WO2013078650A1 - 一种抗原修复液及抗原修复方法 - Google Patents

一种抗原修复液及抗原修复方法 Download PDF

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WO2013078650A1
WO2013078650A1 PCT/CN2011/083255 CN2011083255W WO2013078650A1 WO 2013078650 A1 WO2013078650 A1 WO 2013078650A1 CN 2011083255 W CN2011083255 W CN 2011083255W WO 2013078650 A1 WO2013078650 A1 WO 2013078650A1
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Prior art keywords
soil
antigen retrieval
antigen
solution
itaconic
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PCT/CN2011/083255
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English (en)
French (fr)
Inventor
王小亚
熊玉林
林齐心
Original Assignee
Wang Xiaoya
Xiong Yulin
Lin Qixin
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Application filed by Wang Xiaoya, Xiong Yulin, Lin Qixin filed Critical Wang Xiaoya
Priority to CN2011800241360A priority Critical patent/CN103282778A/zh
Priority to PCT/CN2011/083255 priority patent/WO2013078650A1/zh
Publication of WO2013078650A1 publication Critical patent/WO2013078650A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase

Definitions

  • the present invention relates to the field of biomedicine, and in particular to an antigen retrieval solution for exposing antigens shielded by aldehyde fixation in tissue cells, ie, immunohistochemistry or immunity Antigen repair process during cytochemical staining.
  • an antigen retrieval solution for exposing antigens shielded by aldehyde fixation in tissue cells, ie, immunohistochemistry or immunity Antigen repair process during cytochemical staining.
  • the object of the present invention is to provide an antigenic repair solution and an antigen retrieval method which are effective, widely used, non-toxic, non-irritating odor, non-corrosive, and used for exposure to immunohistochemistry or immunocytochemical staining.
  • An aldehyde-immobilized and shielded antigen is an antigenic repair solution and an antigen retrieval method which are effective, widely used, non-toxic, non-irritating odor, non-corrosive, and used for exposure to immunohistochemistry or immunocytochemical staining.
  • the present invention first provides an antigen retrieval solution which is an aqueous solution of itaconic acid, itaconic anhydride or citraconic acid.
  • the concentration of the itaconic acid, itaconic anhydride or citraconic acid aqueous solution is 0.00001 to 1.0 mol/L, and H is 1.0 to 12.0.
  • the concentration of the itaconic acid, itaconic anhydride or citraconic acid aqueous solution is 0.0001 to 0.1 mol/L, and the pH is 5.0 to 10.0. More preferably, the concentration of the itaconic acid, itaconic anhydride or citraconic acid aqueous solution is 0.001 to 0.01 mol/L, and the pH is 7.0 8.0. Its antigen retrieval effect is stronger than or equal to EDTA and citrate antigen repair effect.
  • the itaconic acid, itaconic anhydride or citraconic acid aqueous solution is such that itaconic acid, itaconic anhydride or citraconic acid is dissolved in distilled water or deionized water.
  • the pH of the itaconic acid, itaconic anhydride or citraconic acid aqueous solution was adjusted with a 0.5 to 8 mol/L NaOH solution.
  • the present invention further provides an antigen retrieval method for performing high temperature or high temperature and high pressure repair on a tissue section using the above antigen retrieval solution.
  • the heating equipment used is a sterilizer, a pressure cooker, an electric cooker, a cooking pot, a microwave oven or a water bath.
  • the outstanding advantages of the present invention are as follows: (1) The antigen retrieval solution of the present invention has a stronger repairing effect than the conventional antigen retrieval solution (0.01 mol/L, pH 6.0 citrate buffer; 0.1 mol/L, pH 9.0). ⁇ pH10.0 Tris-HCl buffer containing 5% urea and EDTA solution of 0.01 mol/L, pH 8.0 ⁇ pH9.0, etc., the staining results show: deep staining, clear tissue morphology, accurate cell localization, no Background dyeing, wrinkle-free film and peeling, reproducibility and stability.
  • the antigen retrieval solution and antigen retrieval method provided by the invention not only have good reproducibility, safety, stability and applicability, but also have higher quality dyeing than other antigen retrieval liquids and antigen retrieval methods. As a result, it is of great significance to improve the reproducibility of clinical pathological diagnosis and reduce the possibility of misdiagnosis and missed diagnosis.
  • the present invention is a very effective antigen retrieval method in immunohistochemistry and immunocytochemistry. It is used to activate antigens that are blocked by aldehyde fixation in tissue cells so that they can immunoreact with the corresponding antibodies.
  • the water used to prepare the antigen retrieval solution should not contain any impurities, preferably deionized water or distilled water.
  • concentration of itaconic acid, itaconic anhydride or citraconic acid in the antigen retrieval solution may be 0.00001 to 1.0 mol/L.
  • the antigen retrieval solution may have a pH ranging from pH 1.0 to pH 12.0.
  • the concentration of the antigen retrieval solution ranges from 0.00001 to 1.0 mol/L, and the pH is ⁇ 1.0 to ⁇ 12.0.
  • the concentration of the antigen retrieval solution ranges from 0.00001 to 1.0 mol/L, and the pH is ⁇ 1.0 to ⁇ 12.0.
  • most of the pathological tissues are treated by aldehyde fixation and paraffin embedding. After aldehyde fixation, the proteins in the tissue cells form a "methylene bridge" crosslink with the aldehyde, thereby shielding the antigenic epitope. Make it impossible to immunoreact with the corresponding antigen.
  • the antigen retrieval step may be after dewaxing, prior to treatment with the endogenous peroxidase blocker; or after the endogenous peroxidase blocker treatment, before the primary antibody is added.
  • Antigen retrieval can take sufficient time to destroy the "methylene bridge" crosslinks caused by aldehyde fixation without destroying the tissue morphology and intracellular antigens.
  • the time used depends on the temperature, the concentration of the antigen retrieval solution, the time of aldehyde fixation, and the thickness of the tissue section.
  • Subjects were formaldehyde-fixed-paraffin-embedded lymphoma, tonsil, malignant mesothelioma, lung adenocarcinoma, thyroid cancer, esophageal squamous cell carcinoma, breast cancer, lung cancer, chronic lymphadenitis, Hodgkin's lymphoma, breast invasive catheter Cancer, lung squamous cell carcinoma, colonic tubular adenocarcinoma tissue (XX Hospital Pathology).
  • the target antigens detected are: CD4 CD8 CD10 CD15 CD30 Bcl-6 WT1 COX2 CyclinDl TTF-1 Ki-67 PCNA ER P21 D2-40 Bob-l p53 ERCC1 TOPO II MLH1 (The target antigens listed are not one-to-one correspondence with tissues) Because a tissue can express multiple target antigens, one target antigen can also be expressed in a variety of tissues).
  • Paraffin tissue sections were subjected to antigen retrieval by conventional xylene dewaxing, gradient alcohol hydration, and tap water washing.
  • the antigen retrieval step is as follows: The slice is placed in an appropriate amount of itaconic acid, itaconic anhydride or citraconic acid antigen repair solution (800-1500 mL, concentration 0.01 mol/L, pH ⁇ 1.0 ⁇ 5.0, respectively). ⁇ 6.0 ⁇ 7.0 ⁇ 8.0 ⁇ 9.0 ⁇ . ⁇ , ⁇ 12.0) heated in a cooking pot at 100 ° C for 20 minutes Or treat at 121 °C for 1.5 minutes in a pressure cooker.
