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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。
OBJECTIVE: To explore the preparation and clonal screening of monoclonal antibodies (mbs) against murine leukocyte antigen (McAbs) with leukocyte as immunogen and to prepare the monoclonal antibody against mouse S100A9 natural protein and to identify the nature of the antibody. METHODS: M Abs were immunized with peripheral blood leukocytes of healthy human beings, and m Abs were prepared by hybridoma technology of B lymphocytes. The hybridoma cells were screened by nonspecific immunocytochemical method by immunocytochemistry and subcloned by limited dilution method. Immunoprecipitation-mass spectrometry MAb was used to identify the mAbs. The cell lines were screened by Western blot. The mouse ascites were induced by ascites method and purified by affinity chromatography. The titer and affinity were determined by ELISA. The specificity and cross Reactivity analysis, immunohistochemical staining of human breast cancer paraffin sections. Results: We obtained 35 immunocytochemical positive polyclonal cells and secreted 11 strains of murine monoclonal antibody against human S100A9 monoclonal antibody, preferably 1 strain for the preparation of ascites fluid and purification of the identified antibody. The antibody titer was 1: 3.18 × 10 ~ 5, The subclass was IgG1, the light chain was kappa chain, the purity of the antibody was above 95%, the affinity constant was 3.54 × 10 ~ 8L / mol, the cross-reactivity with S100A8 was 0.12%, almost no cross-reaction with S100A12 and S100A13, Identify S100A9 protein in human breast cancer tissue. CONCLUSION: The murine anti-human S100A9 monoclonal antibody is successfully prepared. The McAb has high titer, high affinity and high specificity. It can provide the basis for detecting the expression of S100A9 by immunohistochemistry.