抗人CA125单克隆抗体的制备、鉴定及化学发光体系的建立

Preparation and characterization of monoclonal antibodies against human CA125 and establishment of a chemiluminescence immunoassay system

目的制备抗人糖抗原125（CA125）单克隆抗体（mAb），并建立化学发光免疫分析法检测体系。方法将人CA125抗原免疫小鼠，通过细胞融合、筛选后得到杂交瘤细胞株。经细胞扩大培养及纯化获得mAb，鉴定其特异性、纯度、亚型及效价，并建立双抗体夹心ELISA。分别对包被缓冲液、包被浓度和加样模式进行优化和选择后，建立化学发光免疫分析法检测体系，并对其进行评估和血样的测定。结果获得3株抗人CA125的杂交瘤细胞株（30—1—4、31—1-5、43—1-6），不与CA199、CA50、CA724交叉，效价在1：10。以上，用30-1．4和31—1—5建立的化学发光免疫分析法经优化后，检测范围为0～1000U／mL，最低检测限为0．72U／mL，与罗氏试剂检测结果的相关系数大于0．90。结论成功制备了抗人CA125mAb，建立并优化了人CA125的化学发光免疫分析法检测体系，为CA125检测及卵巢癌的诊断奠定了基础。

Objective To prepare the anti-human carbohydrate antigen 125 （CA125） monoclonal antibody （mAb）, and establish a chemiluminescence immunoassay detection system. Methods The BALB/c mice were immunized with human CA125 antigen. The hybddoma cells were obtained by cell fusion and screening. The mAbs were purified using protein A. The specificity, purity, isotype, and affinity of the mAbs were characterized and the sandwich ELISA system was established. The chemiluminescence immunoassay system was established and evaluated after optimization and selection of coating buffer, coating concentration, pipetting mode respectively, and the blood samples were tested with it. Results Three hybridoma cell lines named 30-1-4, 31-1-5 and 43-1-6 were obtained respectively. Anti-CA125 mAbs nearly had no cross- reaction with CA199, CAS0 and CA724. The titres were above 1 ： 10s. The chemiluminescence immunoassay system was established using 30-1-4 and 31-1-5. After optimization, the linear detection of the system covered a range 0 -1000 U/mL, and its lowest detectable limit was 0.72 U/mL The correlation coefficient with the results of the Roche test was above 0.90. Conclusion With the mAbs against human CA125 we prepared, a chemiluminescence immunoassay system for human CA125 detection has been established successfully, which provides a basis for human CA125 detection and ovarian cancer diagnosis.