An immunostaining reagent for detecting mhc-ii expression in gastric cancer biopsy samples

Abstract

The application discloses a kind of gastric cancer biopsy sample MHC-II expression detection immunostaining reagent, belong to the field of bioengineering.The application establishes a kind of immunohistochemical staining section preparation method, specifically including step one early treatment: tissue section baking, dewaxing and rehydration, and subsequent addition antigen solution repair, addition endogenous enzyme inhibitor incubation, addition blocking agent blocking etc.;Step two antibody incubation: addition first second antibody incubation;Step three late treatment: including staining, re-staining, reverse blue and mounting etc..With mixed buffer as endogenous enzyme inhibitor, effectively reduce the background staining intensity, improve the sensitivity and specificity of staining;At the same time, the standard of interpretation of staining results is established, which confirms that MHC-II expression is significantly related to pathological remission of gastric cancer patients, providing an evaluation method for pathological remission of gastric cancer patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to an immunostaining reagent for detecting MHC-II expression of a gastric cancer biopsy sample. BACKGROUND

[0002] Gastric and gastroesophageal junction (G / GEJ) cancer ranks fifth in newly diagnosed malignancies worldwide and fourth in cancer-related deaths. Despite the latest progress in multidisciplinary or multimodal therapy, the prognosis of patients with locally advanced disease is still poor, with a median overall survival (OS) of only 34.4 months and a 5-year OS rate of only 38.7%. According to the guidelines of the Chinese Society of Clinical Oncology (CSCO), perioperative therapy combined with D2 gastrectomy has become the standard treatment for locally advanced G / GEJ cancer (cT3-4aN+M0). However, the optimal perioperative treatment regimen and sequence are still unclear. Although more than 90% of high R0 resection rates have been achieved through perioperative therapy, optimizing the treatment regimen is necessary to improve the clinical outcomes of patients with locally advanced G / GEJ cancer.

[0003] The importance of screening G / GEJ cancer patients to determine which patients may benefit from perioperative immunotherapy. Currently, the biomarkers related to the efficacy of immunotherapy mainly include PD-L1 expression, microsatellite instability (MSI) status, EBV infection status, and tumor-infiltrating lymphocytes (TILs). In theory, cancer patients showing high PD-L1 expression, microsatellite instability-high (MSI-H) status, or EBV infection tend to exhibit enhanced reactivity to immunotherapy. Nevertheless, other patients may not fully respond to immunotherapy. Therefore, it is crucial to further explore increasingly precise biomarkers to determine the most suitable candidates for immunotherapy and improve their efficacy.

[0004] Major histocompatibility complex (MHC) molecules, also known as human leukocyte antigen (HLA) molecules, are currently found to be recognized by the immune system through major histocompatibility complexes (MHCs) to recognize tumor cells, and MHCs 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. The accumulation of HLA-DM and -DO molecules in the endosomal / lysosomal compartment and on the surface of B cells regulates the binding of foreign peptides to class II molecules (HLA-DR) by maintaining a conformation that facilitates peptide exchange, which is expressed in various tumor tissues and is essential for antigen presentation to CD4+ T lymphocytes, and the role of CD4+ T lymphocytes in anti-tumor immunity is also increasingly emphasized. MHC-II is associated with a higher number of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), lack of lymphatic vessel invasion, increased formation of tertiary lymphoid structures, upregulation of genes associated with activation of the IFN-gamma pathway (including the CD274 gene encoding PD-L1), and higher levels of IFNG, IL2 and IL12 mRNA (Th1 cytokines). In general, increased expression of MHC-II or related pathway components in tumor cells is associated with better prognosis and increased anti-tumor immunity, so MHC-II plays an important role in immunotherapy.

[0005] MHC-II expression can be detected by immunohistochemical staining techniques, but 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, the prior art still needs a technology that can obtain high-quality and accurate MHC-II immunohistochemical staining sections in a short time and perfect and accurate pathological section interpretation standards. SUMMARY

[0006] The purpose of the present application is to provide an immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples, to solve the problems of MHC-II expression immunohistochemical staining technology detection, lack of uniform standards and methods for preparation, staining, and interpretation, and the like.

[0007] To solve the above technical problems, the present application specifically provides the following technical solutions:

[0008] The application discloses a use of a mixed buffer in preparation of an endogenous enzyme inhibitor, the mixed buffer containing a hydrazine derivative and amino-polyethylene glycol-galactose, and the mass ratio of the use amount of the hydrazine derivative to the use amount of the amino-polyethylene glycol-galactose in the mixed buffer being 1:0.2-1. The use of the mixed buffer of the hydrazine derivative and the amino-polyethylene glycol-galactose can effectively inhibit the endogenous enzyme, prevent the endogenous enzyme from reacting with a substrate solution, cause false positive, significantly reduce the non-specific background, and improve the sensitivity and specificity of dyeing.

