Evaluation of commercial a-amylase enzyme-linked immunosorbent assay (ELISA) test kits for wheat

Authors: Kiszonas, Alecia; Morris, Craig

Submitted to: Cereal Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/28/2017
Publication Date: 3/6/2018
Citation: Kiszonas, A., Morris, C.F. 2018. Evaluation of commercial a-amylase enzyme-linked immunosorbent assay (ELISA) test kits for wheat. Cereal Chemistry. 95:206-210.

Interpretive Summary: The present report evaluated the potential to use commercial ELISA kits to detect wheat a-amylase. An extensive internet search yielded no commercial ELISA kits for wheat, barley or rice a-amylase. A single kit identified as “plant a-amylase” was found and was included here. Two additional kits targeting human AMY1 and AMY2 were also included. Three wheat samples were chosen to evaluate the ELISA kits. A sample of the soft white winter wheat variety Diva, known to have sound grain, was used as the control. A subsample of Diva was sprouted in the lab and then dried in a low-temperature (130ºC) oven for eight hours. These samples were the ‘PHS Diva’ samples. The soft white winter wheat variety Xerpha had high a amylase levels from a location identified as being affected by both PHS and LMA. As such, Xerpha was considered the ‘PHS/LMA control’ sample. All grain was ground with the Udy grinder and samples were assayed for a-amylase using the Megazyme kit following the manufacturer’s instructions. a-Amylase is one of the most widespread enzymes in nature. Although all share a commonality of catalytic function, an enormous range in primary and secondary structures exist. As such, we consider it highly serendipitous that the AMY2 kit appears to be detecting increased a-amylase levels associated with PHS and LMA.

Technical Abstract: a-Amylase enzyme is associated with preharvest sprouting (PHS) and late-maturity a amylase (LMA) in wheat, and reduces wheat and flour quality. Various means have been developed to measure the presence of a-amylase, thereby predicting end-use quality; most are based on enzyme activity. An alternative technology uses ELISA (enzyme linked immunosorbent assay) to detect the presence of proteins, but not activity. The present report evaluated the potential to use commercial ELISA kits to detect wheat a-amylase. Three kits were evaluated: human AMY1 and AMY2, and a “plant a-amylase” kit. Three wheat samples were chosen to evaluate the ELISA kits, one sound, one lab sprouted, and one experiencing field PHS and LMA. Samples were also assayed for a-amylase activity using the Megazyme method. All three ELISA kits functioned very well based on calibration curves (R2 > 0.99). Of the three, the AMY1 and plant a-amylase kits appeared to show no useful detection of wheat a amylase. The AMY2 kit however, detected a 2.7 fold increase in a-amylase protein in the PHS and PHS/LMA grain lots. As such, it appears that this ELISA kit, and perhaps others, can detect a amylase in wheat and may be useful in predicting PHS and LMA potential damage.