抗SARS冠状病毒单克隆抗体的制备和鉴定

Preparation and Characterization of McAb against SARS-associated Coronavirus

目的建立能稳定分泌抗SARS-CoV单克隆抗体的杂交瘤细胞株。方法用SARS-CoV灭活全病毒液免疫BALB/c小鼠,利用杂交瘤技术建立6株稳定分泌抗SARS-CoV单克隆抗体的杂交瘤细胞株,并进行全面鉴定。结果免疫双扩散证实1C6细胞株分泌的单抗为IgG2a亚类,其余5株细胞分泌的单抗为IgG1亚类;腹水效价均达到10-6以上;6株细胞分泌的单抗均不与麻疹病毒等其它5种呼吸道病毒发生交叉反应;Westernblot证实6株细胞分泌的单抗均识别相对分子质量约150000的S蛋白及其相对分子质量为120000的裂解片段;相加指数证实6株细胞分泌的单抗识别相关的抗原表位;用硫氰酸铵洗脱法比较了6株细胞分泌的单抗的相对亲和力大小,依次为1A4>4F6>1C6>1B10>2H2>1F3;中和试验证实单抗4F6和1F3具有中和活性,中和效价均为1∶10;杂交瘤细胞株连续培养3个月以上及冻存半年后复苏,细胞生长良好,效价稳定。结论已获得抗SARS冠状病毒的单克隆抗体,为SARS的早期诊断及进一步研究奠定了基础。

Objective To establish the hybridoma cell strains stably secreting McAb against SARS-associated coronavirus （SARS-CoV）. Methods Immunize BALB/c mice with inactivated SARS-CoV and establish six hybridoma cell strains stably secret McAbs against SARS-CoV. Perform overall control tests on the six strains. Results Double immunodiffusion test proved the McAbs secreted by strain IC6 as IgG2a subclass,and those secreted by the rest five strains as IgG1 subclass, All the titers of McAbs secreted by the six strains in ascites reached more than 10 -6. None of the six kinds of McAbs showed cross reaction with other respiroviruses. Western blot showed that all the six kinds of McAbs recognized the S protein with a relative molecular weight of 150 000 and its lyric fragment with a relative molecular weight of 120 000. ELISA additivity test proved that all the six kinds of McAbs recognized the corresponding antigen epitopes. The elution with thiocyanate showed that,in the order of relative a.fi＇inity,the six kinds of McAbs were as follows：1A4,4F6,1C6,1B10,2H2 and 1F3. Both the neutralizing titers of McAbs 4F6 and 1F3 were 1： 10. The hybridoma cell strains grew well after continuous culture for more than 3 months or after being stored lyophilized for 6 months, and the titers of secreted McAbs were stable. Conclusion The McAbs against SARS-CoV was obtained. It laid a foundation of early diagnosis and further study of SARS.