Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change

Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity alpha-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated alpha-amylase levels and negatively impact flour quality, resulting in substantial economic losses. The Hagberg-Perten Falling Number (FN) method is the industry standard for measuring alpha-amylase activity in wheatmeal. However, FN does not directly detect alpha-amylase and has major limitations. Developing alpha-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of alpha-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature grain. These were cloned and used to develop E. coli expression lines expressing single isoforms. After assessing amino acid conservation between isoforms, we identified peptide sequences specific to a single isoform (TaAMY1) or that were conserved in all isoforms, to develop monoclonal antibodies with targeted specificities. Three monoclonal antibodies were developed, anti-TaAMY1-A, anti-TaAMY1-B, and anti-TaAMY1-C. All three detected endogenous alpha-amylase(s). Anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4. Thus, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.

Automated summary

Accurate and rapid testing platforms are crucial for early detection and mitigation of late maturity alpha-amylase and preharvest sprouting in wheat. Researchers have successfully developed three monoclonal antibodies with targeted specificities for detecting alpha-amylase using an immunoassay method.