Petri dish-Agar Dot Immunoenzymatic Assay (PADIA) - a new and inexpensive method for the detection and identification of plant viruses

A simple and economical enzyme immunoassay technique using a polystyrene Pe tri dish as the solid support and a substrate mixed with agar to visualize enzymatic activity was developed and standardized for the detection of plan t viruses. Reagents were added as drops into circular areas bounded with a hydrophobic cryomarker pen on the inner surface of the Petri dish. After su bsequent incubation and washing steps with antiserum and alkaline phosphata se conjugate, the substrate (5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium) solution was mixed with warm agar and added to the inner Petri dish surface. Distinct dark blue to purple dots were formed on the ag ar matrix at the sites where the virus was trapped. The technique is tentat ively called `Petri dish-Agar Dot Immunoenzymatic Assay' (PADIA). In additi on to substituting the expensive microtitre plate or nitrocellulose membran e with a plastic Petri dish, PADIA consumes 5-10 times less reagents (antis erum and enzyme conjugate) than conventional ELISA or dot immunobinding ass ay (DIBA). The total assay cost was at least four times less than that of c onventional ELISA. The technique was as sensitive as ELISA. PADIA can be us ed in poorly equipped laboratories with a minimum of input.