[0009] Preferably, the mixed buffer further contains a phosphate buffer, and the amount of the hydrazine derivative in the phosphate buffer is 0.045-0.06 g / ml.

[0010] Preferably, the hydrazine derivative is prepared from 1-hydroxy-2-naphthoyl hydrazine and acetic anhydride, and the mass ratio of the use amount of the 1-hydroxy-2-naphthoyl hydrazine to the use amount of the acetic anhydride is 1:0.45-0.7.

[0011] The application discloses an immunostaining reagent for detecting MHC-II expression of a gastric cancer biopsy sample, which at least comprises an endogenous enzyme inhibitor, and the endogenous enzyme inhibitor comprises a mixed buffer containing a hydrazine derivative and amino-polyethylene glycol-galactose, and the mass ratio of the use amount of the hydrazine derivative to the use amount of the amino-polyethylene glycol-galactose in the mixed buffer is 1:0.2-1.

[0012] Preferably, the immunostaining reagent further comprises a dewaxing and hydrating treatment solution, and the dewaxing and hydrating treatment solution is dimethylbenzene, 95-100% alcohol, 90-95% alcohol or 75-85% alcohol.

[0013] Preferably, the immunostaining reagent further comprises an antigen repair solution, and the antigen repair solution is obtained by mixing citric acid and sodium citrate in water, the mass ratio of the use amount of the citric acid to the use amount of the sodium citrate is 1:6-9, and the amount of the citric acid added into water is 0.0003-0.0005 g / ml.

[0014] Preferably, the immunostaining reagent further comprises a blocking agent, and the blocking agent is sheep serum.

[0015] Preferably, the immunostaining reagent further comprises an enhancing solution.

[0016] Preferably, the immunostaining reagent further comprises a first antibody, and the first antibody is a dilute MHC Class II recombinant rabbit monoclonal antibody, and the dilute MHC Class II recombinant rabbit monoclonal antibody is obtained by mixing and diluting a phosphate buffer and a MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.002-0.015.

[0017] The application further discloses an immunodetection kit based on MHC-II expression, which comprises the immunostaining reagent.

[0018] The application discloses a preparation method of an immunohistochemical staining section.

[0019] Step one: early treatment, adding a deparaffinization and hydration treatment solution to the tissue section, then immersing in an antigen repair solution for repair, then adding an endogenous enzyme inhibitor for incubation, and finally adding a blocking agent for blocking to obtain a treated tissue section; the endogenous enzyme inhibitor is at least one of 1-3% hydrogen peroxide and a mixed buffer solution, and the mixed buffer solution is obtained by dissolving a hydrazine derivative and an amino-polyethylene glycol-galactose in a phosphate buffer solution.

[0020] Step two: antibody incubation, adding a first antibody to the treated tissue section obtained in step one for incubation, then immersing in an enhancer solution for standing, then adding a second antibody for incubation, and finally obtaining a tissue section compound after the incubation is completed;

[0021] Step three: late treatment, adding a color developing agent, a re-staining agent and a counterstain agent to the tissue section compound obtained in step two in sequence for staining, and then performing dehydration and mounting to obtain an immunohistochemical staining section.

[0022] Preferably, the amount of the hydrazine derivative in the phosphate buffer solution in step one is 0.045-0.06 g / ml, and the mass ratio of the use amount of the hydrazine derivative to the use amount of the amino-polyethylene glycol-galactose is 1:0.2-1.

[0023] Preferably, the deparaffinization and hydration treatment solution in step one comprises xylene, 95-100% alcohol, 90-95% alcohol and 75-85% alcohol; the xylene treatment time is 3-10 min, and the 95-100% alcohol, 90-95% alcohol and 75-85% alcohol are treated in sequence for 3-10 min.

[0024] Preferably, the antigen repair solution in step one is obtained by mixing citric acid and sodium citrate in water, the mass ratio of the use amount of the citric acid to the use amount of the sodium citrate is 1:6-9, and the amount of the citric acid added in water is 0.0003-0.0005 g / ml; the blocking agent is a sheep serum.

[0025] Preferably, the use amounts of the deparaffinization and hydration treatment solution, the antigen repair solution, the endogenous enzyme inhibitor and the blocking agent in step one are complete immersion of the tissue section.

