Differentiating biologically relevant from irrelevant IgE binding to food antigens for improved risk assessment and diagnostic studies using a humanized rat basophil cell line (RBL 30/25)EPA Grant Number: R834065
Title: Differentiating biologically relevant from irrelevant IgE binding to food antigens for improved risk assessment and diagnostic studies using a humanized rat basophil cell line (RBL 30/25)
Investigators: Goodman, Richard E.
Institution: University of Nebraska at Lincoln
EPA Project Officer: McOliver, Cynthia
Project Period: May 1, 2009 through March 31, 2011 (Extended to April 30, 2012)
Project Amount: $372,340
RFA: Exploratory Investigations in Food Allergy (2007) RFA Text | Recipients Lists
Research Category: Food Allergy , Health Effects , Health
New genetically engineered food products and novel food ingredients are currently tested by in vitro immunoglobulin E (IgE) binding to evaluate the potential risk of food allergy for consumers. False positive results during premarket approval or post-market testing could raise serious concerns about food safety or cause rejection of safe products and false negatives can increase health risks. Accurate tests are needed to evaluate the potential human health effects, mainly allergenicity, of pesticide proteins introduced into foods through genetic engineering of crops. This project will improve toxicity testing for pesticide proteins introduced into foods through genetic engineering by developing a functional assay to test immune reactions in cell culture and measuring biologically relevant immunoglobulin E (IgE) binding to determine the potential for false positive results using the current binding assay. The proposed tests are expected to demonstrate the utility of in vitro basophil cells to determine whether antigen-specific IgE would likely cause allergic reactions in allergic serum donors or whether binding is ineffective.
The project objectives are to: 1) Develop a standardized basophil activation assay to assess the biological activity of human IgE that is cross-reactive in static binding assays; 2) Use the basophil assay to evaluate whether IgE specific to complex carbohydrates on plant glycoproteins is responsible for false positive results; 3) Identify biologically relevant cross-active proteins (peptides) of related legume seeds that induce basophil histamine release through allergen specific IgE binding.
We will develop a functional assay in a stable cell line (rat basophil cells that express human IgE receptors) as an alternative to the current method of measuring the binding of IgE in the serum of subjects allergic to food proteins. Legume allergic sera collected from the US, EU and India (from an EPA funded study by Goodman et al. (2006-2009)) will be tested in an ex vivo IgE/allergen-induced release assay intended to evaluate the biological relevance of IgE against novel food proteins. Basophil activation will be measured as β-hexosaminidase and histamine release using IgE-stripped human basophils and the humanized RBL cells using legume proteins and extracts to compare sensitivity and specificity.
The standardized allergenicity assay using basophil degranulation (either RBL 30/25 or stripped human donor circulating basophils) that we are developing will improve methods for assessing potential immune system cross-reactivity and overcome some of the problems with the current testing recommended by the Codex Alimentarius. The assay would replace a clinical test done in humans with a cell-based test and improve hazard identification for pesticide proteins. Benefits of this research include improved test methods that can be applied to safety assessment of genetically engineered pesticide proteins in the food supply. Researchers, regulators, and biotechnology scientists in the U.S. and around the world will use this more accurate assay to improve the testing of novel pesticide proteins that may cause food allergy.