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  • Author or Editor: Susan R. Newhart x
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Abstract

The enzyme-linked immunosorbent assay (ELISA) was used for the detection of tobacco ringspot virus (TRSV) in crude leaf extracts of geranium (Pelargonium × hortorum Bailey). An ELISA test protocol using a coating antibody concentration of 10 μg/ml, a 1000-fold dilution of antibody-enzyme conjugate, a substrate reaction time of 30-45 minutes, and a leaf test sample prepared in 20 volumes of phosphate buffer provided the best compromise between test reliability, conservation of reagents, and convenience. Using this protocol, significantly positive absorbante values (A405nm) were obtained with extracts from geraniums latently-infected with TRSV whereas color development form noninfected geraniums was negligible. Potentially, the ELISA represents a rapid, reliable, economical, and convenient method for virus-indexing geraniums commercially.

Open Access

Abstract

Several factors which influence the detection of tobacco ringspot virus (TRSV), by the enzyme linked immunosorbent assay (ELISA) in leaf extracts of geranium (Pelargonuim × hortorum Bailey), were examined. Crude leaf extracts could be used in ELISA to rapidly index geraniums for TRSV, providing measures were taken to minimize the effect of leaf substances which were inhibitory to the ELISA reaction. Optimal detection of TRSV (50 ng/ml) was obtained by extracting leaves in a 20-fold excess (w/v) of either 0.02M phosphate buffer, pH 7.4 containing 0.15M NaCl, 0.05% Tween 20, and 2% polyvinyl pyrrolidone or 0.1M phosphate buffer, pH 7.0 containing 4% polyethylene glycol. Clarification of the extract with polyethylene glycol and NaCl provided a 5-fold higher test sensitivity. ELISA proved useful for the detection of TRSV in both clonal and seedling geranium leaves as well as in geranium seed. The test was found to be more reliable than a bioassay for the detection of virus in the latent stage of infection.

Open Access