Grantee Research Project Results
2008 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, 2007 through September 30,2008
Project Amount: $450,000
RFA: Biotechnology: Potential Allergenicity of Genetically Engineered Foods (2006) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Objective 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.
Objective 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 genetically engineered legumes.
Progress Summary:
The following had been accomplished by the end of the second year of the 3-year project:
1. Institutional Review Board approvals (ethical approvals) were completed in year one for serum collection under the oversight of the University of Nebraska; the Institute of Genomics and Integrative Biology, New Delhi, India; the Istituto Dermopatico Dell’Immacolata, Rome, Italy; and University Hospital of Zurich, Switzerland.
2. Clinical questionnaires developed in the first year were used to collect sera in the first and second years in the United States, Italy, Switzerland and India.
3. Serum collections so far include plasma samples from individuals with peanut or soybean allergy, purchased from SeraCare (under FDA license), for use in methods development, as well as sera collected from 23 subjects from Nebraska, diagnosed as allergic to peanut or soybean. Sera from India included samples from 26 subjects. Five subjects from Switzerland were primarily allergic to peanut and soybean. Twenty subjects donated sera from Italy. Preliminary tests were performed on all of these subjects, and full western blot and ELISA were performed on a few. It was somewhat disappointing that more of the subjects do not have strong IgE binding to proteins from the legume to which they are supposedly allergic, or to other legume proteins. However, there are some useful sera.
4. Seed collections and identity verification have been obtained for the following species (common names): Arachis hypogaea (peanut), Cicer arietinum (chickpea), Lupinus albus (lupine), Glycine max (soybean), Glycine soja (wild soybean), Phaseolus lunatus (lima bean), Phaseolus vulgaris (kidney bean, pinto bean and navy bean), Vigna mungo (black gram), Vigna radiate (mung bean), Vigna unguiculata (cowpea, or blackeyed pea), Lens culinaris (lentil), Pisum sativum (garden pea) and Vicia faba (fava bean) and control materials; Triticum aestivum (wheat) and Oryza sativa (rice). In addition, transgenic Pisum sativum (green pea or field pea) with α-amylase inhibitor has been obtained from TJ Higgins through Larry Murdock for evaluation of IgE binding to α-amylase inhibitor. Purified native soybean glycinin and ß-conglycinin also were obtained to evaluate possible inter-chain IgE epitopes, and to use a s standard in some assays. PHA (phytahemaglutinin), α-amylase inhibitor and phaseolin were purified by members of the Goodman lab, from navy bean (Phaseolus vulgaris) to use as test reagents.
5. Preliminary methods development. The various legume seed and control seed samples were extracted with aqueous and denaturing conditions to evaluate protein solubility differences. Samples were separated by SDS-PAGE, reducing and non-reducing gels, stained to evaluate protein patterns and blotted for incubation with peanut and soy allergic plasma for methods development. Apparent significant IgE cross-reactivity was found with some samples from individuals with allergic symptoms reported only for peanut or peanut and soy, but not other legumes. Therefore, various methods were used to investigate the possibility of IgE binding to common complex plant glycan structures on some proteins. For some subjects essentially all IgE binding to legume protein could be inhibited by intact or highly proteolytically digested protein extract of wheat, indicating dominant binding to the carbohydrate. Inhibition of binding to carbohydrate has been incorporated as a standard method now for screening after identification of apparent specific binding. Additional carbohydrate binding controls including glycoproteins bromolain, horseradish peroxidase and purified MUXF carbohydrate are being studied now to evaluate IgE binding.
6. A sub-study collaboration has been set up with Siemens Diagnostics to use their Immulite system to evaluate IgE binding to extracts of most of the legumes and to carbohydrate in their independent system. Samples of the first plasma and 23 subjects have just been sent for analysis. We also will use our laboratory immunoblotting methods to evaluate the same sera with the extracts of various legumes we have been using. We have a goal to complete the comparative study between our methods of immunoblot with and without detection of IgE binding to carbohydrate with the commercial Immulite system by January 2008.
7. Results of immunoblotting on SDS-PAGE, the use of simple dot blotting (in place of ELISA or native gels), native gel blots, 2D electrophoresis, immunoblots and LC-MS/MS of isolated spots from Phaseolus vulgaris were performed and some of the data were reported at three allergy meetings in 2008. In addition:
- The poster “In vitro diagnosis of food allergy is confounded by IgE binding to cross-reactive carbohydrate determinants”was presented at the March 2008 AAAAI (American Academy of Allergy Asthma and Immunology) meeting in Philadelphia, PA. It demonstrated cases where IgE binding to plant glycoproteins, and specifically to the glycan, were the dominant IgE binding targets for some subjects. Navy bean and kidney bean proteins, both Phaseolus vulgaris, are major targets with PHA representing the dominant IgE binding protein in these bean extracts.