  • a positive control and a negative control were set up, wherein the positive control was divided into EDTA repair (the slice was placed in an EDTA solution at pH 9.0 and heated at 100 ° C for 20 minutes) and the citric acid was repaired (the slice was placed in a citric acid solution at pH 6.0). Heated at 121 °C for 1.5 minutes); the tissue sections in the negative control were not subjected to antigen retrieval; the test solution used for each antigen repair was the same as the positive control. After the antigen retrieval was completed, the peroxidase blocker was added, incubated at room temperature for 10 minutes, and washed with PBS for 3 X 3 minutes.
  • Positive control # is 50 eight repair, positive control ⁇ is citric acid repair. The negative control was not repaired. -: no staining; +: positive; ++: medium positive; +++: strong positive. Soil: The positive intensity is less than +.
  • the experimental results showed that the itaconic acid, itaconic anhydride or citraconic acid antigen repair solution had antigenic repairing effects in the range of pH 1.0 ⁇ 12.0, and almost all pH values obtained a certain degree of staining results.
  • the antigen retrieval solution has good antigen retrieval effect in the range of pH 6.0 ⁇ pH9.0.
  • the immunohistochemical staining degree of almost all tested antigens is stronger than or equal to the control, and the tissue morphology is clear, the cell morphology is complete, and there is no background staining. , cell positioning preparation, is a good antigen repair solution.
  • Example 2 Example 2:
  • Subjects were formaldehyde-fixed-paraffin-embedded lymphoma, tonsil, malignant mesothelioma, lung adenocarcinoma, thyroid cancer, esophageal squamous cell carcinoma, breast cancer, lung cancer, chronic lymphadenitis, Hodgkin's lymphoma, breast invasive catheter Cancer, lung squamous cell carcinoma, colonic tubular adenocarcinoma tissue (XX Hospital Pathology).
  • the target antigens detected were: CD4, CD8, CD10, CD15, CD30, Bcl-6, WT1, COX2, CyclinDl, TTF-1, Ki-67, PCNA, ER, P21, D2-40, Bob-1, p53 , ERCC1, TOPO II MLH1.
  • the target antigens listed do not correspond to tissues one-to-one because one tissue can express multiple target antigens, and one target antigen can also be expressed in multiple tissues).
  • Paraffin sections were subjected to antigen retrieval by conventional xylene dewaxing, gradient alcohol hydration, and tap water washing.
  • the antigen retrieval step is as follows: The slice is placed in an appropriate amount of itaconic acid, itaconic anhydride or citraconic acid antigen repair solution (800-1500 mL, pH 8.0, the concentration is 0.00001, 0.0001, 0.001, 0.01, respectively). 0.1, 1.0 mol/L) Heated at 100 ° C for 20 minutes in a cooking pot or at 121 ° C for 1.5 minutes in a pressure cooker.
  • a positive control and a negative control were set up, wherein the positive control was divided into EDTA repair (the slice was placed in an EDTA solution at pH 9.0 for 10 minutes in 10 CTC) and the citric acid was repaired (the slice was placed in a citric acid solution at pH 6.0 121 Heated at °C for 1.5 minutes); the tissue sections in the negative control were not subjected to antigen retrieval; the test solution used for each antigen repair was the same as the positive control. After the antigen retrieval was completed, the peroxidase blocker was added, incubated at room temperature for 10 minutes, and washed with PBS for 3 x 3 minutes.
  • the immunohistochemical staining degree of almost all tested antigens is stronger than or equal to the control, and the tissue morphology is clear, the cell morphology is complete, and there is no background staining. , cell positioning preparation, is a good antigen repair solution.
  • the itaconic acid, itaconic anhydride or citraconic acid antigenic repair solution with a concentration of 0.00001 ⁇ 1.0mol/L and pH1.0 ⁇ pH12.0 can be used for antigen retrieval; the concentration is 0.001 ⁇ 0.01mol/L,
  • the antigen retrieval solution with pH 7.0 ⁇ pH8.0 has the best effect.
  • CD4 monoclonal antibody, clone 4B12, is primarily used to identify T cell lymphoma and B cell lymphoma and T cell lymphoma subtypes.
  • CD8 monoclonal antibody, clone SP16, is primarily used to identify T cell lymphoma and B cell lymphoma and T cell lymphoma subtypes.
  • CD10 monoclonal antibody, clone 56C6, also known as the common acute lymphoblastic leukemia antigen, is mainly used to identify B cell lymphoma, follicular lymphoma and Butkitt's lymphoma.
  • CD15 monoclonal antibody, clone No. Carb-3, mainly used to identify Hodgkin's disease, thymoma and T-cell type B cell lymphoma.
  • CD30 monoclonal antibody, clone Ber-H2, is a transmembrane single-chain glycoprotein with a molecular weight of 120kDa. It is a receptor for cytokine ligand CD30L. It is mainly used to identify Hodgkin's disease, thymoma, and richness. T cell type B cell lymphoma, embryonal carcinoma and testicular seminoma.
  • Bcl-6 monoclonal antibody, clone P1F6+PG-B6p, is a proto-oncogene that has the function of inducing cell apoptosis. Mainly used to identify diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma and mucosa-associated lymphoma.
  • WT1 monoclonal antibody, clone No. WT49, is a nephroblastoma gene protein, mainly used for sporadic and Diagnosis of familial nephroblastoma.
  • COX2 monoclonal antibody, clone SP21, is an inducible enzyme that is significantly increased in 85-90% of human colon cancers.
  • CyclinDl monoclonal antibody, clone No. SP4, is mainly used to identify mantle cell lymphoma and chronic lymphocytic leukemia/lymphoma.
  • TTF-1 monoclonal antibody, clone 8G7G3/1, is mainly used to identify thyroid cancer, metastatic renal cell carcinoma, small cell lung cancer and Merkel cell carcinoma.
  • Ki-67 monoclonal antibody, clone No. MIB-1, is mainly used to identify high and low non-Hodgkin's disease lymphoma, Berget's lymphoma/leukemia and other lymphomas.
  • PCNA Monoclonal Antibody, clone No. PC10, is a 36kDa nuclear protein associated with the cell cycle, a protein essential for DNA synthesis, and serves as a major marker of cell proliferation index.
  • ER Monoclonal antibody, clone No. SP1
  • clone No. SP1 Monoclonal antibody, clone No. SP1
  • P21 WAF1 monoclonal antibody, clone No. DCS-60.2, is a downstream regulator of p53 protein and a tumor suppressor, mainly used in the study of various malignant tumors.
  • D2-40 Monoclonal antibody, clone No. D2-40, is mainly used to identify mesothelioma, adenocarcinoma, lymphatic endothelium and vascular endothelium.
  • Bob-1 A polyclonal antibody that is required for follicular germinal center formation and immunoglobulin production.
  • P 53 monoclonal antibody, clone DO-7, is a tumor suppressor gene, mainly used to identify uterine serous carcinoma, endometrial carcinoma, malignant mesothelioma, proliferative mesothelioma, pulmonary sarcoma and pneumonia tumor.
  • ERCC1 monoclonal antibody, clone 8F1
  • clone 8F1 is a highly conserved resecting ribozyme in the nucleotide excision repair pathway and may be one of the indicators for the use of cisplatin-assisted chemotherapy in patients.
  • TOPO II Mainly used for the study of drug resistance in various tumor cells.
  • MLH1 monoclonal antibody, clone G168-15, is a mismatch repair gene that can be used to screen for diseases such as hereditary nonpolyposis and rectal cancer.

