一种狂犬病实验室诊断用单克隆抗体制备新路线的探索

目的 探索采用合成肽作为免疫原制备狂犬病实验室诊断用单克隆抗体的可行性.方法 以狂犬病病毒CVS-11核蛋白355-369位B细胞线性抗原表位合成肽与钥孔戚血蓝蛋白（Keyhole Limpe hemocyanin,KLH）大分子耦联后免疫BALB/c小鼠,利用经典杂交瘤细胞技术制备单克隆抗体.采用间接酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）和间接荧光试验（indirect fluorescent assay,IFA）筛选和鉴定杂交瘤细胞株.结果 经过对杂交瘤细胞株上清的间接ELISA和IFA筛选获得阳性杂交瘤细胞株2B1D11,该杂交瘤细胞株产生的抗体经纯化后在IFA中可以有效检出感染犬脑组织和BHK-21细胞的狂犬病病毒.结论 采用合成肽作为免疫原制备狂犬病实验室诊断用抗体在技术上是可行的.

Exploration on a new routine for preparing rabies laboratory diagnostic monoclonal antibodies

Objective To explore the possibility of preparing rabies laboratory diagnostic monoclonal antibodies with synthetic peptides as immunogen.Methods A synthetic peptide located at residues 355 to 369 of rabies virus CVS-11 nucleoprotein was conjugated to the Keyhole Limpe hemocyanin （KLH） macromolecule and used to immunize BALB/c mice to prepare hybridoma cell lines by classical hybridoma technology.Indirect enzyme-linked immunosorbent assay （ELISA） and indirect fluorescent assay （IFA） were used to screen and identify the hybridoma cell lines.Results A positive hybridoma cell line named 2B1D11 was obtained after screening the anti-peptide antibodies in supernatants of hybridoma cell lines by indirect ELISA and IFA.Monoclonal antibody in ascites of BALB/c mice inoculated with 2B1D11 was purified and used successfully to detect the rabies virus in canine brain tissues and BHK-21 cells by IFA.Conclusion It is possible to prepare rabies laboratory diagnostic monoclonal antibodies with synthetic peptides as immunogen.