Improved ELISA for the detection of barley yellow dwarf virus in grasses

BYDV, a phloem restricted luteovirus, occurs in very low concentration in grasses. Several modifications to the virus extraction procedure were tested for their effect on ELISA detection of BYDV from grasses. The most efficient grinding method was to dip plastic bags containing samples in liquid nitrogen prior to crushing with a hand roller. Overnight incubation of samples at 25°C in 0.1 M citrate buffer, pH 6.0, or in 0.1 M phosphate buffer, pH 7.0, prior to testing increased the optical density (OD) obtained without increasing the background. However, the highest OD was obtained when celluclast® was added to the incubation buffer. The end point for detection was in general one to two steps higher with incubation and one further step higher after adding enzyme to the incubation buffer.