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Abstract

提供一种抗原修复液和抗原修复方法，用于免疫组织和细胞的化学染色过程中的抗原修复。所述抗原修复液为衣康酸、衣康酸酐或柠康酸水溶液。所述抗原修复方法为采用上述抗原修复液对组织切片进行高温或高温高压修复。提供的抗原修复液和抗原修复方法具有染色质量高，可重现性、安全性、稳定性好的优点。

Description

一种抗原修复液及抗原修复方法 技术领域 本发明属于生物医学领域， 具体地， 本发明涉及一种抗原修复液， 用于暴露 组织细胞中因醛固定所屏蔽的抗原，即免疫组织化学或免疫细胞化学染色过程中 的抗原修复过程。 技术背景 自从 1991年石善溶教授发明了热抗原修复技术后， 该技术已在全世界得到 广泛的应用， 无数关于病理学和形态学的临床研究文献及著作得到发表。 目前， 最为常用的抗原修复液主要有三种： 0.01mol/L， pH6.0 的柠檬酸缓冲液； 0.1mol/L , pH9.0~pH10.0 含 5%尿素的 Tris-HCl 缓冲液以及 0.01mol/L， pH8.0~pH9.0 的 EDTA 溶液。 特殊情况下采用胰酶或胃酶修复。 少数采用 0.01mol/L, pH7.0含 5%尿素的柠檬酸缓冲液； 常用的抗原修复方法为将组织切 片浸泡于抗原修复液中加热至沸或高压处理适当的时间。但是目前的抗原修复液 对某些抗原的修复效果并不是最佳的， 染色强度也有待提高， 而且在操作过程中 往往要将大部分需要修复的组织切片采用不同的修复方式， 不仅操作繁琐， 而且 浪费试剂和时间。 发明内容 本发明的目的在于提供一种效果好、 应用广、 无毒、 无刺激性气味、 无腐蚀 性的抗原修复液及抗原修复方法，用于免疫组织化学或免疫细胞化学染色过程中 暴露因醛固定而屏蔽的抗原。
本发明首先提供了一种抗原修复液，所述抗原修复液为衣康酸、衣康酸酐或 柠康酸水溶液。
其中所述的衣康酸、 衣康酸酐或柠康酸水溶液的浓度为 0.00001~1.0mol/L， H为 1.0~12.0。
优选地， 所述的衣康酸、 衣康酸酐或柠康酸水溶液的浓度为 0.0001~0.1mol/L, pH为 5.0~10.0。 更优选地， 所述的衣康酸、 衣康酸酐或柠康酸水溶液的浓度为 0.001~0.01mol/L, pH为 7.0 8.0。其抗原修复效果强于或等于 EDTA和柠檬酸抗 原修复效果。
所述的衣康酸、衣康酸酐或柠康酸水溶液是将衣康酸、衣康酸酐或柠康酸溶 于蒸馏水或去离子水中。 用 0.5~8mol/L的 NaOH溶液调节衣康酸、 衣康酸酐或 柠康酸水溶液的 pH值。
本发明另外提供了一种抗原修复方法，采用上述的抗原修复液对组织切片进 行高温或高温高压修复。
采用的加热设备为灭菌锅、 高压锅、 电锅、 蒸煮锅、 微波炉或水浴锅。 本发明的突出优点在于： （1 )本发明的抗原修复液， 其修复效果强于常规的 抗原修复液（0.01 mol/L， pH6.0的柠檬酸缓冲液； 0.1 mol/L， pH9.0~pH10.0含 5%尿素的 Tris-HCl缓冲液以及 0.01 mol/L, pH8.0~pH9.0的 EDTA溶液等）， 染 色结果显示： 染色程度深、 组织形态清晰、 细胞定位准确、 无背景染色、 无皱片 及脱片现象、可重现性和稳定性好。（2) 目前大部分抗体都需采用 EDTA或柠檬 酸修复液分开进行抗原修复，而本发明采用的抗原修复液可对 95%以上的抗原一 起进行抗原修复， 而无需采用不同的抗原修复液分开进行抗原修复， 而且工作液 浓度很低。 因此， 不仅操作简单、 而且节约时间和试剂。 （3)衣康酸、 衣康酸酐 或柠康酸为粉末状、 易溶于水、 无毒、 无挥发性、 无刺激性气味和腐蚀性。
总之， 本发明提供的抗原修复液及抗原修复方法不仅具有良好的可重现性、 安全性、稳定性和应用性， 而且与其它抗原修复液及抗原修复方法相比， 具有更 高质量的染色结果，因此对提高临床病理诊断的重现性和减少误诊和漏诊的可能 性具有重要的意义。 具体实施方式 本发明是免疫组织化学和免疫细胞化学过程中非常有效的抗原修复方法。用 于激活组织细胞中因醛固定而屏蔽的抗原， 使之能与对应的抗体发生免疫反应。
配制抗原修复液所用的水不应含有任何杂质，最好是去离子水或蒸馏水。衣 康酸、 衣康酸酐或柠康酸在抗原修复液中浓度可以为 0.00001~1.0mol/L。
该抗原修复液 pH范围可以为 pH1.0~pH12.0。
特别地，该抗原修复液的浓度范围为 0.00001~1.0mol/L，pH为 ρΗ1.0~ρΗ12.0。 目前， 绝大部分病理组织都是经醛固定和石蜡包埋处理的， 醛固定后， 组织 细胞内的蛋白会和醛形成一种 "亚甲基桥"交联， 从而屏蔽了抗原表位， 使之不 能与相应抗原发生免疫反应。在加热的条件下， 衣康酸、衣康酸酐或柠康酸溶液 可以破坏这种 "亚甲基桥"交联， 使细胞内抗原重新暴露出来， 从而能够与相应 抗体发生免疫反应，进而可进行免疫组织化学或免疫细胞化学染色。抗原修复步 骤可以在脱蜡之后， 内源性过氧化物酶阻断剂处理之前； 也可以在内源性过氧化 物酶阻断剂处理之后， 加入一抗之前。
