猪圆环病毒2型重组Cap蛋白间接ELISA抗体检测方法的建立

Development of an indirect ELISA for detecting antibody to porcine circovirus 2 using recombinant Cap protein

利用pET32a(+)-Cap重组蛋白作为包被抗原,通过反应条件优化,建立了间接ELISA方法用于检测猪圆环病毒2型(PCV2)抗体。结果表明,抗原最适包被浓度为4μg/mL,最佳封闭液为1%BSA,37℃封闭3 h,血清最适稀释度为1∶200,其作用时间为60 min,酶标抗体最适稀释度为1∶20 000,最适作用时间为30 min,37℃显色15 min,判定血清样品P/N≥2.1,且OD450≥0.228为阳性。该方法与猪瘟、猪口蹄疫、猪脑心肌炎、猪呼吸与繁殖综合征病毒阳性血清反应呈阴性。批内和批间重复性试验结果变异系数均值分别为5.65%和5.81%,表明本方法具有较好的特异性和重复性。应用本方法对123份血清样品进行检测,检测结果阳性率78%,与国产商品化试剂盒检测结果对比,符合率为91.9%,表明本试验建立的间接ELISA方法具有较高的敏感性,适于大规模检测PCV2血清抗体的流行病学调查。

An indirect ELISA was performed to detected antibodies to porcine circovirus 2 （PCV2） with recombinant Cap protein. The op- timal concentration of antigen was 4 μg/mL. The sealing buffer was 1% BSA and the sealing time was 3 h at 37℃. The serum sample was diluted by 1 ： 200 and incubated for 60 min at 37 ℃. The dilution of conjugate was 1 ： 20 000 and the reaction time was 30 min. The TMB substrate was added and incubated at 37℃ for 15 min before terminated with stopping solution. The standard of judgment was that the serum sample with P/N ≥2. 1 and the sample with OD450≥0. 228 were positive. The recombinant antigen had no cross reaction with the antibodies against Hog cholera virus （HCV）, foot and mouth disease virus （FMDV）, encephalomyocarditis virus （EMCV） and porcine reproductive and respiratory syndrome virus （PRRSV）. Coefficient variations among wells in one plate and among plates for ELISA were 5.65% and 5.81%, respectively. One hundred and twenty three serum samples were tested with the indirect ELISA and 78% samples were positive, which has 91.9% consistence with the results by commercial ELISA kits. It indicated that the indirect ELISA is not only sensitive and specific, but also suitable for large-scale epidemiological investigation for PCV2 infection.