全人源抗狂犬病病毒单克隆抗体的制备与鉴定

目的:利用人源IgM转基因小鼠制备全人源抗狂犬病病毒单克隆抗体,并对其进行初步鉴定?方法:以灭活的狂犬病病毒CTN株作为抗原免疫人IgM转基因小鼠?采用杂交瘤技术结合酶联免疫吸附试验(ELISA)高通量交叉筛选技术(HTS)制备全人源抗狂犬病病毒单克隆抗体?通过双抗体夹心ELISA鉴定单抗的人源性和抗体型,间接ELISA?斑点杂交(Dot Blot)实验及Western blot检测单抗的特异性,BiaCore X-100测定单抗结合抗原的亲和力?结果:建立了2株稳定分泌抗狂犬病病毒人源性单抗的杂交瘤细胞株,分别命名为9E3和9F2?双抗体夹心ELISA结果显示2株单抗均为人源性免疫球蛋白IgM型?间接ELISA?斑点杂交实验结果表明2株单抗均能特异性识别灭活的狂犬病病毒CTN株?Western blot结果显示2株单抗均能与狂犬病病毒糖蛋白特异性结合?2株单抗与狂犬病病毒CTN株抗原结合的亲和力分别为 2.62 × 10-10 mol/L?4.06 × 10-11 mol/L?结论:筛选制备了2株特异性?高亲和力的全人源抗狂犬病病毒单克隆抗体?本研究为进一步筛选人源抗狂犬病病毒免疫预防性中和抗体及其免疫治疗的应用奠定了基础?

Generation and identification of human monoclonal antibodies to the rabies virus

Objective:To generate human monoclonal antibodies (mAbs) against the rabies virus (RV) by using the mice carrying human immunoglobulin transloci for immunization and further to identify their characters. Methods:The human IgM transgenic mice were immunized with the inactivated RV(CTN strain) as immunogen. The human anti-RV mAbs were screened and produced by using the classical hybridoma technique in a combinatory high throughput screening (HTS) strategy. The fully human mAbs were identified via the double antibody sandwich enzyme-linked immunosorbent assay(ELISA),and the specificity of mAbs was determined by the indirect ELISA,Dot Blot and Western Blot. In addition,the affinity of mAbs was detected by BiaCore X-100 system. Results:Two anti-RV hybridoma cell lines 9E3 and 9F2,which can produce human IgM mAbs and recognize the CTN strain of RV specifically in the indirect ELISA and the Dot Blot assays,have been established. The results of Western Blot demonstrated that the two prepared mAbs specifically recognize glycoprotein(G protein) of the rabies virus. The equilibrium dissociation constants (KD) of 9E3 and 9F2 were 2.62 × 10-10 mol/L and 4.06 × 10-11 mol/L,respectively. Conclusion:Two anti-RV fully human IgM mAbs with specificity and high affinity were generated. The first-phase preparations of mAbs could be the bases for the screening immunoprophylaxis neutralized monoclonal antibody and immunotherapy on the rabies.