狂犬病毒核蛋白单抗和荧光抗体中和试验的建立与应用

Establishment and application of hybridomas producing anti-rabies nucleoprotein McAb and fluorescent antibody virus neutralization test

目的建立狂犬病毒血清中和抗体效价的快速检测方法,以实现狂犬疫苗免疫效果的定量评价,指导高危人群及动物种群的免疫预防。方法采用常规方法建立杂交瘤细胞株并制备抗狂犬病毒核蛋白单抗,以亲和层析法进行纯化,异硫氰酸荧光黄标记制备荧光抗体;以BHK-21细胞系培养狂犬病毒细胞适应株CVS-11,以染毒细胞测定荧光抗体的工作浓度后,通过荧光抗体染色法测定CVS-11细胞半数感染量(TCID50);利用狂犬病毒灭活疫苗免疫犬制备抗狂犬病毒血清,并以国际兽疫局标准品进行标定;参考世界卫生组织及国际兽疫局推荐方法,确定中和试验程序,对51份样品血清进行测定,并与国际狂犬病参考实验室的检测结果进行比较,评价其精确及可靠程度。以Kaber法公式和MicrosoftExcel软件为基础,设计被测样品中和效价计算方法。结果制备的抗狂犬病毒核蛋白单抗的亚型为IgG2a;纯化、标记后的荧光抗体工作浓度为1∶400;CVS-11株的使用量为每孔100TCID50;本研究建立的检测程序对51份人及犬血清测得的中和效价结果与国际参考实验室的测定结果一致;自行设计的计算方法操作简便,计算快捷。结论成功建立了与国际标准相当的狂犬病毒中和抗体效价测定和计算方法。

Objective To develop a rapid method for quantitatively assaying rabies neutralizing antibody titer in serum samples so as to guide vaccination of endangered population and animals based on the titer, and to provide monitoring approach for rabies virus research. Methods Monoclonal antibody against rabies virus nucleoprotein was developed by means of the conventional protocol. The IgG was purified by affinity chromatography and conjugated with FITC. The CVS-11 rabies virus was passaged and yielded on Baby Hamster Kidney （BHK-21） cells. The median tissue culture infectious dose （TCID50 ） was measured by direct immunofluorescent assay. The hyper-immune anti-rabies serum was prepared by immunizing dogs with commercial vaccine and monitored with the standard serum provided by WHO and OIE. A fluorescent antibody virus neutralization （FAVN） assay was established by referring to the protocol of WHO and OIE. 51 serum samples from human and dogs were detected parallelly in this laboratory and the Weybridge Rabies Referrence Laboratory in UK. A simple software was programmed on the basis of Microsoft Excel and Kaeber formula.Results The monoclonal antibody against rabies virus nucleoprotein was successfully produced by hybridoma 9G3. The working concentration of the FITC conjugated McAb is 1 ： 400. The FAVN results of serum samples assayed in this lab with 100 TCID50 CVS-11 and the conjugated McAb was consistent with that in the Weybridge rabies reference laboratory in UK. The software of calculating tool was practical and efficacious. Conclusion Hybridomas producing McAb against rabies virus nucleoprotein and a FAVN procedure for monitoring the neutralizing antibodies of rabies were successfully established and applied.