鼠抗人S100A9天然蛋白单克隆抗体的制备与鉴定

发布时间：2018-10-17 11:34

【摘要】：目的:探索以白细胞为免疫原的鼠抗人白细胞天然蛋白单克隆抗体(monoclonal antibodies,m Abs)制备与克隆筛选鉴定方法,制备鼠抗人S100A9天然蛋白单克隆抗体,并鉴定抗体性质。方法:用健康人外周血白细胞免疫小鼠,利用B淋巴细胞杂交瘤技术制备m Abs,通过免疫细胞化学法对杂交瘤细胞进行非特异性阳性筛选,有限稀释法进行亚克隆,应用免疫沉淀-质谱法进行mAb特异性鉴定,通过Western blot进行细胞株筛选,小鼠体内诱生腹水法制备单抗,亲和层析法纯化,ELISA间接法测定效价及亲和力,Western blot进行特异性鉴定及交叉反应性分析,免疫组化染色人乳腺癌石蜡切片。结果:获得免疫细胞化学检测阳性多克隆细胞35孔,分泌鼠抗人S100A9蛋白单克隆抗体细胞株11株,优选1株制备腹水并纯化鉴定抗体,抗体效价为1∶3.18×10~5,亚类为IgG1,轻链为kappa链,抗体纯度达95%以上,亲和力常数3.54×10~8L/mol,与S100A8的交叉反应率为0.12%,与S100A12和S100A13几乎无交叉反应,组化染色识别人乳腺癌组织中的S100A9蛋白。结论:成功制备鼠抗人S100A9天然蛋白单克隆抗体,该单抗具有高效价、较高亲和力和高特异性,能为其应用于免疫组化检测S100A9蛋白的表达提供依据。
[Abstract]:Objective: to investigate the preparation and clone screening of murine anti-human leukocyte natural protein monoclonal antibody (monoclonal antibodies,m Abs) with leukocyte as immunogen, to prepare mouse anti-human S100A9 natural protein monoclonal antibody and to identify its properties. Methods: the mice were immunized with peripheral white blood cells (PBWs) of healthy persons. M Abs, was prepared by using B lymphocyte hybridoma technique. The hybridoma cells were screened by immunocytochemistry and subcloned by limited dilution method. The specific mAb was identified by immunoprecipitation-mass spectrometry, the cell line was screened by Western blot, and the monoclonal antibody was prepared by inducing ascites in mice. Affinity chromatography was used to purify, ELISA indirect method was used to determine the titer and affinity of, Western blot for specific identification and cross-reactivity analysis, and paraffin sections of human breast cancer were stained with immunohistochemistry. Results: the immunocytochemical analysis showed that the positive polyclonal cells were 35 well, and 11 mouse anti-human S100A9 protein monoclonal antibody cell lines were secreted. One strain was selected to prepare ascites and to purify and identify the antibody. The antibody titer was 1: 3.18 脳 10 ~ (5), and the subclass was IgG1, light chain as kappa chain. The purity of the antibody was over 95%, the affinity constant was 3.54 脳 10 ~ (-1) L / mol, the cross-reaction rate with S100A8 was 0.12 and there was almost no cross-reaction with S100A12 and S100A13. The S100A9 protein in human breast cancer tissue was identified by histochemical staining. Conclusion: the monoclonal antibody against human S100A9 protein was successfully prepared. The monoclonal antibody has high titer, high affinity and high specificity. It can be used to detect the expression of S100A9 protein by immunohistochemistry.
【作者单位】：河南中医药大学中医药免疫学实验室/郑州市抗体组学重点实验室;河南省农业科学院动物免疫学重点实验室;
【基金】：2015年度河南省科技创新人才计划(No.154100510019) 河南中医学院创新团队(No.2014XCXTD01);河南中医学院基本科研业务专项课题(No.2014KYYWF-ZZCX3-06)
【分类号】：R392

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