WO2014117332A1 - 免疫组化用二抗显色系统灵敏度、亲和力的测定方法 - Google Patents

免疫组化用二抗显色系统灵敏度、亲和力的测定方法 Download PDF

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WO2014117332A1
WO2014117332A1 PCT/CN2013/071120 CN2013071120W WO2014117332A1 WO 2014117332 A1 WO2014117332 A1 WO 2014117332A1 CN 2013071120 W CN2013071120 W CN 2013071120W WO 2014117332 A1 WO2014117332 A1 WO 2014117332A1
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secondary antibody
affinity
sensitivity
antibody
concentration
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王小亚
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Wang Xiaoya
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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica

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  • the present invention belongs to the field of biological pathological detection. Specifically, the present invention relates to a method for determining sensitivity and affinity of a secondary antibody colorimetric system for immunohistochemistry. Background technique
  • Immunohistochemistry is the basic principle of applying antigen-antibody reaction. It is used to identify and observe antigens (peptides and proteins) in tissue cells by chemically reacting the coloring reagents (fluorescein, enzymes, metal ions, isotopes) of labeled antibodies. , a technique for positioning, qualitative and quantitative research. The color development process is mostly carried out by the labeled second antibody. Immunohistochemistry is ultimately reflected in the secondary antibody, so the specificity and sensitivity of the secondary antibody determines the specificity and sensitivity of the detection process to some extent. How to make antibodies efficiently bind to antigen and amplify this signal is two important indicators for measuring immunohistochemistry. Around the requirements of signal amplification, new and new are coming out.
  • a representative secondary antibody amplification system mainly uses a biotin and avidin system, a polymer glucan carrier to bind a plurality of secondary antibodies and a horseradish enzyme system.
  • Different companies based on secondary antibody amplification products have different methods in immunohistochemistry due to different production methods. How to evaluate the sensitivity and affinity of secondary antibodies in the process of immunohistochemistry is important for the control of product indicators.
  • the main method currently evaluated is to make an artificial judgment based on the depth of color development on the pathological tissue, which is extremely random.
  • the present invention provides a method for evaluating the sensitivity and affinity of a secondary antibody color development system on a slide.
  • a matrix model of a primary antibody on a glass slide is used as a detection substrate for the secondary antibody, and the sensitivity and affinity of the immunohistochemical secondary antibody color development system are measured.
  • Different concentrations of primary antibodies that can be specifically immunoreactive with the secondary antibody are pre-spotted on a slide, vacuum dried, sealed with paraffin, and then subjected to dewaxing, hydration, repair, secondary antibody reaction, color development, etc. .
  • paraffin sections of different concentrations of primary antibodies that have been embedded are placed on the slides, and then subjected to slicing, scraping, baking, dewaxing, hydration, repair, secondary antibody reaction, and color development.
  • the color rendering results were photographed, and the color rendering result of each spot was calculated by a software identification system to obtain the optical density value.
  • a method for determining sensitivity and affinity of a secondary antibody colorimetric system for immunohistochemistry comprising the following steps: pre-adsorbing a primary antibody capable of specifically immunoreactive with a secondary antibody at a concentration of different dilutions on a slide A concentration of a polypeptide is applied to a slide to form a small round spot; or the embedded different concentrations of primary paraffin sections are pre-placed on the slide and subjected to conventional immunohistochemistry simultaneously with the pathological tissue section.
  • the sensitivity and affinity of the secondary anti-chromogenic system of the immunohistochemical process are determined by the staining result of the specific primary antibody.
  • the pre-adsorption on the slide is adsorbed on the slide for immunohistochemistry by physical adsorption.
  • the conventional immunohistochemical procedure includes: drying, paraffin, dewaxing, hydration, repair, secondary antibody reaction, staining.
  • the paraffin effect is to spot the primary antibody on the slide and dry it, then add the slide to the melted paraffin to ensure that the spot is soaked in the paraffin, and then the slide is taken out to cool and solidify.
  • the staining result of the primary antibody means that the spot color of the primary antibody is gradually deepened from a low to a high concentration; and the pathological tissue section is observed by a microscope, and the lowest color of the target can be displayed according to the pathological tissue slice.
