Grantee Research Project Results
2010 Progress Report: 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: R834065Title: 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: Aja, Hayley
Project Period: May 1, 2009 through March 31, 2011 (Extended to April 30, 2012)
Project Period Covered by this Report: June 1, 2009 through May 31,2011
Project Amount: $372,340
RFA: Exploratory Investigations in Food Allergy (2007) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
The overall purpose of this study is to develop standardized methods to use a humanized rat basophil leukemia (RBL) cell line that has been transformed with the alpha-chain of the human FceRI receptor, as a test system to evaluate potential biological significance of IgE binding from an allergic subject on any test protein/target. Evidence from many allergy studies demonstrates that some subjects have extremely high levels of IgE in their sera that binds specifically to extracts of proteins or purified proteins that the individual can tolerate without experiencing an allergic reaction. Possible reasons for the lack of response center on a lack of effective cross-linking of FceRI receptors on mast cells and basophils through IgE either because only one binding site (epitope) is recognized by that person’s IgE, possible instability of the antigen during cooking or digestion (food proteins), or other unknown reasons. The safety assessment of a new genetically modified organism may include serum IgE tests to evaluate potential risks. If such tests are used, it is important to have a functionality test method, such as basophil histamine release, something short of in vivo challenges in human subjects, to confirm safety or risk.
Objective 1. To develop a standardized method to assess relative biologically active binding of allergenic IgE cross-reactivity using sera from individuals with allergies to diverse legume allergens as a model to evaluate the utility of the RBL cell line 30/25 as a surrogate for in vivo human testing. [NOTE: the RBL line 703/21 cells were subcloned from the original PEI line, 30/25, by Lothar Vogel.]
Objective 2. To evaluate the contribution of IgE to complex carbohydrates on plant glycoproteins in the in vitro RBL 703/21 cell assay with human serum specific to plant glycoproteins.
Objective 3. To identify biologically active proteins (peptides) of related legume seeds that IgE induced basophil histamine release in RBL cell line 703/23 using sera with demonstrable in vitro IgE binding cross-reactivity that is not related to plant glycan recognition.
Progress Summary:
1. S. Pramod (postdoc) and R. Goodman (PI) were trained in procedures to grow the humanized RBL cells [703/21] and perform antigen specific basophil activation assays in the laboratory of S. Vieths at the Paul Ehrlich Institute, Langen, Germany in December, 2008. A stock of 703/21 basophil cells was shipped to the Goodman laboratory at UNL in May, 2009 to establish the cultures.
2. Initial tests at UNL used serum and plasma samples from a number of legume allergic and non-legume allergic subjects. Comparisons of activation were made between703/21 cells and IgE-stripped normal human blood basophils (buffy coat) using the same allergic donor sera and normal controls with stimulation by the same antigenic extracts (e.g. figure 1). Activation in human blood basophils was measured by histamine release, but as beta-hexaminidase in hRBL. Results were similar in responses except, for example, the apparent activation by navy bean extract at ~1:100 of the peanut extract leaves open the question of whether that was due to residual IgE from the PBMC blood donor, potential toxic effects of the extract or specific IgE reactivity from serum donor. In this case we believe the activity with NB was due to residual IgE or possibly a lectin like-activity since the serum IgE to navy bean and kidney bean by the donor was minimal.
Figure 1. Comparison of the serum from peanut allergic donor 728 on activation using IgE stripped
human blood leukocytes by measuring histamine (left panel) and the humanized RBL cells 703/21
by measuring beta-hexosaminidase release (right panel). Extracts and proteins: PN, peanut extract;
NB, navy bean extract; NTP, non-transgenic pea extract; TP, transgenic pea extract; AIgE; pure
hIgE cross-lined with anti-IgE; BSA, bovine serum albumin.
3. Afua Ofori-Anti trained in the PI’s lab as a PhD student, then as a postdoc in the use of the hRBLs and developed and tested the cell assay further with glycoproteins that she purified from common bean (phytohemmaglutinin, or PHA; and alpha-amylase inhibitor αAI). Serum samples were limited, but none of the sera with IgE binding to the carbohydrate moieties on PHA or αAI (See figure 2); activated the hRBL cells in response to purified glycoprotein (the target of significant in vitro IgE binding) or with extracts of common bean (Figure 3).
Figure 2. SDA-PAGE reducing Immunoblots with allergic sera, using hightly
specific mAb-IgE with chemiluminescent substrate. IgE binding to PHA and oIA
detection (serum samples 714 and 715, (circled in pink) was determined to be
carbohydrate-specific based on inhibition assays with protease digested PHA
compared to intact PHA. Protein-specific IgE binding shown in blue. This data
was presented at the EAACI and at the 5th International COwpea Confrence,
both in 2019 as part of two larger presentations on serum IgE binding and
basophil activation.
Figure 3. Beta-hexosaminidase release using hRBL [703/21] cells with diluted serum samples from subject 714 and 719, used in IgE western blots in Figure 2.
