Grantee Research Project Results

Final Report: Appropriate Serum IgE Testing Strategy, Protocols and Serum Donors

EPA Grant Number: R833135
Title: 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 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 GE legumes.

Summary/Accomplishments (Outputs/Outcomes):

The Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) share in the oversight of food safety of genetically modified (GM) food crops. Those crops are produced by specific insertion of genetic material (DNA) from another organism, to provide a new trait, such as protection from insects, tolerance to herbicides, or synthesis of a vitamin or nutrient that was not previously in the food crop. Before the new GM crops are allowed to be grown, consumed or exported, they must be evaluated to ensure they are as safe as the traditionally bred crops of similar varieties. The safety assessment includes evaluation of the GM crops to determine if there is evidence to suspect they are more allergenic than traditionally bred, similar crop varieties. The primary risks of food allergy would be if the gene (DNA) introduced into the food crop encodes an allergenic protein, for example a major allergen from peanut. If that was transferred into rice, eating the rice would be a significant risk for those allergic to peanut and would likely be similar to eating peanut, something they know to avoid. Even if the protein was not from peanut, but if the amino acid sequence, and the structure of the protein was highly similar to a peanut allergen, the risk might be similar to eating peanut. The safety evaluation has been developed to test those possibilities, using blood (serum) from donors who have allergies to the source of the gene (such as peanut) or to a sequence matched protein-allergen (such as an allergenic protein from peanut, lupin, or a tree nut). The tests are very similar to those used for diagnosing allergies. However, the commercially available diagnostic tests are generally developed through a process of extensive testing and validation. That is a costly and time consuming process. Those tests will be used many times over. The safety assessment tests are typically only one-time studies and they cannot practically be validated in the same way. The developer must provide data explaining the source of the introduced new gene (DNA). If the source of the gene is commonly allergenic, the possibility the protein is an allergen must be answered, typically by performing serum IgE comparing the amino acid sequence to those of known allergens.

Previous IgE binding results using reducing-denaturing conditions, non-reducing conditions and native conditions have demonstrated different results for some subjects using the same extracted protein samples from foods. Thus a series of test methods were used to evaluate IgE binding from each serum or plasma sample. Commonly used methods were planned to test each serum sample with 96 serum samples (86 with claimed allergy to at least one legume) obtained from India, Italy, Switzerland and the USA and samples were screened for IgE binding to 14 different legumes in the study (black soybean, black gram, cowpea, chickpea, green pea, kidney bean, lentil, lima bean, lupin, mung bean, navy bean, peanut, pigeon pea and soybean). Donors either claimed food allergy to a specific legume based on specific questionnaire, and/or were tested for high IgE to at least one legume by ImmunoCAP or by skin prick test (SPT) with a legume extract. Screening was performed using native extract dot blot and/or immunoblots using samples separated in SDS-PAGE reducing gels. There were fewer cases of cross-reactivity between legume samples than expected.

Conclusions:

1. The improved (2^nd year) patient questionnaires helped to narrow allergenicity, but there still was ambiguity in many responses.

2. 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. Sera from 20 subjects were donated from Italy. Preliminary tests were performed on all of these subjects, and full western blot and ELISA were performed on a few. In the end, it is somewhat disappointing that more of the subjects do not have strong IgE binding to proteins from the legume they are supposed to be allergic to, or to other legume proteins. However, there are some useful sera.

3. 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 susan), 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 pean or field pea) with alpha-amylase inhibitor has been obtained from TJ Higgins through Larry Murdock for evaluation of IgE binding to alpha-amylase inhibitor. Purified native soybean glycinin and beta conglycinin also were obtained to evaluate possible inter-chain IgE epitopes, and to use as a standard in some assays. PHA (phytahemaglutinin), alpha-amylase inhibitor and phaseolin were purified by members of the Goodman lab, from navy bean (Phaseolus vulgaris), to use as test reagents.

4. 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.

5. 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 their 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 in January 2008.

6. 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 was reported at three allergy meetings in 2008.

a. A poster was presented at the March 2008 AAAAI (American Academy of Allergy Asthma and Immunology) meeting in Philadelphia, PA. “In vitro diagnosis of food allergy is confounded by IgE binding to cross-reactive carbohydrate determinants,” A. Ofori-Anti, S.N. Pramod, L. Chen, H.S. Ariyarathna, R.E. Goodman, demonstrating 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.

b. A poster was presented in June 2008 at the EAACI (European Academy of Allergy and Clinical Immunology). “IgE binding to taxonomically diverse legumes with evaluation of binding to complex carbohydrates of glycoproteins using sera from legume allergic subjects,” Goodman RE, Ofori-Anti AO, Pramod SN, Banik U, Mari A, Ballemer-Weber B, Singh AB, Vieths S, Ferrara R, Taylor SL, was presented in Barcelona, Spain. Sera from India, the US, 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 evaluated easily 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 generally is much more restricted in specificity in direct binding assays.

c. 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 beta 1-6 xylose as more prominent than alpha 1-3 fucose for one patient, but not the other. Further, no binding was observed to glycoproteins hemocyanin or fetuin, as expected.

d. 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 30 September 2008 reporting period and the manuscript will be submitted in June 2009. The title: 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.

e. 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 meetings after that.