在不破坏组织形态和细胞内抗原的前提下，抗原修复可采取足够的时间来破 坏醛固定引起的 "亚甲基桥"交联。 所用的时间取决于温度、 抗原修复液浓度、 醛固定的时间和组织切片的厚度。
采用水浴锅加热， 60~80°C处理 12h以上或过夜。
采用水浴锅加热， 80~98°C处理 30~90min
采用水浴锅加热， 98~100°C处理 20~45min
采用电锅、 蒸煮锅、 微波炉等加热器加热， 10CTC处理 15~20min
采用高压锅加热， 121 °C处理 l~10min
下面结合具体实施例对本发明进行详细说明，以下实施例是为了进一步说明 本发明， 但不应视为限制本发明。
实施例 1 :
研究对象为甲醛固定-石蜡包埋的淋巴瘤、 扁桃体、 恶性间皮瘤、 肺腺癌、 甲状腺癌、 食道鳞癌、 乳腺癌、 肺癌、 慢性淋巴结炎、 霍奇金淋巴瘤、 乳腺浸润 性导管癌、 肺鳞癌、 结肠管状腺癌组织（X X医院病理科）。 检测的目标抗原有： CD4 CD8 CD10 CD15 CD30 Bcl-6 WT1 COX2 CyclinDl TTF-1 Ki-67 PCNA ER P21 D2-40 Bob-l p53 ERCC1 TOPO II MLH1 (所列出的目标抗原并非与组织一一对应， 因为一种组织可以表达多种目标抗 原， 一种目标抗原也可在多种组织中表达）。
石蜡组织切片通过常规二甲苯脱蜡、梯度酒精水化、 自来水冲洗后进行抗原 修复。抗原修复步骤为： 将切片置于适量已稀释成工作液的衣康酸、衣康酸酐或 柠康酸抗原修复液（800-1500mL， 浓度为 0.01mol/L pH分别为 ρΗ1.0 ρΗ5.0 ρΗ6.0 ρΗ7.0 ρΗ8.0 ρΗ9.0 ρΗΙΟ.Ο, ρΗ12.0)于蒸煮锅中 100°C加热 20分钟 或压力锅中 121 °C处理 1.5分钟。 同时设置阳性对照和阴性对照， 其中阳性对照 分为 EDTA修复 （将切片置于 pH9.0的 EDTA溶液中 100°C加热 20分钟） 和柠 檬酸修复（将切片置于 pH6.0的柠檬酸溶液中 121 °C加热 1.5分钟）； 阴性对照中 组织切片不进行抗原修复处理；其中每种抗原修复所用的测试液与阳性对照采用 加热方法相同。抗原修复完成后加入过氧化物酶阻断剂，室温孵育 10分钟， PBS 冲洗 3 X 3分钟。 加入一抗， 室温孵育 1小时， PBS冲洗 3 X 3分钟。 加入二抗， 室温孵育 15分钟， PBS冲洗 3 X 3分钟。 加入 DAB显色液， 室温孵育 5分钟， 自来水冲洗终止显色， 苏木素复染， PBS返蓝。 经梯度酒精脱水干燥， 二甲苯透 明， 中性树胶封片后显微镜检。 实验结果见表 1、 表 2、 表 3。 表 1 不同 pH值衣康酸抗原修复的 IHC染色结果 阳性 PH PH PH PH PH PH PH PH 阴性 对照 1.0 5.0 6.0 7.0 8.0 9.0 10.0 12.0 对照
CD4# +土 ± + +土 +土 ++ ++ + ± -
CD8# ++土 + +土 ++土 +++ +++ +++ ++土 -
CD10# ++土 + + ++土 +++ +++ +++ ++土 + -
CD15# ++土 + +土 ++土 ++土 ++土 ++土 ++土 -
CD30# ++ +土 ++ ++ ++ +土 -
Bcl-6# ++ + ++ ++ ++土 ++土 ++ -
WT1 # + +土 +土 ++ ++ +土 -
COX2# ++ + +土 ++ ++ ++ + -
CyclinDl # ++土 + +土 ++土 ++土 ++土 ++土 ++ + -
TTF-1 # ++ + +土 ++ ++ ++ ++ ++ + -
Ki-67 § ++土 +土 + ++土 +++ +++ +++ ++土 + -
PCNA § ++土 + + ++土 +++ +++ +++ ++ + -
ER § +++ + +++ +++ +++ +++ ++ - § ++ + ++ ++土 ++土 ++土 ++ -
D2-40 § ++ + ++ ++ ++土 ++ +土 -
Bob-1 § ++土 + + ++土 ++土 +++ +++ ++ + - P53 § ++土 + +土 ++土 ++土 +++ ++土 ++ -
ERCC1 § ++土 + +土 ++土 ++土 +++ ++土 ++ + -
TOPO II § ++土 + ++土 ++土 +++ ++土 +土 -
MLH1 § +土 ± + ++ ++ ++ ++ ++ ± - 表 2 不同 pH值衣康酸酐抗原修复的 IHC染色结果 阳性 PH PH PH PH PH PH PH PH 阴性 对照 1.0 5.0 6.0 7.0 8.0 9.0 10.0 12.0 对照
