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The immunodiffusion test, especially the Ouchterlony method has been widely used in plant virus serology. In addition to its simplicity and less equipment required, the main advantage of this method is that it can provide a visible demonstration of the relationships between antigens and antisera. However, its sensitivity in detecting antigen is much lower than that of enzyme-linked immunosorbent assay (ELISA) or immunoelectron microscopy (IEM)1). A conventional method performed in agar gel requires a relatively longer period to obtain the result. Nevertheless, the relatively higher sensitivity, coupled with shorter incubation time, makes the immunodiffusion procedure a useful technique for identification as well as classification of plant viruses. Gelrite (Kelco Div., Merck & Co. Inc., San Diego, Calif.) is an agar-like polysaccharide produced by Pseudomonas elodea. It forms a gel with good thermal stability and better optical clarity in the presence of a cation such as Mg++2). It has been shown to compare favorably with agar gel for the growth of various microorganisms3,4). This investigation was undertaken to examine the suitability of Gelrite as an agar substitute for the immunodifusion technique in plant virus serology. Viruses, antisera, and procedure. Cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), zucchini yellow mosaic virus (ZYMV) isolates and their antisera5-7) were used. Gelrite and a cation in 0.1M Tris-HCl buffer pH 8.0, containing 0.85% NaCl and 0.2% NaN3 with/without 0.3% SDS, were heated to dissolve in a microwave oven. One and a half ml of the gel were spread onto a microscope slide and cooled. Ten ƒÊl of purified virus, infected-leaf sap, or antiserum were dispensed into wells. The slides were incubated at room temperature in a moist chamber. A gel containing 0.5% Bacto-Agar (Difco Lab., Detroit, Michigan) in the same buffer, which has been considered one of the most sensitive system for immunodiffusion5), was uesd as an agar control.

Use of gelrite as a gelling agent in immunodiffusion tests for identification of plant virus antigens.