鼠抗人S100A9天然蛋白单克隆抗体的制备与鉴定  

Development and identification of mouse anti-human S100A9 natural protein

作　　者：米丹阳 段睿[1] 宋军营[1] 孙向东[1] 张钟允[1] 闫敏[1] 袁永[1] 张振强[1] 张改平[2] 刘文第[1] 

机构地区：[1]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,郑州450046 [2]河南省农业科学院动物免疫学重点实验室,河南450002

出　　处：《中国免疫学杂志》2016年第10期1485-1490,共6页Chinese Journal of Immunology

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)

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。Objective： To prepare and identify the mouse anti-human monoclonal antibodies（ mAbs） using leukocytes as immunogens. Methods： The mice were immunized using human peripheral blood leukocytes. Then,use of B lymphocyte hybridoma technology preparation of m Abs,followed screening by immunocytochemistry and limited dilution. The secreted m Abs were identified by immunoprecipitation,mass spectrometry,Western blot,ELISA and immunohistochemistry. Results： The 35 positive polyclonal cells were obtained,of which 11 strains secreted m Abs against S100A9. And one strain was used to prepare monoclonal antibody. The purified m Ab against S100A9 were purified and identified as IgG1 subtype,with the titer,purity and affinity constant was 1 ∶ 3. 18 × 10~5,95% and3. 54× 10~8 L / mol,respectively. This m Ab generally had 0. 12 % crossed reactivity to S100A8,and showed little or no cross reactivity to S100A12 and S100A13. The prepared monoclonal antibodies can specifically recognizes the S100A9 antigen in human breast cancer tissues.Conclusion： Successful preparation of m Ab against S100A9,which can secrete specific m Ab against S100A9 protein with high titers and specificity have been established successfully,which laid the foundation for the immunology application.

关 键 词：白细胞 S100A9 单克隆抗体 免疫学特性 

分 类 号：R392.11[医药卫生—免疫学]