[0026] Preferably, the antigen repair time in step one is 60-120 s; the incubation time of the endogenous enzyme inhibitor is 5-15 min; and the blocking time of the blocking agent is 5-15 min.

[0027] Preferably, in the step two, the first antibody is diluted MHC Class II recombinant rabbit monoclonal antibody, and the second antibody is enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody; the diluted MHC Class II recombinant rabbit monoclonal antibody is obtained by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.002-0.015; the first antibody, the enhancer and the second antibody are used in a complete immersion treatment of the tissue section.

[0028] Preferably, in the step two, the first antibody is incubated at a temperature of 2-6℃ for 6-10h; after adding the enhancer, the temperature is 23-27℃ for 15-30min; and the second antibody is incubated at a temperature of 23-27℃ for 15-30min.

[0029] Preferably, in the step three, the color developing agent is DAB color developing solution, the counterstain agent is hematoxylin, and the decolorizing agent is obtained by mixing sodium bicarbonate in water, and the amount of sodium bicarbonate added in water is 0.001-0.003g / ml; the color developing agent, the counterstain agent and the decolorizing agent are used in a complete immersion treatment of the tissue section complex.

[0030] Preferably, in the step three, after adding the color developing agent, the incubation temperature is 23-27℃ for 1-5min; after adding the counterstain agent, the soaking time is 1-5min; and after adding the decolorizing agent, the soaking time is 1-5min; and the solvent used in the dehydration process is 95-100% alcohol, 90-95% alcohol and 75-85% alcohol, which are sequentially treated for 1-5min.

[0031] The application discloses a gastric cancer biopsy sample immunostaining reagent based on MHC-II expression.

[0032] Preferably, the pre-treatment reagent comprises a dewaxing and hydrating treatment solution, an antigen repair solution, an inhibitor and a blocking agent; the antibody incubation reagent comprises a first antibody, an enhancer and a second antibody; and the post-treatment reagent comprises a color developing agent, a counterstain agent, a decolorizing agent and a dehydration solvent.

[0033] The application discloses a preparation method of an immunohistochemical staining section.

[0034] Step one: pre-treatment, cutting a cancer tissue wax block to obtain a tissue section, treating the tissue section, then immersing the tissue section in an antigen repair solution for high-pressure repair treatment for 60-120s, then drawing the size of the tissue, adding an endogenous enzyme inhibitor for incubation treatment for 5-15min, then adding a blocking agent for blocking treatment for 5-15min, to obtain a treated tissue section.

[0035] Step two: antibody incubation, the first antibody is added to the treated tissue section obtained in step one, and incubated at 2-6°C for 6-10 hours. Then immerse in enhancer solution, and place at 23-27°C for 15-30 minutes. Then add the second antibody to the treated tissue section, and incubate at 23-27°C for 15-30 minutes. After the reaction is completed, the tissue section complex is obtained.

[0036] Step three: post-treatment, under light-proof conditions, add color developing solution to the tissue section complex obtained in step two, and incubate at 23-27°C for 1-5 minutes. After the incubation is completed, immerse in counterstain solution for 1-5 minutes, then immerse in anti-blue solution for 1-5 minutes. Finally, dehydrate the tissue section complex, dry, and mount with mounting medium.

[0037] Preferably, in the above technical solution, the mixed buffer used in step one is obtained by dissolving hydrazine derivative and amino-polyethylene glycol-galactose in phosphate buffer, the amount of hydrazine derivative in the phosphate buffer is 0.045-0.06 g / ml, and the mass ratio of the amount of hydrazine derivative used to the amount of amino-polyethylene glycol-galactose used is 1:0.2-1.

[0038] Preferably, in the above technical solution, the cancer tissue block is a biopsy tissue block of a patient with a first-diagnosed gastric and gastroesophageal junction tumor.

[0039] Preferably, in the above technical solution, the tissue section treatment in step one is specifically as follows: bake the tissue section at 60-75°C for 15-45 minutes, then immerse the tissue section in xylene for 3-10 minutes, repeat the immersion for three times, and then take out the tissue section and immerse it in 95-100% alcohol, 90-95% alcohol, 75-85% alcohol, and water for 3-10 minutes respectively.

[0040] Preferably, in the above technical solution, the antigen retrieval solution used in step one is obtained by mixing citric acid and sodium citrate in water, the mass ratio of the amount of citric acid used to the amount of sodium citrate used is 1:6-9, and the amount of citric acid added to water is 0.0003-0.0005 g / ml.