  • the concentration determination detects the lowest concentration of the primary antibody to determine the sensitivity of the secondary antibody detection system. If there is no obvious color reaction in the spot of one row of the primary anti-spotting concentration, and the spot of the previous concentration is colored and the color is visible to the naked eye, the concentration of the first concentration of the previous concentration of the concentration is the sensitivity of the detection secondary antibody system. value.
  • the specific method for determining the sensitivity and affinity of the secondary anti-chromogenic system of the immunohistochemical process by using the staining result of the specific primary antibody is as follows: photographing the staining result, and performing optical density measurement on the colored spots by using image software, by corresponding one Anti-concentration and optical density, calculate the affinity of the secondary antibody detection system and the primary antibody; when the primary and secondary antibodies show that the optical density value reaches 50% of the saturation value, the concentration of the primary antibody is the affinity of the secondary antibody colorimetric system.
  • the index expressed in g/L, measures the affinity of the secondary antibody by comparing the affinity index of the secondary antibody samples from different sources.
  • the chemical reagents are used to color the coloring reagents (fluorescein, enzymes, metal ions, isotopes) of labeled antibodies to determine and observe the antigens in tissue cells (polypeptides and Protein), a technique for localization, qualitative and quantitative research, how to effectively bind antibodies to antigens and amplify this signal is two important indicators for measuring immunohistochemistry.
  • tissue cells polypeptides and Protein
  • the detection system mainly uses biotin and avidin system, polymer glucan carrier to bind multiple secondary antibodies and horseradish enzyme system, etc., so the specificity and sensitivity of the secondary antibody detection system determine the detection process to some extent. Specificity and sensitivity. Different companies based on secondary antibody amplification detection products have different techniques and methods for production, and their results in immunohistochemistry applications are different. How to evaluate the sensitivity and affinity of the secondary antibody amplification detection system in the immunohistochemistry process is very important for the control of the product index. At present, the main method for the evaluation of secondary anti-magnification detection system is to make artificial qualitative judgment based on the depth of color development in pathological tissues, and the subjective influencing factors are large.
  • this work simulates the immunohistochemical process, immobilizing the primary antibody on the glass slide, and used it as a reference protein for the evaluation criteria of the secondary antibody in pathological tissue staining.
  • the evaluation process is objective, dataable, and accurate.
  • Figure 1 is a staining result of the sensitivity of different concentrations of murine antibody IgG detection secondary antibody colorimetric system in Example 1; wherein from top to bottom is 10 ⁇ ⁇ ⁇ , 2 g / L, 0.4 g / L, 0. 08 g/L, 0. 016 g/L, 0. 0032 g/L, 0. 00064 g/L concentration of murine antibody IgG, and the last row was 1 mg/mL of sheep serum negative control;
  • Figure 2 is a staining result of the sensitivity of the murine antibody IgG detection secondary antibody colorimetric system in Example 2; wherein, from top to bottom, 10 m g / 2 m g / L, 0.4 m / 0.08 m g / L, 0. 016m g / L, 0. 0032m g / 0. 00064m g / L concentration of murine antibody IgG, the last row is 1mg / mL of sheep serum negative control;
  • the mouse antibody IgG obtained by commercial means or obtained by cloning is diluted with l. lmol/L, pH 7.2 phosphate buffer (PBS, the same below) to l ( ⁇ g/L, 2 g/L, 0.4 g / L, 0.08 g / L, 0.016 g / L, 0.0032 g / L, 0.00064 g / L concentration, then in the upper part of the slide near the labeling site, using lmg / mL goat serum as a negative control, each row of points The first concentration of the same concentration of the first anti-spot, each spot 1 ⁇ ⁇ amount, according to the polypeptide concentration from low to high order. After the point is placed in a vacuum oven at 50 ° C for 30 min, removed and placed into the melt The wax solution was taken out for 1 min, cooled until the paraffin was solidified and stored.
  • PBS pH 7.2 phosphate buffer
  • xylene dewaxing and gradient alcohol dehydration xylene I 20min ⁇ xylene II 20min ⁇ 100% alcohol 10min ⁇ 100% alcohol 10min ⁇ 95% alcohol 5min ⁇ 80% alcohol 5min ⁇ 70% alcohol 5min
  • the primary antibody against ER was added dropwise, incubated overnight at 4 ° C in a refrigerator, and washed 3 times with PBS.
