鼠抗人S100A9 天然蛋白单克隆抗体的制备与鉴定

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,mAbs)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mAbs,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,应用免疫沉淀鄄质谱法进行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 3.18 105 ,亚类为IgG1,轻链为kappa 链,抗体纯度达95%以上,亲和力常数3.54 108 L/ 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。

Development and identification of mouse anti-human S100A9 natural protein

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 mAbs,followed screening by immunocytochemistry and limited dilution.The secreted mAbs were identified by immunoprecipitation,mass spectrometry,Western blot,ELISA and immunohistochemistry.Results: The 35 positive polyclonal cells were obtained,of which 11 strains secreted mAbs against S100A9.And one strain was used to prepare monoclonal antibody.The purified mAb against S100A9 were purified and identified as IgG1 subtype,with the titer,purity and affinity constant was 1 3.18 105 ,95% and 3.54 108 L/ mol,respectively.This mAb 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 mAb against S100A9,which can secrete specific mAb against S100A9 protein with high titers and specificity have been established successfully,which laid the foundation for the immunology application.