Maximizing the detection capability of a beet western yellows virus ELISA system

Conditions for maximizing detection of a California isolate of beet western yellows virus (BWYV) were investigated with the double-sandwich enzyme-linked immunosorbent assay (ELISA) system. Within-plate variability was found to account for less than 1% of the total variation observed on individual microtiter plates. Variability across plates was greater than within plates and accounted for less than 10% of the total variation. Significant reductions in absorbance were observed when either coating or conjugated IgGs were incubated for 2 h at 37°C. Incubation of coating IgGs overnight at 4 or 22°C gave good results. No differences in absorbance were observed when antigen sources were incubated at 37, 22, or 4°C. Absorbance at 1, 3 or 6 h incubation of conjugated IgGs at 4 or 22°C was low to moderate but after 18 or 24 h absorbance was markedly increased without increasing background. Efficient extraction of virus from host and vector tissues increased absorbance. Carborundum added to the extraction buffer worked well when tissue samples were ground in a mortar and pestle but highest absorbance resulted when test samples were prepared with a high-speed tissue homogenizer. Background levels were unacceptably high when 10 or more Myzus persicae were pooled for analysis. Since virus can be detected in 3 or even 1 aphid, pooling for detection is unnecessary. Optimization of the BWYV ELISA system made possible the detection of virus quantities less than 2 ng/ml.