  • the sensitivity of the color development system is judged based on the difference in color development of the polypeptide spots. If there is no obvious color reaction in a row of spots, and the spot color of the previous concentration is discernible to the naked eye, the concentration of the first concentration of the previous concentration of the concentration is the sensitivity value of the detection secondary antibody system. In the spots of 10 ⁇ ⁇ ⁇ , 2 g/L, 0.4 g/L, 0.08 g/L, 0.016 g/L, 0.0032 g/L, 0.00064 g/L (Fig. 1), the spot color of 0.0064 g/L could not be judged.
  • the slides were photographed, and the optical density of each spot was analyzed by Photoshop.
  • the concentration was plotted on the abscissa and the optical density was plotted on the ordinate.
  • the concentration of the primary antibody at the time when the optical density value was saturated was 2 g/L. Secret When the degree is half of the saturation value, the corresponding concentration value is 0. 517 g/L. Then the sensitivity of the secondary antibody is
  • affinity index is 0. 517 g / L. Its affinity index is smaller than that of the common secondary antibody system.
  • Solid particles The obtained solid particles were subjected to the same procedure as the pathological tissue, and subjected to gradient alcohol dehydration (dehydration step: 80%, 90%, 95%, 100% ethanol in various concentrations for 2 hours), transparent with xylene , paraffin soaked, and finally paraffin embedded into paraffin specimens. Then, each point of paraffin (that is, IgG with different concentrations mentioned above) was used, and a wax block with a diameter of 3 mm and a length of about 5 mm was taken with a puncher.
  • dehydration step 80%, 90%, 95%, 100% ethanol in various concentrations for 2 hours
