Chen Ji-Ming, Yu Meng, Morrissy Chris, Zhao Yong-Gang, Meehan Greer, Sun Ying-Xue, Wang Qing-Hua, Zhang Wei, Wang Lin-Fa, Wang Zhi-Liang
Chinese National Diagnostic Center for Exotic Animal Diseases, Chinese National Animal Quarantine Institute, Qingdao 266032, China.
J Virol Methods. 2006 Sep;136(1-2):273-6. doi: 10.1016/j.jviromet.2006.05.003.
The indirect ELISA is a simple and useful method for detection of pathogen-specific antibodies in animal sera. However, non-specific or background binding is often a problem, especially when recombinant proteins from Escherichia coli are used. In this study, a comparative indirect ELISA in which the total reactivity and the background binding were determined simultaneously on the same ELISA plate was reported. The background was determined by incubation of the test sera with excess free antigen to block specific binding. The sample was considered positive only when its total reactivity reading was higher than a pre-determined cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Using this approach, an antibody assay for henipaviruses using a recombinant Nipah virus nucleocapsid protein expressed in E. coli was developed. A total of 919 negative serum samples were tested in this assay and the specificity was 95.8%. In addition, eight positive experimental serum samples all tested positive. The use of recombinant protein as the ELISA antigen, instead of inactivated virus antigens, will be of significant advantage for countries where there is no facility of Biosafety level 4 to handle this group of zoonotic viruses.
间接酶联免疫吸附测定（ELISA）是检测动物血清中病原体特异性抗体的一种简单且有用的方法。然而，非特异性或背景结合常常是个问题，尤其是当使用来自大肠杆菌的重组蛋白时。在本研究中，报道了一种比较性间接ELISA，该方法在同一ELISA板上同时测定总反应性和背景结合。背景通过将检测血清与过量游离抗原孵育以阻断特异性结合来确定。仅当样品的总反应性读数高于预先确定的临界值且总反应性与背景读数之比大于2.0时，该样品才被视为阳性。采用这种方法，开发了一种使用在大肠杆菌中表达的重组尼帕病毒核衣壳蛋白检测亨尼帕病毒抗体的检测方法。在此检测方法中总共检测了919份阴性血清样本，特异性为95.8%。此外，8份阳性实验血清样本全部检测为阳性。对于没有生物安全4级设施来处理这类人畜共患病毒的国家而言，使用重组蛋白作为ELISA抗原而非灭活病毒抗原具有显著优势。
