Grantee Research Project Results
2007 Progress Report: Appropriate Serum IgE Testing Strategy, Protocols and Serum Donors
EPA Grant Number: R833135Title: Appropriate Serum IgE Testing Strategy, Protocols and Serum Donors
Investigators: Goodman, Richard E. , Taylor, Steve L. , Chen, Lingyun , Schlegel, Vicki
Institution: University of Nebraska at Lincoln
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2006 through September 30, 2009
Project Period Covered by this Report: October 1, 2006 through September 30,2007
Project Amount: $450,000
RFA: Biotechnology: Potential Allergenicity of Genetically Engineered Foods (2006) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
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 or potentially cross-reactive proteins.
Progress Summary:
Experimental Development: During the first year of the grant, food-grade test materials have been collected and identity verified for 14 species of commonly consumed legumes. Extraction procedures and extracts have been evaluated. The basic protocols of separating the proteins in SDS-PAGE reducing and non-reducing buffers and native gel systems, followed by protein transfer to PVDF have been written and tested using plasma from peanut and soybean allergic subjects. As we found unexpected apparent specific IgE binding to a few proteins in many legumes that are likely to have been consumed in the diets of subjects, but had not been reported as allergens, we tested the sera for IgE binding to common plant glycans using unrelated wheat extracts as a target. Inhibition assay protocols were developed using whole extracts of wheat compared to specific legumes. The results were surprising as binding of some subject samples was completely inhibited by wheat. Procedures were developed to extensively digest the protein of the wheat and different legumes using pepsin and proteinase K. The residual (mostly carbohydrate) was equally able to inhibit binding to the extracts, demonstrating the role of IgE binding to complex carbohydrate for some of the subjects. Procedures have been added to evaluate IgE binding to glycoproteins for each serum donor as part of the subject selection for cross-reactivity. Preliminary tests also havebeen performed to evaluate the specificity of anti-IgE (MAb from Southern Biotech) to detect binding in our assays. We are currently evaluating the use of native gel electrophoresis and immunoblotting to identify IgE binding sites that require secondary and tertiary structure, using purified native glycinin and beta-conglycinin as model antigens. The specific protocols will be posted on our website (www.allergenonline.com) after further testing by collaborators.
Clinical history forms have been developed for each of the collaborative centers (United States, Switzerland, Italy and India). Informed consents and ethical approvals have been obtained. When tested more extensively, the questionnaires and model documents also willbe posted at www.allergenonline.com.
Sera from prospective legume allergic donors has been collected from 23 subjects in the United States; more than 8 in Switzerland. Clinical histories and preliminary IgE levels are being evaluated for a number subjects in India and Italy.
Future Activities:
Serum Evaluation: Testing is just beginning with the sera from clinically well-characterized legume allergic subjects to identify those that will be useful in designing and testing assays to measure cross-reactivity. Each serum sample is being evaluated also by commercial specific IgE assay using ImmunoCAP and/or Immulite assays to help confirm specific IgE binding using different assay formats and test materials for the various legume samples.
ELISA Development: In the second year, protocols will be developed describing qualifying protein coating of extracts and purified proteins, detection reagent selection, blocking optimization and inhibition with specific antigens. Preliminary testing will be performed with peanut and soybean allergic subjects, which we expect to expand to other legumes as subjects with specific allergies are identified and sera become available.
Cross-Reactive Proteins Determined: As subjects become identified who have IgE with demonstrated cross-reactivity to proteins in other legumes, we will attempt to identify the cross-reactive proteins in each of the legumes by LC-MS/MS or other means to characterize the cross-reactive proteins. If full length sequences are known, the identity matches will be evaluated along with the quantitative level of IgE binding with various subjects to help understand the predictive value of bioinformatics comparisons with respect to in vitro IgE binding. The data also will be evaluated in light of clinical histories.
Journal Articles:
No journal articles submitted with this report: View all 23 publications for this projectSupplemental Keywords:
Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, Allergens/Asthma, Biochemistry, peanut allergens, food allergenicity, genetically engineered food, dietary proteins, human exposure, oral allergy syndrome, bioinformatics, data base development, allergic responseRelevant Websites:
http://www.allergenonline.com Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.