  • the sensitivity of the color development system is judged based on the difference in color development of the polypeptide spots. If there is no obvious color reaction in a row of spots, and the spot color of the previous concentration is discernible to the naked eye, the concentration of the first concentration of the previous concentration of the concentration is Detect the sensitivity value of the secondary antibody system. l ( ⁇ g/L, 2 g/L, 0.4 g/L, 0.08 g/L, 0.016 g/L, 0.0032 g/L, 0.00064 g/L in spots (Fig.
  • the detection sensitivity of the secondary antibody system is 0.016 g / L.
  • Figure 2 different concentrations of mouse antibody IgG detection II Dyeing results of sensitivity to color development system; 10m g/L, 2m g/L, 0.4mg/L, 0.08mg/L, 0.016mg/L, 0.0032mg/L, 0.00064mg/ from top to bottom L concentration of murine antibody IgG, the last row is 1 mg/mL sheep serum negative control;
  • the slides were photographed, and the optical density of each spot was analyzed by Photoshop.
  • the concentration was plotted on the abscissa and the optical density was plotted on the ordinate.
  • the concentration of the primary antibody at the time when the optical density value was saturated was 2 g/L. When the density value is half of the saturation value, the corresponding concentration value is 0.737 g/L. Then the sensitivity of the secondary antibody is
  • the affinity index was 0.737 g/L. Its affinity index is smaller than that of the common secondary antibody system.

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Abstract

本发明提供了一种免疫组化用二抗显色系统灵敏度、亲和力的测定方法，将能与二抗发生特异性免疫反应的一抗按不同稀释的浓度预先吸附在玻片上，一个浓度的一种多肽在玻片上经上样后形成一个小圆斑；或是将经过包埋的不同浓度的一抗石蜡切片预先放置在玻片上，与病理组织切片同时经历常规免疫组化操作步骤，以特定一抗的染色结果来判定免疫组化过程二抗显色系统的灵敏度及亲和力。

Description

免疫组化用二抗显色系统灵敏度、 亲和力的测定方法 技术领域：
本发明属于生物病理检测领域， 具体的， 本发明涉及一种免疫组化用二抗显 色系统灵敏度、 亲和力的测定方法。 背景技术
免疫组化技术是应用抗原抗体反应的基本原理， 通过化学反应使标记抗体 的显色剂 （荧光素、 酶、 金属离子、 同位素） 显色， 来确定、 观察组织细胞内抗 原 （多肽和蛋白质）， 对其进行定位、 定性及定量研究的一项技术。 其中显色过 程多为标记的第二抗体承担。免疫组化技术最终要在二抗上面体现， 因此二抗的 特异性及其灵敏度在一定程度上决定了检测过程的特异性和灵敏度。如何使抗体 能与抗原有效地结合并将此信号进行放大， 是衡量免疫组化技术的两项重要指 标。 围绕信号放大这一要求， 不断有新的问世。 目前具有代表性的二抗放大系统 主要用生物素与亲和素系统、 高分子葡聚糖载体结合多个二抗和辣根酶系统等。 不同公司基于二抗放大产品由于生产的方法不同， 其在免疫组化应用中各有差 异。 如何评价二抗在免疫组化过程中的灵敏度、亲和力， 对于产品的指标控制十 分重要。目前评价的主要方法是通过在病理组织上根据其显色的深浅进行人为的 判定， 具有极大的随意性。本发明提供了一种在玻片上评价二抗显色系统灵敏度 及其亲和力的方法。
