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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2.62×10-10mol/L、4.06×10-11mol/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。
OBJECTIVE: To prepare humanized anti-rabies virus monoclonal antibody using human IgM transgenic mice and preliminary identification. Methods: Human IgM transgenic mice were immunized with inactivated rabies virus CTN strain as antigen. Humanized anti-rabies virus monoclonal antibody was prepared by hybridoma technology combined with high-throughput cross-enzyme-linked immunosorbent assay (ELISA). The specificity and specificity of mAb were determined by double antibody sandwich ELISA. Dot Blot and Western blot were used to detect the specificity of mAb. BiaCore X-100 was used to determine the affinity of mAb. Results: Two hybridoma cell lines stably secreting anti-rabies virus human monoclonal antibodies were established and named as 9E3 and 9F2, respectively. Double antibody sandwich ELISA results showed that the two monoclonal antibodies were all human immunoglobulin IgM type. Indirect ELISA and Dot blot hybridization showed that the two McAbs could specifically recognize the inactivated rabies virus CTN strain. Western blot results showed that the two McAbs could specifically bind to rabies virus glycoprotein. The affinities of the two McAbs against rabies virus CTN strain antigen were 2.62 × 10-10 mol / L and 4.06 × 10-11 mol / L, respectively. Conclusion: Two monoclonal antibodies against human rabies virus (IBDV) with high specificity and high affinity were prepared by screening. This study laid the foundation for further screening of anti-rabies virus neutralizing antibodies against human immunodeficiency virus and its immunotherapy.