猪瘟单克隆抗体的制备及ACI-ELISA检测猪瘟病毒的研究

Generation and application of monoclonal antibodies against classical swine fever

本研究用猪瘟石门毒(CSFV-Shimen)免疫BALB/C小鼠,按常规单克隆抗体(McAb)技术方法制作,最终获得4株McAb,分别命名为AC9、CF8、DG5和EC9,4株McAb与基因工程CSFV E2蛋白反应结果表明:AC9、CF8和EC9是抗CSFv E2蛋白的McAb。用AC9和CF8McAb对CSFV进行抗原捕获间接ELISA试验(ACI-ELISA),通过一元McAb和二元McAb CAI-ELISA试验的比较,结果表明AC9与CF8两种McAb有协同作用,其捕获CSFV的能力比一元McAb显著提高。方阵试验结果表明:McAb和血清多抗(PcAb)的最佳工作稀释度分别为1:400和1:200。特异性试验和敏感性试验结果显示本法特异性强,敏感性高。最后用ACI-ELISA与PCR对30份病料的检测结果比较,表明ACI-ELISA与PCR检测结果相符。上述结果说明本研究所获得AC9和CF8可用作猪瘟诊断试剂盒的研制,是检测CSFV的有效方法。

In this study four monoclonal antibodies designated as AC9, CF8, DG5 and EC9 against classical swine fever virus Shimen strain(CSFV-Shimen) were generated by immunization of Balb/C mice with purified CSFV- Shimen, All McAbs except DG5 could react with CSFV E2 protein in indirect ELISA, indicating that AC9, CF8 and EC9 are anti-CSFV E2 protein McAbs. The comparison of antigen-capture indirect ELISA(ACI-ELISA) experiments showed the combination of AC9 and CF8 could enhance reaction sensitivity than AC9 or CF8 alone. Through chessboard test it showed that the most suitable dilution of AC9-CF8 and the CSFV positive standard serum respectively is 1:400 and 1:200. The sensitivity and specificity tests showed the ACI-ELISA has highly sentive and specific reactivity with CSFV. At last, the ACI-ELISA was used to test 30 samples, yielding the same results as PCR. All of above had confirmed the ACI-ELISA is practicable in CSF diagnosis.