[0041] Preferably, in the above technical solution, the endogenous enzyme inhibitor used in step one is at least one of 1-3% hydrogen peroxide and mixed buffer.

[0042] Preferably, in the above technical solution, the blocking agent used in step one is sheep serum.

[0043] Preferably, in the above technical solution, the amounts of the solvent, the antigen retrieval solution, the endogenous enzyme inhibitor, and the blocking agent used in step one are enough to completely immerse the tissue section.

[0044] Preferably, in the above technical solution, the first antibody used in step two is diluted MHC Class II recombinant rabbit monoclonal antibody, which is obtained by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.002-0.015.

[0045] Preferably, in the above technical solution, the second antibody used in step two is an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

[0046] Preferably, in the above technical solution, the use amount of the first antibody, the second antibody and the enhancer in step two is to completely immerse the tissue section.

[0047] Preferably, in the above technical solution, the developing solution used in step three is DAB developing solution, and the counterstaining solution is hematoxylin.

[0048] Preferably, in the above technical solution, the anti-blue solution used in step three is obtained by mixing sodium bicarbonate in water, and the amount of sodium bicarbonate added to water is 0.001-0.003g / ml.

[0049] Preferably, in the above technical solution, the use amount of the developing solution, the counterstaining solution and the anti-blue solution in step three is to completely immerse the tissue section complex.

[0050] Preferably, in the above technical solution, the solvent used in the dehydration process in step three is 95-100% alcohol, 90-95% alcohol and 75-85% alcohol, and the processing time is 1-5min.

[0051] Preferably, in the above technical solution, after each processing is completed, the section needs to be washed and spun dry, that is, the section is completely immersed in phosphate buffer for 3-10min and spun dry, and the section includes the tissue section, the processed tissue section and the tissue section complex.

[0052] Preferably, the application discloses a preparation method of a hydrazine derivative, specifically:

[0053] The 1-hydroxy-2-naphthoic acid is added into methanol, and then concentrated sulfuric acid is added dropwise, and refluxed for 10-15 hours under nitrogen, and then kept at 2-6℃ for 10-15 hours to obtain methyl 1-hydroxy-2-naphthoate. Then the methyl 1-hydroxy-2-naphthoate is added into methanol with NH2NH2·H2O, and refluxed for 1-5 hours at 85-97℃, and then cooled to 23-27℃, and then kept at 2-6℃ for 10-15 hours to obtain 1-hydroxy-2-naphthohydrazide. Finally, the 1-hydroxy-2-naphthohydrazide is added into a mixed solution of acetic anhydride and CHCl3 at 0-4℃, and then warmed to 23-27℃ for 20-28 hours, and then kept at 2-6℃ for 10-15 hours to obtain the hydrazine derivative.

[0054] Preferably, in the preparation of the hydrazine derivative, the amount of 1-hydroxy-2-naphthoic acid added into methanol is 0.1-0.15 g / ml.

[0055] Preferably, in the preparation of the hydrazine derivative, the mass ratio of the amount of concentrated sulfuric acid used to the amount of 1-hydroxy-2-naphthoic acid used is 1:0.25-0.75.

[0056] Preferably, in the preparation of the hydrazine derivative, the mass ratio of the amount of methyl 1-hydroxy-2-naphthoate used to the amount of NH2NH2·H2O used is 1:2-3.5.

[0057] Preferably, in the preparation of the hydrazine derivative, the amount of methyl 1-hydroxy-2-naphthoate in methanol is 0.075-0.1 g / ml.

[0058] Preferably, in the preparation of the hydrazine derivative, the mass ratio of the amount of 1-hydroxy-2-naphthohydrazide used to the amount of acetic anhydride used is 1:0.45-0.7, and the amount of acetic anhydride in the mixed solution of acetic anhydride and CHCl3 is 0.01-0.025 g / ml.

[0059] Preferably, in the immunohistochemical staining method of the present application, glycerol polyoxyethylene ether cocoate buffer solution can be added to replace sheep serum based on the use of hydrazine derivative and amino-polyethylene glycol-galactose mixed buffer solution. The glycerol polyoxyethylene ether cocoate buffer solution effectively reduces and eliminates non-specific sites, prevents non-specific interaction of antibodies or detection reagents with these non-specific sites, thereby improving the specificity of staining, reducing background staining, allowing the antibody to only bind to the target antigen, thereby improving the sensitivity and specificity of staining.

[0060] Preferably, the amount of mixed buffer solution and glycerol polyoxyethylene ether cocoate buffer solution used is to completely immerse the tissue section.