These two sera (714:719) were tested by commercial antigen-specific IgE tests [Immulite, DPC, reported in kU/L] against peanut [13kU/L;7.5kU/L] , CCD
(horseradish peroxidase = HRP [98 kU/L:98 kU/L] and one of the CCDs = MMXF [64 kU/L:48 kU/L]), soybean [22 kU/L:7.5 kU/L] and white bean (~navy
bean [kU/L:28 kU/L]). The angigens for hRBL assays include peanut extract (PN), navy bean extract (NB), kidney bean extract (KB), PHA purified from
navy bean, aAI from navy bean, horseradish peroxidase (HRP), non-transgenic pea (NTP), transgenic, alphy-amyalse-pea (TP), and cross-linking anti
IgE. This data was presented at the EAACI and at the 5th International Cowpea Confrence, both in 2010 as part of two larger presentations on serum
IgE binding and basophil activation.
4. We are testing various parameters to control variance in the hRBL assay that was developed by G. Reese and L Vogel in the PEI in 2005 to 2007. Cell plating density and maturity of the culture impacts responsiveness. Uniformity of cell distribution between plate wells and time of incubation with sera (for IgE binding to the human alpha-chain of FceRI) as well as antigen concentration are important. The baseline (spontaneous release, without antigen) as well as total release (detergent lysis), the use of cross-linking anti-IgE with controls of fixed concentration of purified human IgE from AbCAM as well as release with the allergic donor (or control) subject’s serum IgE may be used as the denominator to estimate antigen specific release. These controls measure different variables. The lysate demonstrates activity of the basophils in terms of intracellular stores of beta-hexosaminidase (parallels histamine). The release with pure-IgE and anti-IgE cross-linking provides a quality control for expression of the FceRI in the cell population. Cross-linking IgE on the hRBLs with the serum donor’s sample provides an estimate of the specific IgE to total IgE within that subject. All three variables should be measured to properly interpret the value of activation elicited by antigen specific release for each subject. Further evaluation is needed and is being conducted with a larger population of subjects allergic to a variety of legumes and other protein sources to provide a more robust assay during the remaining funding period.
5. The preliminary bioinformatics evaluation of the potential allergenicity of GM peas (Pisum sativum) and GM cowpea (Vigna unguiculata) with transgenic aAI by Dr. TJ Higgins identified a modest amino acid sequence match using our AllergenOnline.org database (~45% identity over an 80 amino acid segment, 34.9% overall identity) between the aAI protein and a minor peanut allergen (agglutinin). Initial attempts to find peanut allergic sera with IgE binding to peanut agglutinin (PNA) that could be screened for cross-reactivity to aAI in transgenic legumes lead to the discovery that some of the sera had IgE to CCD, which proved to be irrelevant (above). More importantly, there were few subjects with clear IgE binding to PNA. We screened 34 peanut allergic sera and plasma samples for specific IgE binding to PNA. Only one subject had unequivocal IgE binding to PNA and only in native, non-denatured state. Two other samples had faint (marginal) binding to denatured (SDS-PAGE reducing or non-reducing) gels. These apparently positive IgE binding samples were tested for activation of hRBL using purified PNA and various aAI preparations. When the PNA tested as positive in activation as peanut extract for one serum with extremely high IgE binding to Ara h 2 (major peanut allergen), we tested further with purified allergens and further evaluated the proteins in immunoblots, discovering that the “purified” PNA preparation from Sigma contained low level contamination with Ara h 2. Only subjects with high IgE reactivity to Ara h 2 presented basophil activation with PNA, none had positive basophil activity to pure aAI (figure 4). This demonstrates the need to use high purity antigens that are carefully characterized.
Figure 4, hRBL assay using plasma sample 715 and serum RGLEG 103. ZBoth released beta-hexosamindidase when
stimulated with Ara h 2 and also with PNA. No activity was measured for αAI. Examination of IgE immunoblots of PNA
demonstrated apparently higher IgE binding to Ara h 2 compared to PNA, although the Ara h 2 contamination of PNA
was not evident on stained gells.
Future Activities:
Additional tests for the evaluation of other sera and plasma using other antigens will be performed during the next 5 months in the no-cost extension period. The impact of referencing activation with total release (detergent), pure IgE and anti-IgE and serum donor serum with anti-IgE will be explored. The utility of this test for evaluating heat processing effects on soybean flour products will be tested and possibly activation with maize lipid transfer protein will be added to support, or refute current results that IgE binding to CCD is unimportant, and that the impact of less potent allergens (e.g. PNA) can be over-estimated if contaminated with dominant allergens.
Journal Articles:
No journal articles submitted with this report: View all 7 publications for this projectSupplemental Keywords:
agricultural biotechnology, allergenicity, genetically modified (GM), basophil histamine release, cross-reactive carbohydrate determinants, IgE, FceRIProgress 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.