发明内容
本发明的目的在于提供一种用于免疫组化过程进行二抗显色系统灵敏度和 亲和力测定的技术。
随着免疫组化技术在临床诊断和肿瘤转化诊断中的应用， 医院病理工作者 对免疫组化灵敏度的要求越来越高。选择高灵敏度、 高亲和力、 显色效果明显的 二抗系统， 对免疫组化的检测结果判断具有重要的作用。本发明中， 采用一抗在 载玻片上点样矩阵模型， 作为二抗的检测底物， 进行免疫组化二抗显色系统灵敏 度和亲和力的测定。 将可以与二抗发生特异性免疫反应的不同浓度的一抗预先在玻片上进行点 样， 真空干燥后， 加石蜡封闭， 然后经过脱蜡、 水化、 修复、 二抗反应、 显色等 步骤。 或是经过包埋的不同浓度一抗的石蜡切片预先放置在玻片上， 经过切片、 捞片、烤片、 脱蜡、水化、 修复、 二抗反应、显色等步骤。将显色结果进行拍照， 采用软件识别系统对每个点样点的显色结果进行计算， 获得其光密度值。
为实现上述目的， 本发明提供的技术方案如下：
一种免疫组化用二抗显色系统灵敏度、亲和力的测定方法， 所述测定方法包 括下列工艺步骤：将能与二抗发生特异性免疫反应的一抗按不同稀释的浓度预先 吸附在玻片上，一个浓度的一种多肽在玻片上经上样后形成一个小圆斑； 或是将 经过包埋的不同浓度的一抗石蜡切片预先放置在玻片上，与病理组织切片同时经 历常规免疫组化操作步骤，以特定一抗的染色结果来判定免疫组化过程二抗显色 系统的灵敏度及亲和力。
所述预先吸附在玻片上是以物理吸附的方式， 吸附在免疫组化用的玻片上。 所述常规免疫组化操作步骤包括： 干燥、 石蜡作用、 脱蜡、 水化、 修复、 二抗反应、 染色。
所述石蜡作用是将一抗点样在玻片上并干燥后， 将玻片加入熔化的石蜡中 浸泡， 保证点样位置浸泡于石蜡中， 然后将载玻片取出冷却固化。
所述一抗的染色结果， 是指一抗的斑点颜色从浓度由低到高表现为逐渐加 深的现象； 再通过显微镜对病理组织切片进行观察， 根据病理组织切片上靶位颜 色可显示的最低浓度判定检测一抗的最低浓度， 以确定二抗检测系统的灵敏度。 如某一排一抗点样浓度的斑点无明显的显色反应，而上一浓度的斑点显色且颜色 肉眼可辨， 则该浓度的上一浓度的一抗浓度为检测二抗系统的灵敏度值。
所述以特定一抗的染色结果来判定免疫组化过程二抗显色系统的灵敏度及 亲和力的具体方法为： 对染色结果进行拍照， 用图像软件对呈色斑点进行光密度 测量， 通过对应一抗浓度与光密度， 计算二抗检测系统与一抗的亲和力； 一抗与 二抗结合显示的光密度值达到饱和值 50%时， 其一抗的浓度即为该二抗显色系统 的亲和力指数， 以 g/L表示， 通过比较不同来源二抗样品的亲和力指数衡量二 抗的亲和力高低。
本发明的显著优点： 目前免疫组化方面还未有评价二抗质量的有效方法，更 未有产品面世。本发明提供了一种新型的可用于评价免疫组化用二抗的新技术与 方法， 本发明制备的产品， 用于与免疫组化染色过程一致， 应用简单方便。 免疫 组化（Immunohistochemistry)是应用抗原抗体反应的基本原理，通过化学反应使 标记抗体的显色剂 （荧光素、 酶、 金属离子、 同位素） 显色， 来确定、 观察组织 细胞内抗原 （多肽和蛋白质）， 对其进行定位、 定性及定量研究的一项技术， 如 何使抗体能与抗原有效地结合并将此信号进行放大，是衡量免疫组化技术的两项 重要指标。 围绕信号放大这一要求， 不断有新的技术问世。 目前最常用的是间接 法，通过多个酶标二抗与一抗的不同决定簇反应， 使每个靶点上有更多的酶分子 结合， 从而放大了信号， 具有代表性的二抗放大检测系统主要用生物素与亲和素 系统、高分子葡聚糖载体结合多个二抗和辣根酶系统等， 因此二抗检测系统的特 异性及其灵敏度在一定程度上决定了检测过程的特异性和灵敏度。不同公司基于 二抗放大检测产品由于生产的技术及方法有所不同，其在免疫组化应用的结果中 各有差异。 如何评价二抗放大检测系统在免疫组化过程中的灵敏度、 亲和力，对 于产品的指标控制十分重要。目前二抗放大检测系统评价的主要方法是通过在病 理组织上根据其显色的深浅进行人为定性的判定， 主观影响因素较大。针对二抗 放大检测系统在免疫组化应用的特点，本工作模拟免疫组化过程， 将一抗蛋白固 定于载玻片上， 作为参比蛋白用于病理组织染色中的二抗质控评价标准，评价过 程客观， 可数据化， 结果准确。 附图说明
图 1为实施例 1中不同浓度的鼠抗体 IgG检测二抗显色系统的灵敏度的染色结 果； 其中从上到下一次为 10μβΑ、 2 g/L、 0. 4 g/L、 0. 08 g/L、 0. 016 g/L、 0. 0032 g/L、 0. 00064 g/L浓度的鼠抗体 IgG， 最后一排为 lmg/mL的羊血清阴 性对照；
