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

Abstract

A simple and economical enzyme immunoassay technique using a polystyrene Petri dish as the solid support and a substrate mixed with agar to visualize enzymatic activity was developed and standardized for the detection of plant viruses. Reagents were added as drops into circular areas bounded with a hydrophobic cryomarker pen on the inner surface of the Petri dish. After subsequent incubation and washing steps with antiserum and alkaline phosphatase conjugate, the substrate (5-bromo-4-chloro-3-indolyl phosphate and nitroblue 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 agar matrix at the sites where the virus was trapped. The technique is tentatively called 'Petri dish-Agar Dot Immunoenzymatic Assay' (PADIA). In addition to substituting the expensive microtitre plate or nitrocellulose membrane with a plastic Petri dish, PADIA consumes 5-10 times less reagents (antiserum and enzyme conjugate) than conventional ELISA or dot immunobinding assay (DIBA). The total assay cost was at least four times less than that of conventional ELISA. The technique was as sensitive as ELISA. PADIA can be used in poorly equipped laboratories with a minimum of input.