血清4型禽腺病毒夹心ELISA检测方法的建立

Establishment of sandwich ELISA method for detection of fowl adenovirus serotype 4

为建立一种血清4型禽腺病毒(FAd V-4)夹心ELISA快速检测方法,本研究以FAdV-4多抗作为捕获抗体,FAdV-4 Penton蛋白单克隆抗体作为检测抗体,通过优化反应条件,建立了检测FAdV-4的夹心ELISA方法,对其进行特异性、敏感性、重复性检验和临床样品检测。方阵试验结果显示,FAdV-4多抗最佳稀释度为1∶2000,单抗最佳稀释度为1∶32000。该方法可特异性检测近年流行的高致病性FAdV-4毒株,而对之前的FAdV-4、其他血清型FAdV和常见禽病病原体不发生交叉反应。敏感性结果显示,该法对9.7×10^(2)TCID_(50)/mL FAdV-4仍可检出。重复性结果显示,批内和批间试验的变异系数均小于5.0%。用建立的夹心ELISA方法与荧光PCR同时对100份肛口拭子样品进行检测,两者检测结果相符。上述结果表明,本研究建立的夹心ELISA检测方法特异性强、敏感性高、重复性好,可用于FAdV-4的大批量检测。

In order to establish a sandwich ELISA method for the rapid detection of fowl adenovirus serotype 4(FAdV-4),FAdV-4 polyclonal antibody was used as the capture antibody and FAdV-4 Penton protein monoclonal antibody was used as the detection antibody in this study.By optimizing the reaction conditions,a sandwich ELISA method for the detection of FAdV-4 was established.The specificity,sensitivity and repeatability of the sandwich ELISA method were identified and the clinical samples were tested.The results of square titration showed that the optimal dilution ratio of FAdV-4 polyclonal antibody was 1∶2000 and that of FADV-4 monoclonal antibody was 1∶32000.The method specifically detects recently prevalent highly pathogenic FAdV-4 strains without cross-reactivity to previous FAdV-4,other serotypes of FAdV,and common poultry pathogens.Sensitivity results showed that the method is still detectable for FAdV-4 at 9.7×10^(2) TCID_(50)/mL.Repeatability results showed that the coefficient of variation was less than 5.0%in the intra-batch and inter-batch tests.A total of 100 cloacal and oral swabs were tested simultaneously by the established sandwich ELISA method and real-time PCR,and the test results were consistent with each other.The above results showed that the sandwich ELISA detection method established in this study had strong specificity,high sensitivity and efficient repeatability,and could be used for the detection of FAdV-4 in large quantities.