[0061] Compared with the prior art, the present application has the following beneficial effects:

[0062] The application establishes an immunohistochemical staining section preparation method, specifically comprising the following steps: step one, pre-treatment: including baking, deparaffinization and rehydration of tissue sections, and subsequent addition of antigen solution repair, addition of endogenous enzyme inhibitor for incubation, addition of blocking agent for blocking, etc.; step two, antibody incubation: addition of first and second antibodies for incubation; step three, post-treatment: including staining, re-staining, counterstaining and mounting, etc. The mixed buffer is used as the endogenous enzyme inhibitor, which effectively reduces the background staining intensity and improves the sensitivity and specificity of the staining; meanwhile, the staining result interpretation standard is established, which confirms that the MHC-II expression is significantly related to the pathological remission of gastric cancer patients, and provides an evaluation method for the pathological remission of gastric cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can be obtained from the provided drawings without creative labor.

[0064] Figure 1 It is a schematic diagram of the staining result interpretation standard;

[0065] Figure 2 It is a MHC II expression efficiency diagram. DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0067] The concepts involved in the present application will be described below in combination with the drawings. It should be pointed out here that the following descriptions of the concepts are only to make the content of the present application easier to understand, and do not represent the limitation of the protection scope of the present application; meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0068] Embodiment 1:

[0069] An immunohistochemical staining section preparation method:

[0070] Step one: preliminary treatment, cut the biopsy tissue wax block section of the patient with gastric and gastroesophageal junction tumor as the first diagnosis, obtain the tissue section, bake the tissue section at 72℃ for 20 min, then immerse in xylene for 5 min, repeat the immersion for three times, after the immersion is completed, take out the tissue section and immerse in 100% alcohol, 95% alcohol, 80% alcohol and pure water for 3 min respectively. Then take out the tissue section and immerse in antigen repair solution for high pressure repair for 90 s, after the repair is completed, wash the tissue section, after washing, immerse the tissue section in phosphate buffer for 5 min. Then shake dry the tissue section, add 3% hydrogen peroxide and incubate for 10 min, after incubation, wash the tissue section, after washing, immerse the tissue section in phosphate buffer for 5 min. Then take out the tissue section, add sheep serum and block for 10 min, after blocking, obtain the treated tissue section. The antigen repair solution is obtained by mixing citric acid, sodium citrate and water, the mass ratio of the use amount of citric acid to the use amount of sodium citrate is 1:7.5, the amount of citric acid added in water is 0.0004 g / ml, the use amount of 3% hydrogen peroxide and sheep serum is to completely immerse the tissue section.

[0071] Step two: antibody incubation, add diluted MHC Class II recombinant rabbit monoclonal antibody to the treated tissue section, incubate at 4℃ for 8 h. After the reaction is completed, wash the treated tissue section, then immerse the treated tissue section in phosphate buffer for 5 min. Then shake dry the treated tissue section, immerse in enhancement solution, place at 25℃ for 20 min, then wash, immerse in phosphate buffer for 5 min. After the immersion is completed, shake dry the treated tissue section, add enhancement enzyme-labeled goat anti-rabbit IgG polymer antibody to the treated tissue section, incubate at 25℃ for 20 min, after the reaction is completed, wash, immerse in phosphate buffer for 5 min to obtain the tissue section complex. The use amount of diluted MHC Class II recombinant rabbit monoclonal antibody and enhancement enzyme-labeled goat anti-rabbit IgG polymer antibody is to completely immerse the treated tissue section, the diluted MHC Class II recombinant rabbit monoclonal antibody is obtained by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.01, the MHC Class II recombinant rabbit monoclonal antibody is purchased from Hangzhou Huaan Biotechnology Co., Ltd., the product number is ET1704-13, the enhancement solution and the enhancement enzyme-labeled goat anti-rabbit IgG polymer antibody are derived from a detection kit, the detection kit is purchased from Beijing Zhongsu Jinqiao Biotechnology Co., Ltd., the product number is PV-9001.

[0072] Step three: post-treatment, add DAB developing solution to the tissue section complex under light-proof condition, incubate at 25℃ for 3min, wash with water and spin dry. Add hematoxylin dyeing solution and soak for 2min, wash with water and spin dry. Soak in anti-blue solution for 2min and wash with water. Finally, soak in 100% alcohol, 95% alcohol, 80% alcohol and pure water for 3min each. Dry and seal with sealing agent. The anti-blue solution is obtained by mixing sodium bicarbonate in water, the amount of sodium bicarbonate added in water is 0.002g / ml, the amount of DAB developing solution, hematoxylin dyeing solution and anti-blue solution used is to completely immerse the treated tissue section, and the hematoxylin dyeing solution and sealing agent are purchased from Ningbo Tongsheng Biological Technology Co., Ltd.

