适于HIV-1 p24抗原检测试剂的单抗制备与筛选

Preparation and selection of the monoclonal antibody used for kits to detect the p24 antigen of HIV-1

目的制备抗HIV-1p24单克隆抗体(单抗,McAb),并利用双抗体夹心ELISA法建立HIV-1p24抗原检测试剂。方法以基因工程原核重组抗原HIV-1p24蛋白免疫BALB/c小鼠,利用常规杂交瘤技术和间接ELISA法制备单克隆抗体,单抗经纯化和HRP标记后,利用双抗体夹心ELISA筛选检测p24蛋白的最佳配伍单抗以期建立HIV-1p24抗原检测试剂。结果成功筛选到16株稳定分泌抗HIV-1p24单抗的杂交瘤细胞株,并从中筛选出最佳单抗配伍组合“16G12+2F2b-HRP”,该组合对p24蛋白的检测灵敏度为20pg/mL,对感染性克隆pNL4-3表达的HIV病毒培养上清检测呈阳性,对100份HIV阴性人血清无非特异性反应,显示出良好的检测灵敏度和特异性。结论本研究初步成功地建立了HIV-1p24抗原的检测试剂,并为最终建立HIV第四代诊断试剂(HIV Ag/Ab Assay)奠定了坚实的基础。

To prepare the monoclonal antibody （McAb） used for kits to detect the p24 antigen of HIV-1 by means of double-antibody sandwich ELISA assay, BALB/c mice were immunized with purified recombinant p24 protein of HIV-1, and by routine hybridoma technique, 16 hybridoma cell lines secreting potent McAb against HIV p24 antigen were obtained. The subtype of these 16 McAb was found to be IgG1 and their cross-blocking properties were analyzed , when they reacted with the p24 protein in direct ELISA in order to offer the valuable data for selecting feasible pair of McAb in the detection of the p24 antigen. All the McAbs were used for the detection of p24 antigen by double-antibody sandwich ELISA. In addition, the McAbs were purified and HRP-labelled in advance. Finally, we had successfully screened a pair of McAbs, i.e. 16G12 ＋2F2b-HRP, which exhibited a sensitivity of 20 pg/ml for the detection of p24 antigen. Also, this pair of McAbs could specifically react with the supernatants of the cultured HIV-1 from pNL4-3 transfected clone, but it showed no reactivity with 100 negative sera of patients. It is evident that this pair of McAb shows excellent sensitivity and specificity for the detection of the HIV-1 p24 antigen.