Appropriate Serum IgE Testing Strategy, Protocols and Serum DonorsEPA Grant Number: R833135
Title: Appropriate Serum IgE Testing Strategy, Protocols and Serum Donors
Investigators: Goodman, Richard E. , Chen, Lingyun , Schlegel, Vicki , Taylor, Steve L.
Institution: University of Nebraska at Lincoln
EPA Project Officer: McOliver, Cynthia
Project Period: October 1, 2006 through September 30, 2009
Project Amount: $450,000
RFA: Biotechnology: Potential Allergenicity of Genetically Engineered Foods (2006) RFA Text | Recipients Lists
Research Category: Food Allergy , Health Effects , Health
1) To increase the value of serum screening methods by devising a comprehensive strategy and model protocols to identify specific IgE binding to proteins with various sequential, conformational or glycan epitopes that may be relevant to human allergic responses to primary allergens and potentially cross-reactive proteins. Our hypothesis is that specific independent assays are needed to identify IgE binding to potential sequential, conformational and/or glycan epitopes on the introduced (transgenic) proteins in genetically engineered (GE) organisms in order to provide a scientifically valid assessment of potential allergenicity. 2) To demonstrate the range of IgE binding/cross-reactivity results that can be expected from specific and narrowly defined targeted serum screening using taxonomically diverse, cross-reactive legumes with sera from donors allergic to specific legumes (food), a major legume allergen (Ara h 2) and α-amylase inhibitor, a glycoprotein expressed in three species of GE legumes. Our hypothesis is that IgE cross-reactivity, suspected because of sequence similarity or the presence of asparagine-linked glycan, can only be effectively tested using sera from donors allergic to the identified allergenic source, or narrowly defined taxonomically related species, using well defined IgE binding assays.
We are developing model protocols that will be tested with peanut allergic sera and controls with antigens in peanuts, soybeans and control plant materials under native, denaturing and denaturing/reducing conditions to evaluate overall IgE binding. Cross-inhibition assays will be described and tested to demonstrate specificity of binding. Samples with specific glycans (e.g. bromelain) will be used in direct binding and inhibition to evaluate carbohydrate epitope contributions. The potential value of using sera from individuals allergic to very closely related taxa, as opposed to the broadly defined relationships recommended by FAO/WHO (2001), will be tested using a range of food legumes (e.g. peanut, soybean, lupin, lentil, chickpea, and cowpea), with appropriately allergic subjects (allergic to the specific sources and from the U.S., EU and India). Most legumes are allergenic, and have evolutionarily related major proteins, but there are limited reports of cross-reactivity across taxonomic tribes. Specific proteins (e.g. Ara h 2 and alpha amylase inhibitor from kidney bean) will be used to demonstrate specificity of binding across taxa. Homologues of many major seed storage proteins vary from approximately 35% to 60% between certain legumes and these tests should help understand the relationship between sequence identity and cross-reactive IgE binding.
Based on previous tests with other subjects and allergens, we expect to see individual variation of binding to reduced, denatured and native peanut allergens. We expect the assay sensitivity of the model methods will be sufficient to detect allergen when diluted to below one-tenth, preferably to one-hundredth the maximum loading in order to identify potentially relevant cross-reactive binding to proteins of similar concentration. We expect to be able to differentiate between IgE binding to peptide and glycan epitopes and conformational epitopes that may be destroyed under reducing and denaturing conditions. If results are as expected, the protocols and strategy should enable many laboratories to produce similarly valid test results that will differentiate between proteins that might pose a significant risk, or low risk of allergy to foods derived from the specific GE organism. Examples of representative cross-reactive proteins and species are needed to provide guidance for consistent regulatory interpretation.