A two-step ELISA for rapid,reliable detection of potato viruses

The reliability of the standard double antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA) was compared with a shorter, two-step DAS procedure in which sample and conjugate were mixed and incubated together in one step. The two assays were compared using beet western yellows virus and potato leafroll, M, S, X, and Y viruses. The two-step procedure was more sensitive,i.e., it detected small quantities of virus with greater statistical reliability than the standard procedure. At high virus concentrations, the standard produced stronger ELISA reactions than the two-step assay, but both assays were reliable. Since all of the viruses tested withstood high incubation temperatures, the incubation period for the two-step procedure could be reduced to 1 hr at 30 or 37 C. Therefore, assays could be completed within 2 hr using the two-step procedure compared with 2 days for the standard procedure. Reliable results were achieved with samples prepared by grinding tissues in buffer or, more simply, by adding pure, pressure extracted juice directly to conjugate in assay wells. Coating plates with gamma globulins or with F(ab′)₂ fragments of gamma globulins gave equally reliable results with all viruses except potato leafroll, where coating with gamma globulins was superior.