CD4# +土 ± + +土 +土 ++ ++ + ± -
CD8# ++土 + ++土 ++土 +++ ++土 ++土 + -
CD10# ++土 + + ++土 +++ +++ ++土 ++土 + -
CD15# ++土 + +土 ++土 ++土 ++土 ++土 ++土 -
CD30# ++ - +土 +土 ++ +土 +土 -
Bcl-6# ++ + ++ ++ ++土 ++ ++ -
WT1# + - +土 +土 ++ ++ +土 -
COX2# ++ + +土 ++ ++ ++ + -
CyclinDl # ++土 + +土 ++土 ++土 ++土 ++土 ++ + -
TTF-1 # ++ + +土 ++ ++ ++ ++ ++ + -
Ki-67 § ++土 + + ++土 +++ +++ ++土 ++土 +土 -
PCNA§ ++土 + + ++土 +++ +++ ++土 ++ -
ER§ +++ + +++ +++ +++ +++ ++ - § ++ + ++ ++土 ++土 ++土 ++ -
D2-40 § ++ + ++ ++ ++ ++ +土 -
Bob-1 § ++土 + + ++土 ++土 +++ ++土 ++ + - p53 § ++土 + +土 ++土 ++土 +++ ++土 ++ + -
ERCC1 § ++土 + +土 ++土 ++土 +++ ++土 ++ + -
TOPO II § ++土 + + ++土 ++土 +++ ++土 +土 -
MLH1 § +土 + ++ ++ ++ ++ ++ - 表 3 不同 pH值柠康酸抗原修复的 IHC染色结果 阳性 PH PH PH PH PH PH PH PH 阴性 对照 1.0 5.0 6.0 7.0 8.0 9.0 10.0 12.0 对照
CD4# +土 ± + +土 +土 ++ ++ + ± -
CD8# ++土 + +土 ++土 +++ +++ +++ ++土 -
CD10# ++土 + ++土 +++ +++ ++土 ++ + -
CD15# ++土 + +土 ++土 ++土 ++土 ++土 ++ -
CD30# ++ +土 ++ ++ +土 +土 - -
Bcl-6# ++ + ++ ++ ++土 ++土 ++ -
WT1 # + - + +土 +土 +土 ± -
COX2# ++ + +土 ++ ++ ++ + -
CyclinDl # ++土 + +土 ++ ++土 ++土 ++土 ++ + -
TTF-1 # ++ + +土 ++ ++ ++ ++ ++ + -
Ki-67 § ++土 + + ++土 +++ +++ +++ ++土 + -
PCNA § ++土 + + ++土 +++ +++ +++ ++ + -
ER § +++ + ++ +++ +++ ++土 ++ - § ++ + ++ ++土 ++土 ++土 ++ -
D2-40 § ++ + +土 ++ ++土 ++ +土 -
Bob-1 § ++土 + + ++土 ++土 +++ +++ ++ + - p53 § ++土 + +土 ++土 ++土 +++ ++土 ++ -
ERCC1 § ++土 + +土 ++土 ++土 +++ ++土 ++ + -
TOPO II § ++土 + + ++ ++土 +++ ++ +土 -
MLH1 § +土 + + +土 ++ ++ + - 备注： 阳性对照#为50了八修复， 阳性对照§为柠檬酸修复。 阴性对照为不修复。 -: 无染色； +: 阳性； ++: 中等阳性； +++: 强阳性。 土： 阳性强度小于 +。
实验结果显示，衣康酸、衣康酸酐或柠康酸抗原修复液在 pH1.0~pH12.0 范围内都具有抗原修复作用， 几乎所有的 pH值都获得了一定程度的染色结果。 该抗原修复液在 pH6.0~pH9.0范围内的抗原修复效果良好，几乎所有被测抗原的 免疫组化染色程度都强于或等于对照， 且组织形态清晰、细胞形态完整、无背景 染色、 细胞定位准备， 是一种良好的抗原修复液。 实施例 2:
研究对象为甲醛固定-石蜡包埋的淋巴瘤、 扁桃体、 恶性间皮瘤、 肺腺癌、 甲状腺癌、 食道鳞癌、 乳腺癌、 肺癌、 慢性淋巴结炎、 霍奇金淋巴瘤、 乳腺浸润 性导管癌、 肺鳞癌、 结肠管状腺癌组织（X X医院病理科）。 检测的目标抗原有： CD4、 CD8、 CD10、 CD15、 CD30、 Bcl-6、 WT1、 COX2、 CyclinDl、 TTF-1、 Ki-67、 PCNA、 ER、 P21醫、 D2-40、 Bob-l、 p53、 ERCC1、 TOPO II MLH1。 (所列出的目标抗原并非与组织一一对应， 因为一种组织可以表达多种目标抗 原， 一种目标抗原也可在多种组织中表达）。