[0073] Example 2

[0074] Preparation of hydrazine derivative: 1-hydroxy-2-naphthoic acid is added into methanol to obtain a mixture, then concentrated sulfuric acid is added dropwise into the mixture, and refluxed under nitrogen for 14h, and then placed at 4℃ for 12h to obtain methyl 1-hydroxy-2-naphthoate. Then methyl 1-hydroxy-2-naphthoate and NH2NH2·H2O are added into methanol, and refluxed at 95℃ for 3h, cooled to room temperature, and then placed at 4℃ for 12h to obtain 1-hydroxy-2-naphthohydrazide. Finally, 1-hydroxy-2-naphthohydrazide is added into a mixed solution of acetic anhydride and CHCl3 at 0℃, warmed to 25℃ for reaction for 24h, and then placed at 4℃ for 12h to obtain the hydrazine derivative. The amount of 1-hydroxy-2-naphthoic acid added into methanol is 0.12g / ml, the mass ratio of the amount of concentrated sulfuric acid to the amount of 1-hydroxy-2-naphthoic acid is 1:0.5, the mass ratio of the amount of methyl 1-hydroxy-2-naphthoate to the amount of NH2NH2·H2O is 1:2.5, the amount of methyl 1-hydroxy-2-naphthoate in methanol is 0.089g / ml, the mass ratio of the amount of 1-hydroxy-2-naphthohydrazide to the amount of acetic anhydride is 1:0.59, and the amount of acetic anhydride in the mixed solution of acetic anhydride and CHCl3 is 0.019g / ml.

[0075] A method for preparing an immunohistochemical staining section: in this embodiment, the preparation of the immunohistochemical staining section is compared with that in Example 1, the difference is that 3% hydrogen peroxide is replaced by a mixed buffer, the mixed buffer is obtained by dissolving a hydrazine derivative and amino-polyethylene glycol-galactose in a phosphate buffer, the amount of the hydrazine derivative in the phosphate buffer is 0.054g / ml, the mass ratio of the amount of the hydrazine derivative to the amount of amino-polyethylene glycol-galactose is 1:0.55, the amount of the mixed buffer used is to completely immerse the tissue section, and other conditions and parameters are the same as in Example 1.

[0076] Example 3

[0077] The preparation of the hydrazine derivative is the same as in Example 2.

[0078] A method for preparing an immunohistochemical staining section: the preparation of the immunohistochemical staining section in this embodiment is compared with that in Example 2, the difference is that the mass ratio of the amount of hydrazine derivative used and the amount of amino-polyethylene glycol-galactose used is 1:0.8, and other conditions and parameters are the same as in Example 2.

[0079] Example 4:

[0080] The preparation of hydrazine derivative is the same as in Example 2.

[0081] A method for preparing an immunohistochemical staining section: the preparation of the immunohistochemical staining section in this embodiment is compared with that in Example 2, the difference is that the mass ratio of the amount of hydrazine derivative used and the amount of amino-polyethylene glycol-galactose used is 1:0.25, and other conditions and parameters are the same as in Example 2.

[0082] Example 5:

[0083] The preparation of hydrazine derivative is the same as in Example 2.

[0084] A method for preparing an immunohistochemical staining section: the preparation of the immunohistochemical staining section in this embodiment is compared with that in Example 2, the difference is that the sheep serum is replaced by glycerol polyoxyethylene ether coconut acid ester buffer, and the amount of glycerol polyoxyethylene ether coconut acid ester in the phosphate buffer is 0.15 g / ml.

[0085] Example 6:

[0086] The preparation of hydrazine derivative is the same as in Example 2.

[0087] A method for preparing an immunohistochemical staining section: the preparation of the immunohistochemical staining section in this embodiment is compared with that in Example 2, the difference is that the sheep serum is replaced by glycerol polyoxyethylene ether coconut acid ester buffer, and the amount of glycerol polyoxyethylene ether coconut acid ester in the phosphate buffer is 0.25 g / ml.

[0088] Comparative Example 1:

[0089] A method for preparing an immunohistochemical staining section: the preparation of the immunohistochemical staining section in this embodiment is compared with that in Example 2, the difference is that no hydrazine derivative is used in the mixed buffer, and other conditions and parameters are the same as in Example 2.

