Dot-immunobinding assay for zucchini yellow mosaic virus using polyclonal and monoclonal antibodies.

Abstract

The application of dot-immunobinding assay (DIA) for detecting zucchini yellow mosaic virus (ZYMV) was described. The virus showed the same binding activity to nitrocellulose membrane (NCM) in three kinds of coating buffers tested. Antigenicity of ZYMV in DIA could be maintained in leaf extract-dotted NCM which had been stored at -20C, 6C, or 20C at least for six months. The methods were developed for sending samples using infected leaves extract-dotted NCM in replace of fresh leaves and for simplifying the procedure for direct deposition of the samples onto NCM. Seven protocols of DIA tested proved to be applicable to discriminate between healthy and infected pumpkin, with limits of detection ranging from 10-3∼10-5 dilution of leaf extracts. Rabbit polyclonal antibody against ZYMV cross-reacted with watermelon mosaic virus 2 (WMV-2) in two-step direct-DIA and indirect-DIA but not in DAS-DIA. It did not also cross-react with papaya ringspot virus, type W (PRSV-W) in three DIA protocols. When the three DIA protocols were used with monoclonal antibody against ZYMV, the assays resulted in the same sensitivity as those obtained by using polyclonal antibody, and could also be used to discriminate ZYMV from PRSV-W and WMV-2.