Detection of cucumber mosaic cucumovirus in weed species: a cautionary report on nonspecific reactions in ELISA

The reliability of enzyme-linked immunosorbent assay (ELISA) to detect cucu mber mosaic cucumovirus (CMV) coat protein (CP) in three weed species (Taxa cum officinale, Gnaphalium obtusifolium, and Conyza canadensis) was compare d with other detection methods, including Western blot analysis and a bioas say. Analysis of extracts prepared from these species by double antibody sa ndwich (DAS) ELISA yielded high absorbance values (A(405nm)) that were indi cative of CMV concentrations of purified CMV standards of greater than 50 m g/mL. However, results from an ELISA procedure designed such that no viral antigen should be detected suggested that the strong ELISA absorbance value s were nonspecific reactions with material(s) in the weed extracts. These f indings were substantiated when no CMV CP was detected by Western blot anal ysis or infectious CMV was detected in bioassays involving inoculation of e xtracts onto an indicator host (Cucurbita pepo 'Crookneck' (squash)). In bo th DAS ELISA and indirect ELISA procedures, the nonspecific reaction was sh own to result from the interaction of weed extract and alkaline phosphatase -conjugated immunoglobulin (including anti-CMV and goat anti-rabbit). Addit ion of bovine serum albumin to the extraction buffer for DAS ELISA or to th e extraction buffer and at each antibody step for indirect ELISA dramatical ly reduced the nonspecific reactions.