[0090] Comparative Example 2:

[0091] The preparation of hydrazine derivative is the same as in Example 2.

[0092] A method for preparing an immunohistochemical staining section: the preparation of the immunohistochemical staining section in this embodiment is compared with that in Example 2, the difference is that no amino-polyethylene glycol-galactose is used in the mixed buffer, and other conditions and parameters are the same as in Example 2.

[0093] Experimental Example 1:

[0094] Evaluation of background staining intensity, the immunohistochemical staining sections prepared from Examples 1-6 and Comparative Examples 1-2 were evaluated for background staining intensity. "-": indicates no staining; "±": weakly positive and very weak locally; "+": indicates diffuse and weakly positive; "++": diffuse and moderately positive. "+++ ": diffuse and strongly positive.

[0095] Table 1 Results of evaluation of background staining intensity of immunohistochemical staining sections

[0096]

[0097] The results of background staining intensity are shown in Table 1. The background staining intensity of the immunohistochemical staining sections prepared by the immunohistochemical staining method of Example 2 is lower than that of Example 1, indicating that the use of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid effectively reduces the background staining intensity, thereby reducing the interference on subsequent interpretation. The background staining intensity of the immunohistochemical staining sections prepared by the immunohistochemical staining method of Example 5 is lower than that of Example 2, indicating that on the basis of the use of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid, the use of glycerol polyoxyethylene ether cocoate can further improve the accuracy and specificity of staining and reduce the background staining intensity. The background staining intensity of the immunohistochemical staining sections prepared by the immunohistochemical staining method of Comparative Examples 1 and 2 is higher than that of Example 2, indicating that hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid need to work together, and the use of either of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid alone has no significant effect on reducing the background staining intensity.

[0098] Experimental Example 2:

[0099] Evaluation of gastric cancer tumor pathology based on MHC II expression. First, the biopsy tissue blocks of patients with first diagnosis of gastric and gastroesophageal junction tumors were obtained, and immunohistochemical staining sections were obtained according to the preparation method of the immunohistochemical staining sections in Example 1, and the staining results were interpreted according to the interpretation criteria and staining results were divided into negative and positive expression populations. The interpretation criteria are as follows:

[0100] 0: no coloring or ≤10% of cancer cells showing incomplete, weak cell membrane staining;

[0101] 1+: >10% of cancer cells showing incomplete, weak cell membrane staining;

[0102] 2+: >10% of cancer cells showing weak to moderate intensity complete cell membrane staining, and ≤10% of cancer cells showing strong and complete cell membrane staining;

[0103] 3+: 10% of cancer cells present strong, complete and uniform cell membrane staining.

[0104] As shown in Figure 1 , negative expression is scored as 0 or 1+, and positive expression is scored as 2+ or 3+.

[0105] Then, pathological remission evaluation was performed on the negative and positive expression populations, respectively. 235 patients with gastric cancer who underwent radical resection after neoadjuvant therapy (chemotherapy + immunotherapy) were analyzed by chi-square test. Pathological complete remission rate (pCR) refers to the absence of residual tumor cells under an optical microscope (including lymph nodes, stage ypT0N0M0), and major pathological remission rate (mPR) refers to tumor regression grade (TRG) of 0 or 1. The pathological remission evaluation criteria are shown in Table 1.

[0106] Table 2. Tumor pathological remission evaluation criteria

[0107]

[0108] The evaluation results are shown in Figure 2 , and the expression level of MHC II is significantly correlated with pCR and mPR, indicating the potential importance of MHC II in the pathological remission evaluation of gastric cancer.

[0109] Example 3:

[0110] To detect the sensitivity and specificity, 50 paraffin blocks of biopsy tissues from patients with gastric and gastroesophageal junction tumors (gastric cancer group) and 50 paraffin blocks of tissues from patients without gastric and gastroesophageal junction tumors (non-gastric cancer group) were taken. Immunohistochemical staining sections were prepared according to the preparation method of immunohistochemical staining sections in Examples 1-6 and Comparative Examples 1-2. The staining results of the immunohistochemical staining sections were interpreted according to the interpretation criteria in Example 2, and the sensitivity and specificity were statistically calculated.

