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目的利用活体骨髓瘤细胞与杂交瘤技术,采用间接免疫荧光分析法(IFA)筛选出更多可识别人呼吸道合胞病毒(RSV)的单克隆抗体,并对其体外性质进行鉴定,以期为RSV的被动免疫治疗以及临床诊断奠定基础。方法以RSV通过滴鼻途径免疫小鼠,同时采用实体瘤技术体内制备骨髓瘤细胞。取免疫后小鼠的脾细胞与骨髓瘤细胞进行细胞融合,利用IFA方法筛选经有限稀释法亚克隆获得的可稳定分泌抗RSV单克隆抗体(McAb)的杂交瘤细胞株。通过间接ELISA和Western blot等方法初步分析McAb对RSV蛋白的特异性结合能力,通过免疫酶法(IE)蚀斑减少中和实验和融合抑制实验分析McAb体外保护作用。结果获得4株稳定分泌抗RSV单克隆抗体的杂交瘤细胞株,分别为2A7、3E10、1F10及3G4,经间接ELISA以及Westernblot初步鉴定,获得的4株单克隆抗体可特异性识别RSV蛋白,McAbs 2A7和3E10可分别识别融合糖蛋白(F)蛋白二聚体和单体,McAbs 1F10和3G4可识别黏附糖蛋白(G)蛋白。其中McAbs 2A7与1F10可以在体外抑制RSV对HEp-2细胞的感染,具有中和活性,同时具有融合抑制活性。结论利用IFA法成功筛选了能特异性识别RSV的4种单克隆抗体,初步鉴定显示两种单抗可分别识别F蛋白二聚体和单体,另两种单抗可识别G蛋白。IE法蚀斑减少实验显示两种单抗在体外具有中和活性和融合抑制活性,表明获得的单抗对RSV感染具有一定的预防作用。
OBJECTIVE: To screen out more monoclonal antibodies recognizing human respiratory syncytial virus (RSV) by indirect immunofluorescence assay (IFA) using live myeloma cells and hybridoma technology and to identify their in vitro characteristics for the purpose of RSV Passive immunotherapy and clinical diagnosis to lay the foundation. Methods Mice were immunized with RSV via the nasal route while myeloma cells were prepared in vivo using solid tumor technology. The spleen cells of the mice after immunization were fused with myeloma cells, and IFA method was used to screen the hybridoma cell lines stably secreting anti-RSV monoclonal antibody (McAb) obtained by sub-cloning by limiting dilution method. The specific binding ability of McAb to RSV protein was analyzed by indirect ELISA and Western blot. The in vitro protective effect of McAb was analyzed by immunoenzyme (IE) plaque reduction neutralization assay and fusion inhibition assay. Results Four hybridoma cell lines stably secreting anti-RSV monoclonal antibodies were obtained, 2A7, 3E10, 1F10 and 3G4, respectively. The indirect ELISA and Western blot were used to identify the four hybridoma cell lines. The four McAbs specifically recognized RSV protein, McAbs 2A7 and 3E10 recognize the fusion glycoprotein (F) protein dimer and monomer, McAbs 1F10 and 3G4 recognize the adhesion glycoprotein (G) protein, respectively. Among them McAbs 2A7 and 1F10 could inhibit RSV infection in HEp-2 cells in vitro, had neutralizing activity and had fusion inhibition activity. CONCLUSION: Four monoclonal antibodies that can specifically recognize RSV were successfully screened by IFA. Preliminary identification showed that the two McAbs could recognize F protein dimer and monomer respectively, and the other two McAbs could recognize G protein. IE method of plaque reduction experiments showed that the two monoclonal antibodies in vitro with neutralizing activity and fusion inhibitory activity, indicating that the obtained monoclonal antibody has a preventive effect on RSV infection.