H5亚型禽流感病毒抗原捕捉ELISA诊断方法的建立
摘要
本研究采用纯化的高致病性禽流感病毒H5N1亚型A/Duck/zhejiang/11/00 (DZJ/1100)毒株作为免疫原,灭活后加入弗氏完全佐剂和弗氏不完全佐剂制成疫苗多次免疫山羊,获得抗H5亚型禽流感病毒的高免血清。在实验室已有的H5亚型单抗的基础上,建立了以多克隆抗体(羊血清)作为捕获抗体,H5亚型HA特异性单克隆抗体为检测抗体的检测H5亚型禽流感病毒的抗原捕捉ELISA方法。通过对各个反应条件进行优化,获得的最佳工作条件为:羊血清1:1600倍稀释;单抗1:10000稀释;酶标抗体最适工作浓度为1:5000倍稀释;单抗反应时间1.5h;酶标抗体作用时间1.5h。
用建立的抗原捕捉ELISA方法对已知的NDV、EDS76、IBV、IBDV、ILTV、MDV和APV禽类病毒以及其他14个HA亚型禽流感病毒进行交叉反应性试验,结果表明:基于单抗检测的H5亚型禽流感病毒抗原捕捉ELISA方法和其他禽类病毒以及其他亚型禽流感病毒均没有明显的交叉反应性,与阴性对照没有明显差异,说明该方法对H5亚型流感病毒具有高度的特异性。与HA和鸡胚接种试验相比,本实验建立的双抗体夹心ELISA方法较HA敏感2倍以上,但不如鸡胚接种试验敏感。
利用优化的ELISA反应条件对人工感染的临床样本进行检测,通过对脾脏、肾脏、心脏、肺脏等多份阴阳性样本的检测最终确定以脾脏作为临床检测的靶器官,并将0.15(OD490nm)作为阴阳性判定标准;同时以心脏、肝脏和肺脏作为辅助检测器官,并将0.1(OD490nm)作为阴阳性判定标准。
将反应用各种试剂及包被的反应板组装成试剂盒,同时保存于室温、4℃和-20℃,定期进行检测,结果表明:试剂盒在室温保存时极不稳定,保存期限仅为1个月,4℃可保存4个月,可在-20℃稳定保存8个月以上。
In this study, hyper-immunized goat serum was prepared by immunizing for three times with inactive purified H5N1 AIV (DZJ/1100) mixed with Freund’s adjuvant or incomplete Freund’s adjuvant. The antigen capture ELISA was developed using polyclonal anti-sera (goat serum against AIV) as capture antibody and H5 specificity monoclonal antibody as detecting antibody for detecting H5 AIV. The optimal conditions were determined: capture anti-sera was diluted for 1:1600, H5 AIV specificity McAb was diluted for 1:10000 and the enzyme-labeled antibody was diluted for 1:5000. The optimal reactive time of the detecting antibody and conjugate were one and half an hour separately in the assay.
NDV、EDS76、IBV、ILTV、MDV、APV and H1-H15 AIV were detected by the sandwich ELISA and the result proved that there was no crossing-reaction between the antigen capture ELISA and other avian viruses and others fourteen AIV except H5. It proved that the H5 AIV antigen capture ELISA had excellent specificity.
Comparing with HA test and egg inoculation test, the sensitivity of the antigen capture ELISA was twice better than that of HA test, but it was less than that of egg inoculation test.
Many experimently infected clinical samples were detected by the sandwich ELISA under the most optimal conditions. Finally, spleen, lung, heart and liver were defined as target organ when clinical samples were detected by the ELISA and the cut-off value of spleen was 0.15 and others were 0.1.
The ELISA kit which consisted of coated microplate and reactive solutions were kept in room temperature, 4℃and -20℃seperately. The detection results showed that kits had bad stability in room temperature and the period of validity was only one month. At the same time, the period of validity in 4℃was 4 months and over 8 months in -20℃.
用建立的抗原捕捉ELISA方法对已知的NDV、EDS76、IBV、IBDV、ILTV、MDV和APV禽类病毒以及其他14个HA亚型禽流感病毒进行交叉反应性试验,结果表明:基于单抗检测的H5亚型禽流感病毒抗原捕捉ELISA方法和其他禽类病毒以及其他亚型禽流感病毒均没有明显的交叉反应性,与阴性对照没有明显差异,说明该方法对H5亚型流感病毒具有高度的特异性。与HA和鸡胚接种试验相比,本实验建立的双抗体夹心ELISA方法较HA敏感2倍以上,但不如鸡胚接种试验敏感。
利用优化的ELISA反应条件对人工感染的临床样本进行检测,通过对脾脏、肾脏、心脏、肺脏等多份阴阳性样本的检测最终确定以脾脏作为临床检测的靶器官,并将0.15(OD490nm)作为阴阳性判定标准;同时以心脏、肝脏和肺脏作为辅助检测器官,并将0.1(OD490nm)作为阴阳性判定标准。
将反应用各种试剂及包被的反应板组装成试剂盒,同时保存于室温、4℃和-20℃,定期进行检测,结果表明:试剂盒在室温保存时极不稳定,保存期限仅为1个月,4℃可保存4个月,可在-20℃稳定保存8个月以上。
In this study, hyper-immunized goat serum was prepared by immunizing for three times with inactive purified H5N1 AIV (DZJ/1100) mixed with Freund’s adjuvant or incomplete Freund’s adjuvant. The antigen capture ELISA was developed using polyclonal anti-sera (goat serum against AIV) as capture antibody and H5 specificity monoclonal antibody as detecting antibody for detecting H5 AIV. The optimal conditions were determined: capture anti-sera was diluted for 1:1600, H5 AIV specificity McAb was diluted for 1:10000 and the enzyme-labeled antibody was diluted for 1:5000. The optimal reactive time of the detecting antibody and conjugate were one and half an hour separately in the assay.
NDV、EDS76、IBV、ILTV、MDV、APV and H1-H15 AIV were detected by the sandwich ELISA and the result proved that there was no crossing-reaction between the antigen capture ELISA and other avian viruses and others fourteen AIV except H5. It proved that the H5 AIV antigen capture ELISA had excellent specificity.
Comparing with HA test and egg inoculation test, the sensitivity of the antigen capture ELISA was twice better than that of HA test, but it was less than that of egg inoculation test.
Many experimently infected clinical samples were detected by the sandwich ELISA under the most optimal conditions. Finally, spleen, lung, heart and liver were defined as target organ when clinical samples were detected by the ELISA and the cut-off value of spleen was 0.15 and others were 0.1.
The ELISA kit which consisted of coated microplate and reactive solutions were kept in room temperature, 4℃and -20℃seperately. The detection results showed that kits had bad stability in room temperature and the period of validity was only one month. At the same time, the period of validity in 4℃was 4 months and over 8 months in -20℃.
引文
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