禽流感病毒NS1抗体间接阻断ELISA检测方法的建立

Establishment of an indirect-blocking ELISA for detection of antibodies against NS1 protein of avian influenza virus

以禽流感病毒(AIV)重组NS1蛋白作为检测抗原,兔抗NS1血清为阻断抗体,建立了检测AIVNS1抗体的间接阻断ELISA方法。经筛选确定,抗原的最佳包被浓度为0.6μg/mL,阻断抗体的最佳稀释度为1∶10000,待检血清最佳稀释度为1∶10。用该方法对42份AIV感染禽类血清样本和50份用AIV灭活疫苗免疫的禽类血清样本进行检测,结果显示,该方法的敏感性为95.2%,特异性为90.0%。交叉反应试验证明,该方法与新城疫等8种其他禽病阳性血清不发生交叉反应。临床检测结果显示,186份AIV灭活疫苗免疫禽血清样本的阴性率为89.2%,20份基因工程疫苗免疫禽血清样本的阴性率为95.0%,42份健康非免疫禽血清样本的阴性率为100%。表明,建立的间接阻断ELISA方法可用于区分AIV自然感染禽和疫苗免疫禽。

An indirect blocking ELISA was established using a recombinant avian influenza virus（AIV） NS1 protein as the diagnostic antigen, and rabbit antiserum against the NS1 protein as the blocking antibody. The optimal concentration of the coating antigen was O. 6μg/mL,the optimal dilution of the blocking antibody was 1 ： 10 000,and the optimal dilution of the serum was 1 ： 10. Forty-two AIV-infected samples and 50 vaccinated samples were detected by the established indirect-blocking ELISA, indicating that the sensitivity of the method was 95.2% ,and the specificity was 90.0%. Cross-reaction detection showed no cross-reaction to the other avian positive sera. The clinical detections showed that the negative rates of 186 antiserum samples against the inactivated vaccine, 20 antiserum samples against the recombinant live vaccine and 42 serum samples from healthy avians without vaccination were 89.2% ,95.0% and 100% respectively. The results indicated that this method could be used for differentiating naturally-infected poultry from vaccinated chickens.