- The poster “IgE binding to taxonomically diverse legumes with evaluation of binding to complex carbohydrates of glycoproteins using sera from legume allergic subjects” was presented in June 2008 at the EAACI (European Academy of Allergy and Clinical Immunology) in Barcelona, Spain. Sera from India, the United States, Italy and Switzerland were tested and those with IgE binding to CCD were shown to be CCD specific using inhibition assays with western blots as well as inhibition of direct binding in ELISA. The CCD binding was dominant target in three of seven legume allergic subjects. The CCD binding was easily evaluated with irrelevant antigens, HRP, Bromelain and negative controls Hemocyanin and Fetuin and verified by inhibition with variously digested HRP, wheat, peanut and kidney bean digests. For some subjects, CCD binding appears to account for most IgE binding to extracts of peanut, soybean, white bean, kidney bean, chick pea, lentil lupine and wheat. Peptide specific IgE binding is generally much more restricted in specificity in direct binding assays.
- A poster was presented in April 2008 at the Molecular Allergology meeting in Salzburg, Austria, on evaluation of IgE binding to CCD vs. peptide determinants. A number of subjects with clear evidence of clinical allergy exhibited IgE binding to peptides as well as to CCD, although the CCD binding was not indicated as the cause of binding to the foods of reactivity. One curious sample from India, a young girl of ~ 7 years with strong systemic reactions to cowpea and black gram exhibited strong IgE binding to a number of proteins in those legume extracts, and much less abundant binding to proteins in other legumes, though clearly present. Inhibition assays demonstrated that IgE was specific primarily to cowpea and to a slightly lesser extent to black gram. In addition there were clear differences in IgE binding to HRP vs bromelain between 2 subjects with high CCD IgE, demonstrating some specificity related to binding to ß(1,2)-xylose as more prominent than α(1,3)-fucose for one patient, but not the other. Further, no binding was observed to glycoproteins hemocyanin or fetuin, as expected.
- A manuscript has been drafted and is being revised demonstrating the need to prove specificity of the anti-IgE used to perform these assays as some, like a commonly used polyclonal antibody from Sigma, has enough binding to IgG antibodies to provide false positive IgE binding results. Some additional experiments have been carried out since the September 30, 2008 reporting period and it is about to be submitted (in June 2009). The title of the draft manuscript is "Evaluation of three commercial anti-human IgE antibodies to assess binding specificity to human IgE versus IgG and the potential impact on allergen identification." (Authors: SN Pramod, AB Singh, S Vieths, SL Taylor, T Holzhauser RE Goodman).
- The data generated so far have been presented at some allergy meetings including the ILSI-HESI-EPA meeting in Washington, DC in October 2008 and a number of subsequent meetings.
8. Where are we relative to the plan?
- Clinical questionnaires, informed consents and IRB approvals are completed.
- Detailed QA, is an ongoing process. Appropriate record keeping requirements have been discussed with all participants at University of Nebraska-Lincoln and other institutions. Training about records and quality assurance in the PI's lab will continue this summer as well.
- Protocols for protein determinations, gel electrophoresis and blotting, dot-blotting and ELISA have been developed and incorporated into our work.
9. Although we have obtained a number of sera from the different geographical locations that meet the clinical criteria we set, in vitro IgE tests have shown that many of the subjects have little or no detectable IgE binding to any legume in the test, even though they have reported allergic reactions to the source material. Dr. Singh, our Indian collaborator, was at the University of Nebraska-Lincoln for training in February and March of 2008 (Borlaug fellowship), and I have visited his laboratory on three occasions to discuss IgE binding methods with him and his graduate students. It is difficult to do comparisons of IgE cross-reactivity because we have few qualified sera. Therefore, we continue looking for additional sera and are attempting to improve the sensitivity of our assays for sensitivity and specificity.
10. Siemen’s diagnostics have helped in testing “native” proteins that we produced in their protein testing panel, beside their normal antigens for allergy testing.
11. Plan for Year 3
- University of Nebraska-Lincoln continues to develop methods as per the plan. Ms. Ofori-Anti is looking to identify human subjects out of our current group who have IgE binding to peanut (and specifically to peanut agglutinin). She and Dr. Pramod Siddanokoppalu also are purifying phaseolin, PHA and α-amylase inhibitor as specific reagents for cross-reactivity. We are continuing to evaluate IgE binding to blots of native gels as well as using dot blots as a “native” protein allernative. We are using purified multi-meric soy glycinin and ß-conglycinin as model proteins and a variety of plasma samples.
- We are performing parallel tests with some of the Indian sera, using similar methods as Dr. Singh’s lab at the Institute of Genomics and Integrative Biology.
- We will continue to screen sera for IgE to the α-amylase inhibitor of Phaseolus vulgaris to determine whether we see any IgE binding. This is an important test for the α-amylase inhibitor genetically modified plant that TJ Higgins has developed. So far, we have not found specific IgE binding to any protein in the right MW range of α-amylase inhibitor in extracts of legumes.
- Develop ELISA assays that are useful for semi-quantitative analysis.
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.