石蜡组织切片通过常规二甲苯脱蜡、梯度酒精水化、 自来水冲洗后进行抗原 修复。抗原修复步骤为： 将切片置于适量已稀释成工作液的衣康酸、衣康酸酐或 柠康酸抗原修复液（800-1500mL， pH为 8.0，浓度分别为 0.00001、 0.0001、 0.001、 0.01、 0.1、 1.0mol/L) 于蒸煮锅中 100°C加热 20分钟或压力锅中 121 °C处理 1.5 分钟。 同时设置阳性对照和阴性对照， 其中阳性对照分为 EDTA修复（将切片置 于 pH9.0的 EDTA溶液中 10CTC加热 20分钟）和柠檬酸修复（将切片置于 pH6.0 的柠檬酸溶液中 121 °C加热 1.5 分钟）； 阴性对照中组织切片不进行抗原修复处 理； 其中每种抗原修复所用的测试液与阳性对照采用加热方法相同。抗原修复完 成后加入过氧化物酶阻断剂，室温孵育 10分钟， PBS冲洗 3 X 3分钟。加入一抗， 室温孵育 1小时， PBS冲洗 3 X 3分钟。加入二抗， 室温孵育 15分钟， PBS冲洗 3 X 3分钟。 加入 DAB显色液， 室温孵育 5分钟， 自来水冲洗终止显色， 苏木素 复染， PBS返蓝。经梯度酒精脱水干燥，二甲苯透明， 中性树胶封片后显微镜检。 实验结果见表 4、 表 5、 表 6。
表 4 不同浓度衣康酸抗原修复的 IHC染色结果 阳性 0.00001 0.0001 0.001 0.01 0.1 1.0 阴性 对照 mol/L mol/L mol/L mol/L mol/L mol/L 对照
CD4# +土 ± ± +土 +土 ++ + -
CD8# ++土 + +土 ++土 +++ +++ +土 -
CD10# ++土 + +土 ++土 ++土 +++ + -
CD15# ++土 + + ++土 ++土 ++土 + - CD30# ++ - +土 ++ ++ -
Bcl-6# ++ + ++ ++ ++土 -
WT1 # + - +土 ++ ++ -
COX2# ++ + +土 ++ ++ -
CyclinDl # ++土 +土 ++ ++土 ++土 + -
TTF-1 # ++ +土 ++ ++ ++ -
Ki-67 § ++土 + ++ ++土 +++ +++ +土 -
PCNA § ++土 + +土 ++土 +++ +++ + -
ER§ +++ +土 +++ +++ +++ + - § ++ + ++土 +++ ++土 -
D2-40 § ++ + ++土 +++ ++土 + -
Bob-1 § ++土 + + +++ ++土 +++ + - p53 § ++土 + ++ ++土 +++ +++ +土 -
ERCCl § ++土 + +土 +++ +++ +++ +土 -
TOPO II § ++土 + ++土 ++土 ++土 +土 -
MLH1 § +土 - ± ++ ++ ++ ± - 表 5 不同浓度衣康酸酐抗原修复的 IHC染色结果 阳性 0.00001 0.0001 0.001 0.01 0.1 1.0 阴性 对照 mol/L mol/L mol/L mol/L mol/L mol/L 对照
CD4# +土 ± ± +土 +土 ++ + -
CD8# ++土 + +土 ++土 ++土 ++土 +土 -
CD10# ++土 + +土 ++土 ++土 ++ + -
CD15# ++土 + + ++土 ++土 ++土 + -
CD30# ++ - +土 ++ ++ -
Bcl-6# ++ + ++ ++ ++土 -
WT1 # + - +土 ++ ++ -
COX2# ++ + +土 ++ ++ -
CyclinDl # ++土 + +土 ++ ++土 ++土 + -
TTF-1 # ++ + +土 ++ ++ ++ - Ki-67 § ++土 +土 ++ ++土 +++ +++ +土 -
PCNA § ++土 + +土 ++土 +++ +++ + -
ER§ +++ +土 +++ +++ ++土 + - § ++ + ++土 +++ ++土 -
D2-40 § ++ + ++土 +++ ++土 + -
Bob-1 § ++土 + + +++ ++土 ++土 + - p53 § ++土 + ++ ++土 +++ ++土 +土 -
ERCCl § ++土 + +土 +++ +++ ++土 +土 -
TOPO II § ++土 + ++土 ++土 ++土 +土 -
MLH1 § +土 ± ± ++ ++ ++ ± - 表 6 不同浓度柠康酸抗原修复的 IHC染色结果 阳性 0.00001 0.0001 0.001 0.01 0.1 1.0 阴性 对照 mol/L mol/L mol/L mol/L mol/L mol/L 对照
CD4# +土 ± + +土 +土 +土 + -
CD8# ++土 + +土 ++土 +++ ++土 +土 -
CD10# ++土 + + ++土 +++ ++土 + -
CD15# ++土 + +土 ++土 ++土 ++土 + -
CD30# ++ - +土 ++ ++ -
Bcl-6# ++ + ++ ++ ++土 -
WT1 # + +土 +土 ++ - -
