犬流感病毒重组核蛋白抗体间接ELISA检测方法的建立

Development of an indirect ELISA for detecting antibodies against nucleoprotein of canine influenza virus

为建立H3N2亚型犬流感病毒（cIv）血清学检测方法，本研究利用H3N2亚型CIV重组核蛋白（rNP）作为检测抗原，通过优化反应条件，建立了检测CIV核蛋白血清抗体的间接ELISA方法。通过检测10份SPF犬血清样品确定阴阳性临界值为0．288。该方法检测犬瘟热病毒、犬细小病毒、犬副流感毒、犬腺病毒II型、狂犬病病毒、犬弓形虫、犬弓首蛔虫、犬复孔绦虫的阳性血清均为阴性，具有良好的特异性，但与H1N1、H3N8和H9N2亚型CIV有交叉反应。该方法检测H3N2CIV血清抗体的灵敏度为血凝抑制试验（HI）的3～12．5倍；而且其批内批间变异系数为1．85％～6．57％，重复性良好。通过对H3N2CIV攻毒犬血清进行分析，表明该检测方法具有滞后性，检测到CIV抗体的时间晚于HI试验。利用本研究建立的方法和以H3N2CIV为诊断抗原的HI检测方法对450份血清样品进行检测，结果显示该方法对血清样品具有初筛作用，两者阳性符合率为58-3％，阴性符合率为100％。本研究可以结合其它血清学方法为CIV流行病学调查进行快速、高效的检测。

In order to rapidly detect serum antibody against canine influenza virus （CIV）, an indirect ELISA was developed with the purified recombinant NP protein of H3N2 CIV as coating antigen. The reaction conditions of the ELISA were optimized and the cut-off value of the assay was set as 0.288 determined by 10 SPF canine serum samples. The sensitivity of the ELISA was 3 to 12.5 times as that of hemagglutination inhibition test （HI）. The assay had no cross-reaction with other related positive serum against canine viruses, bacteria and parasites, but had cross-reaction with serum against H1N1, H3N8, and H9N2 subtypes of C1V. The assay was reproducible with coefficient variation of 1.85% to 6.57%. In addition, a total of 450 clinical sera were tested by the established assay, compared with HI test used H3N2 CIV as diagnostic antigen. The positive and negative coincident rates were 58.3% and 100%, respectively. Thus, the developed ELISA is potential to be applied in epidemiological study and surveillance of canine influenza.