Detection and identification of plant viruses by ELISA using crude sap extracts and unfractionated antisera

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.