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ARS Home » Pacific West Area » Pullman, Washington » WHGQ » Research » Publications at this Location » Publication #408905
Research Project: Wheat and Barley Adaptation to a Changing Climate - Discovery of Genetic and Physiological Processes for Improved Crop Productivity and Quality

Location: Wheat Health, Genetics, and Quality Research

Title: Development of novel monoclonal antibodies to wheat alpha-amylases associated with grain quality problems that are increasing with climate change

Author
item HAUVERMALE, AMBER - Washington State University
item MATZKE, COURTNEY - Washington State University
item BOHALIGA, GAMILA - Washington State University
item PUMPHREY, MICHAEL - Washington State University
item Steber, Camille
item MCCUBBIN, ANDREW - Washington State University

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2023
Publication Date: 11/8/2023
Citation: Hauvermale, A.L., Matzke, C., Bohaliga, G., Pumphrey, M., Steber, C.M., McCubbin, A.G. 2023. Development of novel monoclonal antibodies to wheat alpha-amylases associated with grain quality problems that are increasing with climate change . Plants. 12(22). Article 3798. https://doi.org/10.3390/plants12223798.
DOI: https://doi.org/10.3390/plants12223798

Interpretive Summary: This study explores the development of an immunoassay as an alternative to the Hagberg-Perten falling numbers method for detecting alpha-amylase in wheat grain. Wheat grain yield and quality can be compromised by the presence of the starch-degrading enzyme alpha-amylase as a result of preharvest sprouting or of late-maturity alpha-amylase (LMA). Too much alpha-amylase can result in cakes that fall and in sticky bread and noodles. Three of the four wheat alpha-amylases were found to be expressed in grain. Antibodies were raised to these antibodies, and found to detect alpha-amylase in a wide range of wheat varieties. This work demonstrated the plausibility of using two antibody combintations to detect alpha-amylase, making it possible to develp a quick and easy "dipstick" lateral flow immunoassay.

Technical Abstract: Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity a-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated a-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 a-amylase activity in wheatmeal. However, FN does not directly detect a-amylase and has major limitations. Developing a-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed expression of a-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature seeds. 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 a-amylase(s), anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.