7. Where are we relative to the plan?

a. Clinical questionnaires, informed consents and IRB approvals are done.

b. Detailed QA, is an ongoing process. Appropriate record keeping requirements have been discussed with all participants at UNL and other institutions. Training about records and quality assurance in my lab will continue this summer as well.

c. Protocol development. Protocols for protein determinations, gel electrophoresis and blotting, dot-blotting and ELISA have been developed and incorporated into our work.

8. Although we have obtained a number of sera from all geographies, 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 here 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 makes it difficult to do comparisons of IgE cross-reactivity as we have few qualified sera, so we continue looking for additional sera and are attempting to improve the sensitivity of our assays for sensitivity and specificity.

9. Siemen’s diagnostics have helped in testing “native” proteins that we produced in their protein testing panel, beside their normal antigens for allergy testing.

10. Year 3 plan.

a. UNL methods development continues as per the plan. Ms. Ofori-Anti is looking to identify human subjects out of our current group that have IgE binding to peanut (and specifically to peanut agglutinin). She and Dr. Pramod Siddanokoppalu also are purifying phaseolin, PHA and alpha-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 alternative. We are using purified multi-meric soy glycinin and beta-conglycinin as model proteins and a variety of plasma samples.

b. We are performing parallel tests with some of the Indian sera, using similar methods as Dr. Singh’s lab in the Institute of Genomics and Integrative Biology.

c. We will continue to screen sera for IgE to the alpha-amylase inhibitor of Phaseolus vulgaris to determine whether we see any IgE binding. This is an important test for the alpha-amylase inhibitor GM plant that TJ Higgins has developed. So far we have not found specific IgE binding to any protein in the right MW range of AAI in extracts of legumes.

d. ELISA assays are developed that should be useful for semi-quantitative analysis.

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Publications Views
Other project views:	All 23 publications	5 publications in selected types	All 5 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Goodman RE, Vieths S, Sampson HA, Hill D, Ebisawa M, Taylor SL, van Ree R. Allergenicity assessment of genetically modified crops—what makes sense? Nature Biotechnology 2008;26(1):73-81.	R833135 (Final)	Abstract from PubMed Abstract: Nature Biotechnology Exit
Journal Article	Goodman RE. Performing IgE serum testing due to bioinformatics matches in the allergenicity assessment of GM crops. Food and Chemical Toxicology 2008;46(Suppl 10):S24-S34.	R833135 (Final)	Abstract from PubMed Full-text: ScienceDirect-HTML Exit Abstract: ScienceDirect-Abstract Exit Other: ScienceDirect-PDF Exit
Journal Article	Goodman RE, Panda R, Ariyarathna H. Evaluation of Endogenous Allergens for the Safety Evaluation of Genetically Engineered Food Crops: Review of Potential Risks, Test Methods, Examples and Relevance. Journal of Agricultural and Food Chemistry 2013;61(35):8317-8332.	R833135 (Final)	Abstract from PubMed Full-text: ACS-Full Text PDF Exit Abstract: ACS-Abstract & Full Text HTML Exit
Journal Article	Panda R, Ariyarathna H, Amnuaycheewa P, Tetteh A, Pramod SN, Taylor SL, Ballmer-Weber BK, Goodman RE. Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety. Allergy 2013;68(2):142-151.	R833135 (Final)	Abstract from PubMed Full-text: Wiley-Full Text PDF Exit Abstract: Wiley-Abstract Exit
Journal Article	Panda R, Tetteh AO, Pramod SN, Goodman RE. Enzymatic hydrolysis does not reduce the biological reactivity of soybean proteins for all allergic subjects. Journal of Agricultural and Food Chemistry 2015;63(43):9629-9639.	R833135 (Final) R834065 (Final)	Abstract from PubMed Abstract: ACS-Abstract Exit Other: University of Nebraska-Abstract Exit

Supplemental Keywords:

agricultural biotechnology, genetically modified, GM, allergenicity, IgE, serum testing, cross-reactivity, risk assessment, 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 response

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