Mowat W P, Dawson S
J Virol Methods. 1987 Feb;15(3):233-47. doi: 10.1016/0166-0934(87)90101-7.
A simple and rapid procedure of enzyme immunoassay (PTA-ELISA) was used to detect and identify viruses in individual plants. Virus antigen in crude leaf extracts was adsorbed directly to a solid-phase support, allowed to react with unfractionated antiserum and the antigen-antibody complex detected with a general purpose conjugate of protein A and enzyme. Viral antigens were trapped most effectively by high bonding polystyrene microtitre plates loaded with leaf extracts prepared in carbonate buffer at pH 9.6. With protein A-alkaline phosphatase conjugate and the substrate p-nitrophenyl phosphate as the antibody-detection system, 18 plant viruses in 8 virus groups were detected reliably and nonspecific reactions did not occur. However, when the substrate 3,3',5,-tetramethyl benzidine was used in conjunction with protein A-horseradish peroxidase conjugate, nonspecific reactions were given by leaf extracts from some uninfected or virus-infected plant species. Where less sensitivity is required than is provided by versions of ELISA that rely on antibody-captured antigen, this method provides a simple and rapid means of detecting and identifying viruses in crude sap extracts with the aid of unfractionated antisera.
采用一种简单快速的酶免疫测定方法（PTA - ELISA）来检测和鉴定单株植物中的病毒。粗叶提取物中的病毒抗原直接吸附到固相载体上，与未分级的抗血清反应，然后用蛋白A和酶的通用偶联物检测抗原 - 抗体复合物。用pH 9.6的碳酸盐缓冲液制备的叶提取物加载到高结合力聚苯乙烯微量滴定板上时，病毒抗原捕获效果最佳。以蛋白A - 碱性磷酸酶偶联物和对硝基苯磷酸酯作为抗体检测系统，可靠地检测出了8个病毒组中的18种植物病毒，且未出现非特异性反应。然而，当底物3,3',5 - 四甲基联苯胺与蛋白A - 辣根过氧化物酶偶联物一起使用时，一些未感染或病毒感染植物物种的叶提取物会出现非特异性反应。在对灵敏度要求低于依赖抗体捕获抗原的ELISA方法的情况下，该方法借助未分级抗血清为检测和鉴定粗汁液提取物中的病毒提供了一种简单快速的手段。
