抗人CA50单克隆抗体的制备、鉴定及其应用

Preparation and characterization of monoclonal antibodies against human CA50 and establishment of chemiluminescence immunoassay system

目的制备抗人糖抗原50（CA50）单克隆抗体（m Ab）,鉴定其免疫学特性并建立化学发光免疫分析法检测体系。方法将人CA50抗原免疫BALB/c小鼠,利用杂交瘤技术进行细胞融合,筛选后得到稳定分泌抗CA50抗体的杂交瘤细胞株。经细胞扩大培养及纯化获得m Ab后,建立化学发光免疫分析法检测体系,并对其线性范围、准确度、灵敏度、重复性进行评估,同时进行血样的测定。结果筛选出4株抗人CA50的杂交瘤细胞株,效价均在1∶108以上,2株配对抗体不与CA125、CA153、CA199、CA724交叉。建立的化学发光免疫分析法检测范围为0~500 U/m L,回收率为107.08%,灵敏度为0.83 U/m L,重复性CV值〈10%,与参比试剂检测血样的结果比较符合率为96.7%,Kappa值为0.911。结论成功制备了抗人CA50m Ab,建立了人CA50的化学发光免疫分析法检测体系。

Objective To prepare the anti-human carbohydrate antigen 50（CA50） monoclonal antibody （mAb）, characterize its immunological features and establish a chemiluminescence immunoassay system with it. Methods BALB/c mice were immunized with human CA50 antigen for preparing mAb using hybridoma technique. Stable anti-CA50-secreting hybridoma cell lines were obtained after screening. The mAbs were pudfied using protein A after expanding culture. The immunoassay system was established and evaluated in its linear range, accuracy, sensitivity, reproducibility, and the blood samples were tested with it. Results Four hybridoma cell lines were obtained respectively and their titers were above 1：10^8. Anti-CA50 mAbs nearly had no cross-reaction with CA125, CA153, CA199 and CA724. The linear detection of the chemiluminescence immunoassay system covered a range 0 - 500 U/mL. The recovery rate for accuracy was 107.08% and its sensitivity was 0.83 U/mL. The assay was highly repeatable [ coefficient of variation （CV） 〈 10% ]. The correlation coefficient with the test of reference reagent was 0.96. Conclusion The monoclonal antibodies against human CA50 had been screened and a chemiluminescence immunoassay system for human CA50 detection had been established successfully.