抗重组人乳腺珠蛋白单克隆抗体的制备及其特异性鉴定

Preparation of monoclonal antibodies against hMAM and identification of its specificity

目的通过杂交瘤技术获得特异性抗hMAM单克隆抗体,为进一步研制基于hMAM的乳腺癌早期诊断试剂盒奠定基础。方法以纯化的重组蛋白为抗原免疫Balb/c小鼠,运用杂交瘤技术制备、间接ELISA法及免疫组化法筛选出能稳定分泌特异性抗hMAM的单克隆抗体细胞株。结果通过杂交瘤技术获得了2株能分泌特异性抗hMAM单克隆抗体的杂交瘤细胞株hMAM-A2及hMAM-B7,其分泌的单克隆抗体亚类均属于IgG1,Western blotting结果显示2株细胞分泌的单克隆抗体特异性强能与34kDa处的hMAM抗原形成单一的蛋白质显色条带,免疫组化结果显示其仅与乳腺癌组织呈阳性反应,与其他肿瘤组织不发生交叉反应。结论利用鼠-鼠杂交瘤技术成功的制备了2株稳定分泌高效价、高特异性抗hMAM的杂交瘤细胞株。

Objective To acquire monoclonal antibodies against hMAM by hybridoma technique,as a basis for breast cancer early diagnosis kit. Methods Balb/c mice were immunized with purified hMAM. Hybridoma technique,ELISA test and immunohisto chemical method were used to prepare and screen the hybridoma cells lines of monoclonal antibodies against purified hMAM. Results Two hybridoma cell lines were obtained, named hMAM-A2 and hMAM-B7 respectively, which produce monoclonal antibodies specifically against purified hMAM. Antibody subtypes were identified to be IgG1. Western blotting showed they were made of only single protein band in 34kDa. The results of immunohistochemical test verified that specific reaction were observed between the gifted mouse anti- hMAM monoclonal antibody and breast cancer tissue,without any cross reaction with other cancer tissue. Conclusion Two hybridoma cell lines have been prepared successfully, which stably secrete high-titer and high specific monoclonal antibodies against purified hMAM.