[0111] Table 3. Statistical calculation results of sensitivity and specificity

[0112]

[0113] The sensitivity and specificity statistical calculation results are shown in Table 3. The immunohistochemical staining sections prepared according to Example 2 are interpreted, and the sensitivity and specificity are statistically calculated. The sensitivity and specificity are higher than those of Example 1, indicating that the use of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid effectively improves the binding capacity with the target antigen; the sensitivity and specificity calculated according to Example 5 are higher than those of Example 2, indicating that on the basis of using hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid, the use of glycerol polyoxyethylene ether coconut oil acid ester can further reduce the background intensity and non-specificity of staining, thereby improving the sensitivity and specificity; the sensitivity and specificity calculated according to Comparative Examples 1 and 2 are lower than those of Example 2, indicating that hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid need to act together, and the use of any one of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid alone has no obvious effect on improving the sensitivity and specificity of staining.

[0114] The above-described examples and / or embodiments are merely used to illustrate the preferred examples and / or embodiments of the present application and do not limit the embodiments of the present application in any form. Any person skilled in the art can make some changes or modifications to other equivalent examples without departing from the scope of the technical means disclosed in the present application, and such changes or modifications should be considered as the same technical or embodiment as the present application.

[0115] The principles and embodiments of the present application are described using specific examples. The above example is only used to help understand the method and core idea of the present application. The above description is only the preferred embodiment of the present application. It should be noted that due to the limitation of language expression, there are infinite specific structures. For ordinary skilled persons in the art, some improvements, refinements or changes can be made without departing from the principles of the present application, and the above technical features can be combined in an appropriate manner. These improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be considered as the protection scope of the present application.

Claims

1. Use of a mixed buffer containing a hydrazine derivative and amino-polyethylene glycol-galactose in the preparation of an endogenous enzyme inhibitor, wherein the mass ratio of the amount of the hydrazine derivative to the amount of the amino-polyethylene glycol-galactose in the mixed buffer is 1:0.2-1; and the hydrazine derivative is prepared from 1-hydroxy-2-naphthoyl hydrazine and acetic anhydride, wherein the mass ratio of the amount of the 1-hydroxy-2-naphthoyl hydrazine to the amount of the acetic anhydride is 1:0.45-0.

7.

2. Use of a mixed buffer according to claim 1 for the preparation of an endogenous enzyme inhibitor, characterized in that: The mixed buffer further contains a phosphate buffer, and the amount of the hydrazine derivative in the phosphate buffer is 0.045-0.06 g / ml.

3. An immunostaining reagent for detecting MHC-II expression in a gastric cancer biopsy sample, comprising at least an endogenous enzyme inhibitor, wherein the endogenous enzyme inhibitor comprises a mixed buffer containing a hydrazine derivative and amino-polyethylene glycol-galactose, wherein the mass ratio of the amount of the hydrazine derivative to the amount of the amino-polyethylene glycol-galactose in the mixed buffer is 1:0.2-1; and the hydrazine derivative is prepared from 1-hydroxy-2-naphthoyl hydrazine and acetic anhydride, wherein the mass ratio of the amount of the 1-hydroxy-2-naphthoyl hydrazine to the amount of the acetic anhydride is 1:0.45-0.

7.

4. The immunostaining reagent for detecting MHC-II expression in a gastric cancer biopsy sample according to claim 3, characterized in that: The immunostaining reagent further comprises a deparaffinization and hydration treatment solution, which is xylene, 95-100% alcohol, 90-95% alcohol, or 75-85% alcohol.

5. The immunostaining reagent for detecting MHC-II expression in a gastric cancer biopsy sample according to claim 3, characterized in that: The immunostaining reagent further comprises an antigen retrieval solution, which is prepared by mixing citric acid and sodium citrate in water, wherein the mass ratio of the amount of the citric acid to the amount of the sodium citrate is 1:6-9, and the amount of the citric acid added to the water is 0.0003-0.0005 g / ml.

6. The immunostaining reagent for detecting MHC-II expression in a gastric cancer biopsy sample according to claim 3, characterized in that: The immunostaining reagent further comprises a blocking agent, which is goat serum.

7. The immunostaining reagent for detecting MHC-II expression in a gastric cancer biopsy sample according to claim 3, characterized in that: The immunostaining reagent further comprises an enhancer solution.

8. The immunostaining reagent for detecting MHC-II expression in a gastric cancer biopsy sample according to claim 3, characterized by: The immunostaining reagent further comprises a first antibody, which is a diluted MHC Class II recombinant rabbit monoclonal antibody, and the diluted MHC Class II recombinant rabbit monoclonal antibody is prepared by mixing a phosphate buffer and a MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.002-0.

015.

9. An immunostaining kit for detecting MHC-II expression in a gastric cancer biopsy sample, comprising the immunostaining reagent of any one of claims 3-8.

Images