摘要
用基因工程表达的NBcAg免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞Sp2/0融合,以ELISA抑制法检测抗体,获得28株分泌抗-HBc McAb的杂交瘤细胞系。对其中7株进行了克隆化,培养上清抗体滴度1:320～1:2560,免疫小鼠腹水和血清滴度1:10万～1:80万。Ig类型测定2株IgG1、2株IgG2a、1株IgG3、2株IgM。杂交瘤细胞系在体外连续传代培养6个月及液氮冻存的细胞系经复苏后仍能稳定地分泌McAb。得到的McAb只与HBcAg反应且能被HBcAg特异阻断,证明其特异性高。用ELISA间接法测定了McAb的相对亲和力,从50％最大结合的浓度分析结果,7株McAb的相对亲和力为2E6>2F5>2B11>2C10>1A7>1B7>1H7,浓度范围为0.6～10μg/ml。2E6和2C10 2株McAb与HRP结合,用ELISA双抗休夹心法筛选出2F5、2B111和H7 3株McAb适合作为包被抗体。2F5与2E6-HRP配对,建立了快速McAb-ELIS抑制法测定患者血清中抗-HBc。用本法测定了222份临床患者血清标本,结果与“华美”试剂盒测定结果基本一致。
Seven hybridoma cell lines secreting anti-HBcAg monoclonal antibodies (McAbs) were established by fusing cells of mouse myeloma line Sp2/0 with splenocytes from BALB/c mice immunized with recombinant HBcAg. These hybridoma cell lines secreted high titer of McAbs stably by cultivating in vitro for six months in turn and resuscitating from freezing in liquid nitrogen. The McAbs obtained were characterized immunologically. By analysing the 50% maximum binding of the McAbs, the relative affinities of them measured by indirect ELISA could be ranked as following 2E6>2F5 >2B11>2C10>1A7>lB7>1H7 at the protein concentration of 0.6-10μg/ml. Two McAbs (2E6 and 2C10) were labelled with horseradish peroxidase (HRP) and used to develop a rapid inhibiting McAb-ELISA for detection of anti-HBcAg antibody.Anti-HBcAg antibodies in 222 clinical serum samples were detected by the McAb-ELISA and a commercially available kit (Sino-American Biotechnology Company), the results obtained showed no difference between them.
关键词
单克隆抗体
乙型肝炎
核心抗原
HBcAg
antibody, monoclonal
relative affinity
antibody labelled enzyme
