Development of a field enzyme-linked immunosorbent assay (ELISA) for detection of α-amylase in preharvest-sprouted wheat

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for detection of alpha-amylase in preharvest sprouted wheat and adapted to rapid field-use formats requiring 15-20 min to perform. Polyclonal and monoclonal antibodies were prepared to detect a mixture of high and low pi isozymes of alpha-amylase and high pI isozymes only. All antibodies detected alpha-amylase on immunoblots of either a crude wheat extract or of purified enzyme, but only the polyclonal antibodies functioned in a sandwich ELISA. Depending on the antibody combination, the tube ELISA detected either the high and low pI isozymes of alpha-amylase or the high pi isozymes only with a detection limit of approximate to 0.5-1.0 ng/mL of amylase. Wheats with falling numbers (FN) of <350 sec could be discriminated from sound wheats, with decreasing FN producing increasing assay color. Using 130 wheat grain samples, ELISA absorbances for detection of both high and low pI isozymes and of high pI isozymes only were highly positively correlated with amylase enzyme activity and negatively correlated with FN. The correlations were similar for detection of both isozyme families and for detection of high pi isozymes only. Analyses of three sets of wheat samples from different environments demonstrated that the relationship between ELISA absorbance and PN had little dependence on wheat cultivar. The precision of sample analysis using the field ELISA was similar to the precision of FN test apparatus.