Reducing agents interfere with the detection of lettuce necrotic yellows virus in infected plants by immunoblotting with monoclonal antibodies.

A procedure is described for the detection of lettuce necrotic yellows virus (LNYV) nucleocapsid protein (N) or envelope glycoprotein (G) by immuno-blotting with their respective monoclonal antibodies. The antigens can be detected in 1-10 mg of fresh tissue from systemically infected Nicotiana glutinosa leaves showing prominent symptoms. The composition of the extraction buffer played a crucial role in the recovery of antigenically active proteins. Procedures involving tissue extraction in the presence of the reducing agents 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) failed to detect either the N or G antigen. For optimum detection of the N or G antigens, leaf tissue was ground with 10 mM phosphate buffer, pH 7.6 (PB), containing 3% (w/v) sodium dodecyl sulphate (SDS) and 1 mM of the protease inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK). The extract was then mixed with an equal volume of dissociation buffer containing 2-ME before electrophoresis and immunoblotting.