图 2为实施例 2中不同浓度的鼠抗体 IgG检测二抗显色系统的灵敏度的染色结 果；其中从上到下一次为 10m g/ 2m g/L、 0. 4m g/ 0. 08m g/L、 0. 016m g/L、 0. 0032m g/ 0. 00064m g/L浓度的鼠抗体 IgG， 最后一排为 lmg/mL的羊血清阴 性对照； 具体实施方式
下面通过具体实施示例对本发明的技术方案做进一步说明，但是 不能以此限制本发明的范围。
实施例 1:
通过商业途径购买或通过克隆方式获得的鼠抗体 IgG，用 0. lmol/L, pH7.2 的磷酸盐缓冲液（PBS， 下同）分别稀释成 l(^g/L、 2 g/L、 0.4 g/L、 0.08 g/L、 0.016 g/L、 0.0032 g/L、 0.00064 g/L的浓度， 然后在玻片上部靠近贴标签的 部位， 采用 lmg/mL山羊血清作为阴性对照， 每排点四个浓度一样的一抗斑点， 每个斑点点 1μβ量，按多肽浓度从低到高的顺序依次点样。点完后放在真空干燥 箱中 50° C干燥 30min， 取出后放入融化的蜡液中 lmin取出， 冷却至石蜡凝固 后保存。
检测时， 公认的二甲苯脱蜡， 梯度酒精脱水 （二甲苯 I 20min→二甲苯 II 20min→100%酒精 10min→100%酒精 10min→95%酒精 5min→80%酒精 5min→70% 酒精 5min)，PBS冲洗 3次，每次 5min;取出后正常羊血清工作液封闭， 37°C lOmin, 倾去勿洗。 滴加抗 ER的一抗， 4°C冰箱孵育过夜， PBS冲洗 3次。 每次 5min;加 入福州迈新生物科技开发有限公司购买的 Elivision二抗显色系统，根据二抗系 统说明书推荐的方式添加， 使其覆盖各个斑点，， 室温孵育 15min， 磷酸盐缓冲 液冲洗 3次， 每次 3min。 然后加入 DAB显色液， 室温孵育 5min， 自来水冲洗终 止显色。 经梯度酒精脱水干燥， 中性树胶封片后观察。
根据多肽斑点显色差异， 判断显色系统的灵敏度。 如某一排斑点无明显的 显色反应， 且上一浓度的斑点颜色肉眼可辨， 则该浓度的上一浓度的一抗浓度即 为检测二抗系统的灵敏度值。 10μβΑ、 2 g/L、 0.4 g/L、 0.08 g/L、 0.016 g/L、 0.0032 g/L、 0.00064 g/L的斑点中 （图 1)， 0.0064 g/L斑点颜色无法判断且 与阴性对照斑点无差异， 而 0.0032 g/L颜色肉眼可辨， 与阴性对照差异显著， 则该二抗系统的检测灵敏度为 0.0032 g/L。 图 1 不同浓度的鼠抗体 IgG检测二抗显色系统的灵敏度的染色结果； 其中从 上到下一次为 1(WL、 2 g/L、 0.4 g/L、 0.08 g/L、 0.016 g/L、 0.0032 g/L、 0.00064 g/L浓度的鼠抗体 IgG， 最后一排为 lmg/mL的羊血清阴性对照；
将玻片照相， 通过 Photoshop分析每个斑点的光密度， 以浓度为横坐标， 光密度值为纵坐标， 得出其光密度值饱和时的斑点的一抗浓度为 2 g/L， 其光密 度值为饱和值一半时对应的浓度值为 0. 517 g/L。 则该二抗的灵敏度为
0. 0032 g/L， 亲和力指数为 0. 517 g/L。 其亲和力指数小于普通二抗系统的
1. ( g/L。 说明该二抗显示系统的亲和力强 （亲和力指数越小， 表明一抗与二抗 的结合能力越强）。
实施例 2
称取 0. 2g脱脂奶粉，溶解于 lOmL双蒸水中，再分别加用 PBS稀释的 l(^g/L、 2 g/L、 0. 4 g/L、 0. 08 g/L、 0. 016 g/L、 0. 0032 g/L、 0. 00064 g/L的浓度小 鼠 IgG， 充分搅拌溶解。 再分别称取 0. 2g葡聚糖加入到上述溶液中， 搅拌后分 别加入 20 L乙酸， 不断搅拌使葡聚糖彻底溶解， 4°C放置除去葡聚糖溶液中的 气泡。 用一次性注射针头将葡聚糖溶液打到 1摩尔 /L的氢氧化钠溶液中固化， 形成白色固体颗粒。获得的固体颗粒进行与病理组织同样的程序， 经过梯度酒精 脱水 （脱水的步骤： 80%、 90%、 95%、 100%各种浓度的乙醇分别脱水 2小时），用 二甲苯透明，石蜡浸泡，最后石蜡包埋制作成石蜡标本。然后将每一点的石蜡（即 分别包埋有不同上述浓度的 IgG)， 用打孔器取直径为 3毫米、 长为 5毫米左右 的蜡块， 按照每个浓度一排放置 （每排 4个）， 共放置 7排， 最后靠近标签的一 排放置 lmg/mL的预先包埋的羊血清作为阴性对照， 点好样后将整个玻片放入融 化的石蜡中。