Kulkarni Diwakar D, Venkatesh Govindarajalu, Tosh Chakradhar, Patel Priyanka, Mashoria Anita, Gupta Vandana, Gupta Sourabh, D Senthilkumar
a ICAR - National Institute of High Security Animal Diseases, Anand Nagar , Bhopal , India.
b Pinnacle Biomedical Research Institute , Bhopal , India.
J Immunoassay Immunochem. 2016;37(2):154-66. doi: 10.1080/15321819.2015.1074922.
The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.
基于重组病毒蛋白的间接酶联免疫吸附测定（ELISA）是一种经济高效、安全、特异且快速的病毒感染诊断工具。尼帕病毒核衣壳（NiV-N）蛋白在大肠杆菌中表达，并通过基于组氨酸标签的亲和色谱法进行纯化。选择N蛋白是基于其免疫优势以及在不同尼帕病毒株中的保守性。使用重组NiV-N蛋白作为抗原，连同阴性和阳性对照，优化了针对猪血清的间接免疫球蛋白G（IgG）酶联免疫吸附测定（ELISA）。使用脱脂奶粉和鸡血清封闭背景读数。用间接ELISA和蛋白质印迹法检测了来自印度不同邦的总共1709份猪血清样本。仅当其总反应性读数高于0.2的临界值且总反应性与背景读数的比值大于2.0时，该检测才被视为阳性。由于蛋白质印迹法的特异性较高，因此将其用作比较间接ELISA结果的标准检测方法。与蛋白质印迹法相比，间接ELISA的灵敏度和特异性分别为100%和98.7%。基于重组N蛋白的ELISA可用于在没有高防护实验室来处理这种人畜共患病毒的发展中国家筛查大量血清样本以进行流行病学调查。
