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

Abstract

The reliability of enzyme-linked immunosorbent assay (ELISA) to detect cucumber mosaic cucumovirus (CMV) coat protein (CP) in three weed species (Taraxacum officinale, Gnaphalium obtusifolium, and Conyza canadensis) was compared with other detection methods, including Western blot analysis and a bioassay. Analysis of extracts prepared from these species by double antibody sandwich (DAS) ELISA yielded high absorbance values (A4O5nm) that were indicative of CMV concentrations of purified CMV standards of greater than 50 mg/mL. However, results from an ELISA procedure designed such that no viral antigen should be detected suggested that the strong ELISA absorbance values were nonspecific reactions with material(s) in the weed extracts. These findings were substantiated when no CMV CP was detected by Western blot analysis or infectious CMV was detected in bioassays involving inoculation of extracts onto an indicator host (Cucurbita pepo ‘Crookneck’ (squash)). In both DAS ELISA and indirect ELISA procedures, the nonspecific reaction was shown to result from the interaction of weed extract and alkaline phosphatase-conjugated immunoglobulin (including anti-CMV and goat anti-rabbit). Addition of bovine serum albumin to the extraction buffer for DAS ELISA or to the extraction buffer and at each antibody step for indirect ELISA dramatically reduced the nonspecific reactions.