COX2# ++ + +土 ++ ++ -
CyclinDl # ++土 + +土 ++土 ++土 ++土 + -
TTF-1 # ++ + +土 ++ ++ ++ -
Ki-67 § ++土 +土 + ++土 +++ ++土 +土 -
PCNA § ++土 + + ++土 +++ ++土 + -
ER§ +++ + +++ +++ +++ + - § ++ + ++ ++土 ++土 -
D2-40 § ++ + ++ ++ ++土 + - Bob-1 § ++土 + + ++土 ++土 ++ + - p53 § ++土 + +土 ++土 +++ ++土 +土 -
ERCC1 § ++土 + +土 ++土 ++土 ++ +土 -
TOPO II § ++土 + ++土 ++土 +++ +土 -
MLH1 § +土 + ++ ++ ++ 土 ― 备注： 阳性对照#为50了八修复， 阳性对照§为柠檬酸修复。 阴性对照为不修复。 -: 无染色； +: 阳性； ++: 中等阳性； +++: 强阳性。 土： 阳性强度小于 +。 实验结果显示，衣康酸、衣康酸酐或柠康酸抗原修复液在浓度为 0.00001~1.0 mol/L范围内都具有抗原修复作用， 几乎所有的浓度都获得了一定程度的染色结 果。 该抗原修复液浓度在 0.001~0.01mol/L范围内的抗原修复效果良好， 几乎所 有被测抗原的免疫组化染色程度都强于或等于对照， 且组织形态清晰、细胞形态 完整、 无背景染色、 细胞定位准备， 是一种良好的抗原修复液。
综上， 浓度为 0.00001~1.0mol/L， pH1.0~pH12.0的衣康酸、衣康酸酐或柠康 酸抗原修复液都可用于抗原修复； 其中浓度为 0.001~0.01mol/L， pH7.0~pH8.0的 抗原修复液效果最好。
所用一抗信息：
CD4: 单克隆抗体， 克隆号 4B12, 主要用于鉴别 T细胞淋巴瘤和 B细胞淋巴瘤 以及 T细胞淋巴瘤亚型的确认。
CD8: 单克隆抗体， 克隆号 SP16, 主要用于鉴别 T细胞淋巴瘤和 B细胞淋巴瘤 以及 T细胞淋巴瘤亚型的确认。
CD10: 单克隆抗体， 克隆号 56C6, 也称共同型急性淋巴细胞白血病抗原， 主要 用于鉴别 B细胞淋巴瘤、 滤泡性淋巴瘤和 Butkitt's淋巴瘤。
CD15: 单克隆抗体， 克隆号 Carb-3， 主要用于鉴别霍奇金氏病、 胸腺瘤和富于 T细胞型 B细胞淋巴瘤。
CD30: 单克隆抗体， 克隆号 Ber-H2， 是一种分子量 120kDa的跨膜单链糖蛋白， 是细胞因子配体 CD30L的受体， 主要用于鉴别霍奇金氏病、 胸腺瘤、 富于 T细 胞型 B细胞淋巴瘤、 胚胎性癌和睾丸精原细胞瘤。
Bcl-6: 单克隆抗体， 克隆号 P1F6+PG-B6p， 是一种原癌基因， 具有诱导细胞凋 亡的功能。 主要用于鉴别弥漫大 B 细胞淋巴瘤、 滤泡性淋巴瘤、 套细胞性淋巴 瘤和粘膜相关性淋巴瘤等。
WT1 : 单克隆抗体， 克隆号 WT49, 是肾母细胞瘤基因蛋白， 主要用于散发性和 家族性肾母细胞瘤的诊断。
COX2: 单克隆抗体， 克隆号 SP21 , 为诱导性酶， 在 85~90%的人结肠癌中表达 显著增高。
CyclinDl : 单克隆抗体， 克隆号 SP4， 主要用于鉴别套细胞淋巴瘤和慢性淋巴细 胞白血病 /淋巴瘤。
TTF-1 : 单克隆抗体， 克隆号 8G7G3/1 , 主要用于鉴别甲状腺癌、 转移性肾细胞 癌、 肺小细胞癌和 Merkel细胞癌。
Ki-67: 单克隆抗体， 克隆号 MIB-1 , 主要用于鉴别高度和低度非霍奇金氏病淋 巴瘤、 伯吉特淋巴瘤 /白血病和其它淋巴瘤。
PCNA: 单克隆抗体， 克隆号 PC10, 是和细胞周期相关的 36kDa的核蛋白， 是 细胞 DNA合成所必需的蛋白， 作为细胞增殖指数的主要标记物。
ER: 单克隆抗体， 克隆号 SP1， 主要用于鉴别乳腺癌激素水平、 乳腺来源转移 癌和其它部位来源转移癌。
P21WAF1: 单克隆抗体， 克隆号 DCS-60.2, 是 p53蛋白下游调控因子， 也是一种 肿瘤抑制因子， 主要用于各种恶性肿瘤的研究。
D2-40: 单克隆抗体， 克隆号 D2-40, 主要用于鉴别间皮瘤、 腺癌、 淋巴管内皮 和血管内皮。
Bob-1 : 多克隆抗体， 是滤泡生发中心形成和免疫球蛋白产生所必需的一种转录 活化因子。
P53: 单克隆抗体，克隆号 DO-7,是一种抑癌基因，主要用于鉴别子宫浆液性癌、 内膜样癌、 恶性间皮瘤、 增生性间皮、 肺肉瘤和肺炎性假瘤。
ERCC1 : 单克隆抗体， 克隆号 8F1 , 是核苷酸切除修复通路中高度保守的切除性 核酶， 可能成为患者是否应用顺铂辅助化疗的指标之一。
TOPO II : 主要用于各种肿瘤细胞耐药的研究。
MLH1 : 单克隆抗体， 克隆号 G168-15, 是一种错配修复基因， 可用于对遗传性 非息肉病性结、 直肠癌等疾病的筛选。

