抗腺病毒五邻体单克隆抗体的制备及双抗夹心ELISA方法的建立

Preparation of monoclonal antibodies against adenoviral penton and development of a sandwich ELISA

为获得稳定性好、特异性强、效价高的抗腺病毒五邻体单克隆抗体,用腺病毒五邻体基因工程表达抗原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤细胞融合,以腺病毒五邻体为筛选抗原对融合后的杂交瘤细胞间接ELISA进行筛选.结果表明:试验获得5株杂交瘤细胞,标记为3C4、4C4、4D7、4F2和4H3,4D7免疫球蛋白亚类为IgG2a,其余4株为IgG1;对5株细胞连续3个月体外传代试验和小鼠腹水连续传代试验,5株细胞都能持续稳定分泌单克隆抗体,对细胞上清和腹水采用间接ELISA进行效价测定,细胞上清效价稳定维持在2 000左右,腹水效价稳定维持在200 000以上,且与引起腹泻的鼠伤寒沙门氏菌和轮状病毒无交叉反应;对其中可以配对的2株单抗4D7、4F2建立双抗夹心ELISA,可以应用于临床检测腺病毒.

In order to obtain fine stability,specificity,better titer of monoclonal antibodies （McAbs）a-gainst adenoviral penton,adenoviral penton antigen was used after genetically engineered expressed to im-munize BALB/c mice after fused spleen cells of the immunized mice and SP2/0 cells.The results showed that five hybridomas were gotten after using indirect ELISA to screen hybridoma by fusing used adenovirus penton antigen,marked 3C4,4C4,4D7,4F2 and 4H3.The subcategory Ig of 4D7 was IgG2a,the others were IgG1 .Five hybridomas serially passaged experiments in vitro for 3 months and serially passaged mouse as-cites,indicating all hybridomas secreted McAb sustaining.Cell titers remained around 2 000 and ascites ti-ters remained 200 000 stabled after indirect ELISA.No cross-reaction was happened with Salmonellatyph-imurium and rotavirus which caused diarrhea.It is suggested that sandwich ELISA with two mated Abs 4D7 and 4F2 can be applied to clinical detection of adenovirus.