等石蜡冷却后， 将放置有 8排石蜡条的石蜡块， 按照公认的标准的 程序各组织取一片进行切片、 载玻片贴片。 烤片， 68°C， 20min, 公认的二甲苯 脱蜡，梯度酒精脱水 仁甲苯 I 20min→二甲苯 II 20min→100%酒精 10min→100% 酒精 10min→95%酒精 5min→80%酒精 5min→70%酒精 5min )， PBS冲洗 3次， 每 次 5min; 取出后进行抗原修复： 置 0. 01M枸橼酸缓冲液（ΪΉ6. 0)中煮沸（95°C, 15-20min), 自然冷却 20min以上， 再用冷水冲洗缸子， 加快冷却至室温， PBS 冲洗 3次， 每次 5min。 正常羊血清工作液封闭， 37°C 10min， 倾去勿洗。 加入迈 新生物科技开发有限公司购买的 Maxviosn二抗显色系统， 根据二抗系统说明书 推荐的方式添加，使其覆盖各个斑点，室温孵育 15min，磷酸盐缓冲液冲洗 3次， 每次 3min。 然后加入 DAB显色液， 室温孵育 5min， 自来水冲洗终止显色。 经梯 度酒精脱水干燥， 中性树胶封片后观察。
根据多肽斑点显色差异， 判断显色系统的灵敏度。 如某一排斑点无明显的 显色反应， 且上一浓度的斑点颜色肉眼可辨， 则该浓度的上一浓度的一抗浓度为 检测二抗系统的灵敏度值。 l(^g/L、 2 g/L、 0.4 g/L、 0.08 g/L、 0.016 g/L、 0.0032 g/L、 0.00064 g/L的斑点中 （图 2)， 0.0032 g/L斑点颜色无法判断且 与阴性对照斑点无差异， 而 0.016 g/L颜色肉眼可辨， 与阴性对照差异显著，则 该二抗系统的检测灵敏度为 0.016 g/L。 图 2不同浓度的鼠抗体 IgG检测二抗显色系统的灵敏度的染色结果；其中从上 到下一次为 10m g/L、 2m g/L、 0.4m g/L、 0.08m g/L、 0.016m g/L、 0.0032m g/L、 0.00064m g/L浓度的鼠抗体 IgG， 最后一排为 lmg/mL的羊血清阴性对照；
将玻片照相， 通过 Photoshop分析每个斑点的光密度， 以浓度为横坐标， 光密度值为纵坐标， 得出其光密度值饱和时的斑点的一抗浓度为 2 g/L， 其光密 度值为饱和值一半时对应的浓度值为 0.737 g/L。 则该二抗的灵敏度为
0.016 g/L， 亲和力指数为 0.737 g/L。 其亲和力指数小于普通二抗系统的
1. (^g/L。 说明该二抗显示系统的亲和力强 （亲和力指数越小， 表明一抗与二抗 的结合能力越强）。

Claims

权利要求书
1、一种免疫组化用二抗显色系统灵敏度、亲和力的测定方法，其特征在于: 所述测定方法包括下列工艺步骤：将能与二抗发生特异性免疫反应的一抗按不同 稀释的浓度预先吸附在玻片上，一个浓度的一种多肽在玻片上经上样后形成一个 小圆斑； 或是将经过包埋的不同浓度的一抗石蜡切片预先放置在玻片上， 与病理 组织切片同时经历常规免疫组化操作步骤，以特定一抗的染色结果来判定免疫组 化过程二抗显色系统的灵敏度及亲和力。
2、根据权利要求 1所述的一种免疫组化用二抗显色系统灵敏度、 亲和力的 测定方法， 其特征在于： 所述预先吸附在玻片上是以物理吸附的方式， 吸附在免 疫组化用的玻片上。
3、根据权利要求 1所述的一种免疫组化用二抗显色系统灵敏度、 亲和力的 测定方法， 其特征在于： 所述常规免疫组化操作步骤包括： 干燥、 石蜡作用、脱 蜡、 水化、 修复、 二抗反应、 染色。
4、根据权利要求 2所述的一种免疫组化用二抗显色系统灵敏度、 亲和力的 测定方法， 其特征在于： 所述石蜡作用是将一抗点样在玻片上并干燥后， 将玻片 加入熔化的石蜡中浸泡，保证点样位置浸泡于石蜡中， 然后将载玻片取出冷却固 化。
5、根据权利要求 1所述的一种免疫组化用二抗显色系统灵敏度、 亲和力的 测定方法， 其特征在于： 所述一抗的染色结果， 是指一抗的斑点颜色从浓度由低 到高表现为逐渐加深的现象； 再通过显微镜对病理组织切片进行观察， 根据病理 组织切片上靶位颜色可显示的最低浓度判定检测一抗的最低浓度，以确定二抗检 测系统的灵敏度。
6、根据权利要求 1所述的一种免疫组化用二抗显色系统灵敏度、 亲和力的 测定方法，其特征在于： 所述以特定一抗的染色结果来判定免疫组化过程二抗显 色系统的灵敏度及亲和力的具体方法为： 对染色结果进行拍照， 用图像软件对呈 色斑点进行光密度测量，通过对应一抗浓度与光密度， 计算二抗检测系统与一抗 的亲和力；一抗与二抗结合显示的光密度值达到饱和值 50%时，其一抗的浓度即 为该二抗显色系统的亲和力指数， 以 g/L表示， 通过比较不同来源二抗样品的
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