Abstract
Indirect enzyme-liked immunsorbent assay (I-ELISA) on plates not precoated with antibody was evaluated for detecting zucchini yellow mosaic virus (ZYMV) in either purified preparation or infected plants. The suitable conjugate, method of bovine serum albumin treatment, and extraction medium for the I-ELISA are described. Alkaline phosphatase labeled affinity-purified goat anti-rabbit IgG (H+L) (commercial conjugate) was superior to our prepared conjugate of the alkaline phosphatase because the former conjugate resulted in enhancement of specific absorbance and reduction of non-specific absorbance. In the assay with commercial alkaline phosphatase conjugate, this procedure was more sensitive than double antibody sandwich (DAS)-ELISA, capable of detecting purified ZYMV at minimum concentration of 1-5ng/ml compared with 5-10ng/ml for DAS-ELISA. The dilution end points of crude edxtracts for identical leaves assayed with I-ELISA and DAS-ELISA were 106-107 and 105-106, respectively. It was found that non-precoated I-ELISA with commercial alkaline phosphatase conjugate is a useful tool for disease diagnosis of field samples. Since the endogenous peroxidase activity in ZYMV infected pumpkin was very high, the use of peroxidase labeled affinity-purified goat anti-rabbit IgG in non-precoated I-ELISA for detecting ZYMV in crude extracts could not be recommended.
