solution, Comparative Example 3 uses xylene and sodium di-n-pentyl sulfosuccinate, while Example 5 uses xylene, flavonol aminobenzoate derivative, erucamide benzoic acid benzyl trimethyl ammonium salt and sodium di-n-pentyl sulfosuccinate; compared with Example 6, the sensitivity of Example 5 for detecting MCH-II is higher than that of Example 6, because the amount of sodium di-n-pentyl sulfosuccinate used in the dewaxing solution is different. This shows that adding sodium di-n-pentyl sulfosuccinate in the dewaxing solution can further improve the dewaxing effect on paraffin sections, thereby improving the sensitivity of detecting MCH-II expression.

[0092] 3. Specificity of detecting MHC-II expression in tumor cells of gastric cancer tissue

[0093] The expression of MHC-II in normal cells of gastric tissue was detected according to the methods of Examples 1-6 and Comparative Examples 1-3, respectively, the specificity of detecting MCH-II expression of each group was 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.

[0094] Table 2 Specificity (%) of detecting MCH-II expression

[0095]

[0096] From Table 2, the specificity of detecting MCH-II expression of Example 2 is higher than that of Example 1, which is because that in the dewaxing solution, xylene is used in Example 1, and xylene, flavonol aminobenzoate derivative and erucylamide benzyltrimethylammonium benzoate are used in Example 2, which shows 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 in the dewaxing solution, xylene, flavonol aminobenzoate derivative and erucylamide benzyltrimethylammonium benzoate are used in Example 2, xylene and flavonol aminobenzoate derivative are used in Comparative Example 1, and xylene and erucylamide benzyltrimethylammonium benzoate are used 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 amount of flavonol aminobenzoate derivative and erucylamide benzyltrimethylammonium benzoate used in the dewaxing solution is different. This shows that the synergistic use of xylene, flavonol aminobenzoate derivative and erucylamide benzyltrimethylammonium benzoate in the dewaxing solution makes the specificity of detecting MCH-II expression better.

[0097] The specificity of detecting MCH-II of Example 5 is higher than that of Example 2, which is because that in the dewaxing solution, xylene, flavonol aminobenzoate derivative, erucylamide benzyltrimethylammonium benzoate and sodium di-n-pentyl sulfosuccinate are used in Example 5, and xylene, flavonol aminobenzoate derivative and erucylamide benzyltrimethylammonium benzoate are used in Example 2; the specificity of detecting MCH-II of Example 5 is higher than that of Comparative Example 3, which is because that in the dewaxing solution, xylene and sodium di-n-pentyl sulfosuccinate are used in Comparative Example 3, and xylene, flavonol aminobenzoate derivative, erucylamide benzyltrimethylammonium benzoate and sodium di-n-pentyl sulfosuccinate are used in Example 5; the specificity of detecting MCH-II of Example 5 is higher than that of Example 6, which is because that the amount of sodium di-n-pentyl sulfosuccinate used in the dewaxing solution is different. This shows that adding sodium di-n-pentyl sulfosuccinate in the dewaxing solution can further improve the dewaxing effect on paraffin sections, thereby improving the specificity of detecting MCH-II expression.

[0098] 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.

[0099] 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 are specific examples of the present application, and are 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 detection reagent for detecting MHC-II expression in gastric cancer tumor cells, comprising the following components in parts by weight: 10-30 parts paraffin section preparation solution, 2-10 parts dewaxing solution, 2-10 parts sodium citrate antigen retrieval solution, and 20-40 parts immunohistochemical staining solution; wherein the dewaxing solution comprises the following components in parts by weight: 10-30 parts xylene, 5-20 parts flavonol aminobenzoate derivative, 5-20 parts benzyltrimethylammonium succinate, and 2-10 parts sodium di-n-pentyl succinate sulfonate, wherein the flavonol aminobenzoate derivative has a nitrogen-containing group and a benzene ring, and the benzyltrimethylammonium succinate has a long-chain unsaturated alkane and a nitrogen-containing group; wherein the flavonol aminobenzoate derivative is composed of 3-hydroxyflavone and p-aminobenzoic acid in 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine. The product is obtained by esterification under catalysis; the benzyltrimethylammonium succinate is obtained by reacting erucic acid with oxaloyl chloride and p-aminobenzoic acid, and then with benzyltrimethylammonium hydroxide; the preparation method of sodium di-n-pentyl succinate sulfonate is as follows: maleic anhydride and n-pentanol are used as raw materials, and p-toluenesulfonic acid is used as catalyst to react for 1-4 hours at a reaction temperature of 80-100℃. Then, the pressure is reduced to 0.08-0.1 MPa and the reaction is carried out for 2-5 hours. Sodium carbonate solution is added dropwise to adjust the pH of the reaction solution to neutral. Sodium bisulfite and deionized water are added and the reaction is carried out at 120-160℃. After the reaction is completed, methanol is added to completely dissolve the reaction product. The product is filtered, rotary evaporated, and the precipitate is dried at 80-120℃ for 6-24 hours. After drying, activated carbon is added, and the mixture is stirred for 3-6 hours. The precipitate is then filtered and rotary evaporated to obtain sodium di-n-pentyl succinate sulfonate.

2. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 1, characterized in that, The mass ratio of erucic acid to oxaloyl chloride is 1:1-2.

3. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 1, characterized in that, The paraffin section preparation solution comprises the following components in parts by weight: 10-50 parts formalin solution, 10-50 parts 80% ethanol, 10-50 parts 90% ethanol, 10-50 parts 100% ethanol, 20-40 parts xylene, and 10-30 parts paraffin.

4. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 3, characterized in that, The formalin solution has a mass fraction of 2-10%.

5. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 1, characterized in that, The sodium citrate antigen repair solution is prepared by mixing citric acid, sodium citrate and pure water.

6. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 5, characterized in that, The mass ratio of citric acid to sodium citrate is 1:5-10.

7. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 5, characterized in that, The volume ratio of citric acid to pure water is 1g:2-3L.

8. The detection reagent for detecting MHC-II expression in gastric cancer tissue tumor cells according to claim 1, characterized in that, The immunohistochemical staining solution comprises the following components in parts by weight: 10-50 parts of 80% ethanol, 10-50 parts of 95% ethanol, 10-50 parts of 100% ethanol, 30-70 parts of phosphate buffer solution, 10-30 parts of hydrogen peroxide, 10-30 parts of sheep serum, 2-10 parts of enhancement solution, 1-5 parts of MHC Class II recombinant rabbit monoclonal antibody, 1-5 parts of enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody, 1-5 parts of diaminobenzidine chromogenic solution, 1-5 parts of hematoxylin staining solution, and 1-5 parts of reverse blue solution.

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