基于免疫彩色纳米微球的犬瘟热病毒抗体检测凝集试验

Immuno-colored nanospheres based agglutination test for the detection of antibody against CDV

为建立以红色聚苯乙烯纳米微球为载体,快速检测幼犬犬瘟热病毒(CDV)母源抗体或免疫犬抗体水平的间接凝集试验,本研究表达纯化了CDV H截短的重组蛋白rsH2,并以rsH为致敏原与纳米微球偶联制备了免疫彩色纳米微球;通过间接凝集反应条件的优化,特异性、敏感性、稳定性试验和临床样品的检测,建立了一种快速检测CDV抗体的间接凝集试验。特异性结果显示,致敏微球仅与CDV阳性血清发生凝集反应,不与狂犬病病毒、犬腺病毒和犬细小病毒的阳性血清发生凝集反应,特异性强;凝集试验可检测血清中和抗体的最低效价为1∶35;不同批次制备的致敏微球和4℃保存90 d的致敏微球的凝集反应特异性一致,凝集程度无明显差异,重复性稳定性好;该凝集试验对45份血清样品的检测结果与CDV抗体检测试剂盒检测结果的阳性符合率为100%。上述结果表明,本研究建立的检测CDV抗体的间接凝集试验具有良好的特异性、敏感性和稳定性,为CDV抗体检测提供了一种快速、简便、准确、经济的检测方法。

The aim of this study is to develop an indirect agglutination test based on immuno-colored nanospheres bound with recombinant antigen for detecting the maternal antibodies status against canine distemper virus of puppies prior to vaccination or seroconversion in vaccinated dogs.Truncated recombinant protein rsH of canine distemper virus(CDV) H protein was expressed,purified,and bound with colored polyphenylethylene nanospheres,then the immuno-colored nanospheres was prepared.The indirect agglutination test was established by optimization of reaction conditions,the tests of specificity,sensitivity,stability,and detection of clinical samples.The agglutination reaction was observed only between sensitized nanospheres and positive serum against CDV,but was not positive serum against CPV-2,CAV,RBAV.The minimum neutralizing antibody titer of 1 ∶ 35 against CDV was detected by this agglutination test while the reaction displayed the agglutination degree of 50%(++).The sensitized nanospheres prepared in different batches or stored at 4 ℃ for 90 days had the same specificity,stability and repeatability,and there was no significant difference in agglutination degree.The coincidence of detecting positive results of 45 samples by this agglutination test and commercial kit was 100%.These results indicated that the indirect agglutination test established in this study may be a useful method to determine the serum antibodies status against CDV in puppies.