Claims

权利要求书
1. 一种抗原修复液， 其特征在于： 所述抗原修复液为衣康酸、 衣康酸酐或柠康 酸水溶液。
2. 根据权利要求 1所述的抗原修复液， 其特征在于， 所述的衣康酸、 衣康酸酐 或柠康酸水溶液的浓度为 0.00001~1.0mol/L， pH为 1.0~12.0。
3. 根据权利要求 1所述的抗原修复液， 其特征在于， 所述的衣康酸、 衣康酸酐 或柠康酸水溶液的浓度为 0.0001~0.1mol/L， pH为 5.0~10.0。
4. 根据权利要求 1所述的抗原修复液， 其特征在于， 所述的衣康酸、 衣康酸酐 或柠康酸水溶液的浓度为 0.001~0.01mol/L， pH为 7.0~8.0。
5. 根据权利要求 1所述的抗原修复液， 其特征在于， 所述的衣康酸、 衣康酸酐 或柠康酸水溶液是将衣康酸、 衣康酸酐或柠康酸溶于蒸馏水或去离子水中。
6. 根据权利要求 2、 3或 4所述的抗原修复液， 其特征在于， 用 0.5~8mol/L的 NaOH溶液调节衣康酸、 衣康酸酐或柠康酸水溶液的 pH值。
7. 一种抗原修复方法， 其特征在于： 采用权利要求 1-6中任一项所述的抗原修 复液对组织切片进行高温或高温高压修复。
8. 根据权利要求 7所述的抗原修复方法， 其特征在于， 采用的加热设备为灭菌 锅、 高压锅、 电锅、 蒸煮锅、 微波炉或水浴锅。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9506928B2 (en) 2013-12-12 2016-11-29 University Of Houston System Methods for retrieving antigens using aldehyde scavenging agents
US10203269B2 (en) 2013-12-12 2019-02-12 University Of Houston System Method of retrieving elements from fixed tissue
CN110506206A (zh) * 2017-03-25 2019-11-26 柏林夏瑞蒂医科大学 作为肾脏疾病的生物标志物的尿液流式细胞术

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116046503B (zh) * 2023-04-03 2023-06-23 迈杰转化医学研究(苏州)有限公司 一种能够增强特异性染色效果并降低染色背景的抗原修复液及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006007841A2 (en) * 2004-07-23 2006-01-26 Dakocytomation Denmark A/S Heat-induced antigen retrieval methods and compositions therefor
WO2009085574A2 (en) * 2007-12-28 2009-07-09 Spring Bioscience Corporation Microwave antigen retrieval in non-aqueous solvents

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3797418B2 (ja) * 2001-05-29 2006-07-19 茂樹 並松 抗原賦活化法及びその抗原賦活剤
WO2008095501A1 (en) * 2007-02-09 2008-08-14 Dako Denmark A/S Horizontal antigen retrieval

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006007841A2 (en) * 2004-07-23 2006-01-26 Dakocytomation Denmark A/S Heat-induced antigen retrieval methods and compositions therefor
WO2009085574A2 (en) * 2007-12-28 2009-07-09 Spring Bioscience Corporation Microwave antigen retrieval in non-aqueous solvents

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAUL, A.P. ET AL.: "A New Antigen Retrieval Technique for Human Brain Tissue", PLOS ONE, vol. 3, no. ISS.10, 13 October 2008 (2008-10-13), pages 1 - 7, XP055070671 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9506928B2 (en) 2013-12-12 2016-11-29 University Of Houston System Methods for retrieving antigens using aldehyde scavenging agents
JP2017502272A (ja) * 2013-12-12 2017-01-19 ユニバーシティー オブ ヒューストン システム 普遍的な抗原の賦活化化合物とその使用の方法
EP3080610A4 (en) * 2013-12-12 2018-02-28 University of Houston System Universal antigen retrieval compounds and methods of use
US10203269B2 (en) 2013-12-12 2019-02-12 University Of Houston System Method of retrieving elements from fixed tissue
CN110506206A (zh) * 2017-03-25 2019-11-26 柏林夏瑞蒂医科大学 作为肾脏疾病的生物标志物的尿液流式细胞术

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