Evaluation of Wheat-Specific Peptide Targets for Use in the Development of ELISA and Mass Spectrometry-Based Detection Methods

Authors

Jessica Humphrey, University of Nebraska-LincolnFollow

First Advisor

Joseph L. Baumert

Date of this Version

11-2022

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Food Science and Technology

Under the supervision of Professor Joseph L. Baumert

Lincoln, Nebraska, November 2022

Comments

Copyright 2022, Jessica Humphrey

Abstract

Individuals with IgE-mediated wheat allergy and celiac disease must avoid the consumption of wheat. Those with celiac disease must also avoid rye and barley in addition to wheat as these make up the gluten-containing grains. To ensure products are accurately labeled, ELISA methods may be used to monitor compliance with food allergen and gluten-free regulations. Currently, available ELISA methods for wheat detection utilize monoclonal antibodies that target peptides from the gluten fraction of proteins, and therefore, also detect rye and barley. Limitations such as differing antibody reactivity and unequal gluten content among wheat, rye, and barley have led to inconsistent results. A newer method, the RIDASCREEN^® Total Gluten ELISA kit, uses several monoclonal antibodies to detect gluten proteins across the gluten-containing grains. To evaluate this ELISA kit in comparison with the RIDASCREEN^® Gliadin ELISA kit, different concentrations of gluten-containing grains spiked into flour backgrounds were analyzed. The results indicated that the Total Gluten kit quantified the gluten concentration more accurately across the spiked samples compared to the Gliadin kit. While the Total Gluten kit improves upon the limitations mentioned, the food industry remains unable to distinguish the gluten-containing grains to determine which grain caused a positive analytical result. This creates a challenge under food allergen regulation because wheat is a priority allergen. Therefore, the food industry needs a wheat-specific ELISA method. To accomplish this, this study utilized combined experimental and bioinformatics approaches which resulted in the identification of nine wheat-specific and ELISA-compatible peptides. Upon further analysis, it was deemed that these peptides may not be robust in an ELISA detection method. Because mass spectrometry-based methods can distinguish between single amino acid variances in peptides, however, the same dataset could be used to identify peptides for a mass spectrometry method. Using a similar filtration strategy combined with a series of targeted mass spectrometry analyses, three specific peptides which detect wheat and one specific peptide for each of the other grains, rye, barley, and oat were identified.

Advisor: Joseph L. Baumert