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 α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-amylase levels and negatively impact flour quality, resulting in substantial economic losses....
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-11-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/12/22/3798 |
| _version_ | 1797457968966402048 |
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| author | Amber L. Hauvermale Courtney Matzke Gamila Bohaliga Mike O. Pumphrey Camille M. Steber Andrew G. McCubbin |
| author_facet | Amber L. Hauvermale Courtney Matzke Gamila Bohaliga Mike O. Pumphrey Camille M. Steber Andrew G. McCubbin |
| author_sort | Amber L. Hauvermale |
| collection | DOAJ |
| description | Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-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 α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-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 <i>E. coli</i> 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 α-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. |
| first_indexed | 2024-03-09T16:31:22Z |
| format | Article |
| id | doaj.art-5d623903f8ff4505bc273deb021558f5 |
| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
| last_indexed | 2024-03-09T16:31:22Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Plants |
| spelling | doaj.art-5d623903f8ff4505bc273deb021558f52023-11-24T15:01:49ZengMDPI AGPlants2223-77472023-11-011222379810.3390/plants12223798Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate ChangeAmber L. Hauvermale0Courtney Matzke1Gamila Bohaliga2Mike O. Pumphrey3Camille M. Steber4Andrew G. McCubbin5Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USASchool of Biological Sciences, Washington State University, Pullman, WA 99164, USADepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USADepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USADepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USASchool of Biological Sciences, Washington State University, Pullman, WA 99164, USAAccurate, rapid testing platforms are essential for early detection and mitigation of late maturity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-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 α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-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 <i>E. coli</i> 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 α-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.https://www.mdpi.com/2223-7747/12/22/3798wheatalpha-amylasemonoclonal antibodiesimmunoassayenzyme-linked immunosorbent assaypreharvest sprouting |
| spellingShingle | Amber L. Hauvermale Courtney Matzke Gamila Bohaliga Mike O. Pumphrey Camille M. Steber Andrew G. McCubbin Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change Plants wheat alpha-amylase monoclonal antibodies immunoassay enzyme-linked immunosorbent assay preharvest sprouting |
| title | Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change |
| title_full | Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change |
| title_fullStr | Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change |
| title_full_unstemmed | Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change |
| title_short | Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change |
| title_sort | development of novel monoclonal antibodies to wheat alpha amylases associated with grain quality problems that are increasing with climate change |
| topic | wheat alpha-amylase monoclonal antibodies immunoassay enzyme-linked immunosorbent assay preharvest sprouting |
| url | https://www.mdpi.com/2223-7747/12/22/3798 |
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