Identification of Antigens for Food Allergy and Food Allergen Detection

Authors

Ellenor Sell, University of Nebraska-LincolnFollow

First Advisor

Philip E. Johnson

Second Advisor

Joseph L. Baumert

Committee Members

Rick Goodman, Heather Hallen-Adams

Date of this Version

5-2025

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College of 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 Professors Philip E. Johnson and Joseph L. Baumert

Lincoln, Nebraska, May 2025

Comments

Copyright 2025, Ellenor Sell. Used by permission

Abstract

Antibody-based allergen testing is an essential part of allergen management in the food industry. These methods directly detect one or more molecules of the allergenic foodstuff (target’ molecule(s)) and the presence of the allergenic foodstuff is inferred. The identity of these target molecules is crucial to the performance of the method and the interpretation of results. The goal of this study was to develop protein/antibody capture methodology for the identification of antibody binding partners using mass spectrometry.

This study is broken down into three separate projects: the characterization of the antigen-antibody complexes present in sandwich ELISAs, LFDs, and human sera. For the ELISA project, three commercial peanut ELISAs were optimized for analysis by LCMS. Data-dependent acquisition (DDA) analysis of a hazelnut RBioPharm^® RIDASCREEN^® ELISA and a soy RBioPharm^® RIDASCREEN^® ELISA were used to demonstrate the feasibility of the work. Subsequent PRM analysis, using a method for the detection and quantification of peanut allergens, was used to confirm antibody binding targets. We could detect and quantify binding targets for the antibodies immobilized on the ELISA plate. As expected, these differed by kit. Interestingly, thermal processing of peanut affected the profile of antibody binding partners.

In the LFD project, a Neogen^® Reveal^® 3-D for Peanut LFD was exposed to 1,000 ppm, and each distinct section of the LFD was analyzed by PRM. Results appeared to reflect migration of proteins across the membrane as well as antibody specificity. Interestingly, different peanut proteins have different migration characteristics, a finding that may improve selection of potential targets for future methods.

In order to examine the feasibility of characterizing antigen-specific antibodies within human sera, two antibody-coupling tools were tested for their ability to pull the antigen-antibody complex from sera. DDA analysis was performed on peanut protein exposed IgG coupled to ThermoScientific^TM Invitrogen^TM Dynabeads, and silver staining was performed on peanut exposed IgE coupled to ThermoScientific^TM CaptureSelect^TM IgE Affinity Matrix. Neither of these tools appeared suitable for the identification of binding partners, even using monoclonal IgE in large quantities.

Advisors: Philip E. Johnson and Joseph L. Baumert