串联表达猪繁殖与呼吸综合征病毒GP5表位蛋白间接ELISA方法的建立

Development of an Indirect ELISA Assay for Detecting Antibody to Porcine Reproductive and Respiratory Syndrome Virus with Recombinant GP5-encoding-epitopes Protein

利用猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)GP5抗原表位串联表达重组蛋白作为包被抗原,建立检测PPRSV抗体的间接ELISA方法。重组蛋白最佳包被浓度为7.5μg/mL,最佳封闭液为5%脱脂奶粉,37℃封闭2h;血清最适稀释度为1∶100,37℃作用2h;兔抗猪IgG/辣根酶(HRP)(1∶3000),37℃作用2h;37℃避光显色15min读取D450nm值。结果经统计学分析得出,S/P值≥0.254为阳性,S/P值≤0.212为阴性。所建立的ELISA方法检测其他5种猪常见病原阳性血清,其D450nm值均小于0.212。利用建立的ELISA方法对临床免疫勃林格殷格翰猪繁殖与呼吸综合征活疫苗4周后的猪血清70份进行检测,其D450nm值均大于0.85,表明本研究建立的重组GP5表位蛋白间接ELISA方法可用于临床样品的监测。

An indirect ELISA assay was constructed to detect porcine reproductive and respiratory syndrome virus （PRRSV） antibodies with recombinant GP5-encoding-epitopes protein. The optimal concentration of antigen was 7.5 ug/mL; The blocking buffer was 5% defatted milk powder with the blocking time 2 h,at 37℃ ;The serum sample was diluted in 1 ： 100 with incubated for 2 h at 37 ℃ ;The dilution of rabbit-anti-porcine IgO labeled by HRP was diluted in 1 ： 3000 with incubated for 2 h at 37℃ ;The TMB substrate was added and incubated at 37℃ for 15 min and then terminated with stopping solution. The standard of judgment was that the serum sample D450 am value with S/P≥0. 254 was positive, and the sample D450 nm value with S/P≤0. 212 was negative. The recombinant antigen had no cross reaction with antibodies of other five porcine diseases. 70 serum samples collected from pigs which were vaccinated with Ingelvac PRRS MLV were detected by this assay, the D450nm value of which were higher than 0.85. The results demonstrated that the establishment indirect ELISA method could be used for monitoring PRRSV antibody.