Development of a Biological Assay to Determine the Allergenic Potential of Foods

Abstract

Abbreviations: ELISA = enzyme-linked immunosorbent assay; PCR = polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfatepolyacrylamide gel electrophoresis; FcεRI = high affinity receptor for IgE; RBL = rat basophilic leukemia; PBS = phosphate-buffered saline; TBS = tris-buffered saline.Abstract: Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.

A novel method for quantifying ingested food allergens in human sera

“…The quantitative outcome was compared and confirmed by ELISA results of the same samples. 10 Our purpose was, through novel integration of BHRA with logarithmic modelling of the area under the curve (AUC), to develop a reliable method to quantify the total amount of allergenic food proteins in circulation. This was achieved by spiking of serum with peanut and hazelnut extracts at different concentrations followed by the integrated AUC method to estimate allergen concentrations in serum.…”

Recent advances in food allergy

“…A critical issue with these cell lines has been the cytotoxicity of certain human sera for rat cells, requiring high dilutions and resulting in insufficient sensitisation. A complete review on recent developments in RBLs can be found in and their use in allergenicity assessment of food products in (VOGEL et al, 2006).…”

Use of a rat basophil leukemia (RBL) cell-based immunological assay for allergen identification, clinical diagnosis of allergy, and identification of anti-allergy agents for use in immunotherapy

“…The human F ce RI allowed these cells to bind IgE from sera of allergic subjects and subsequently be activated in allergen-specific manners. Mediator release by humanized RBL cells provided an appropriate cell-based assay to measure the biological activity of allergens in vitro and, over time, one that proved to be sensitive, specific, and reproducible (Blanc et al, 2009;Dibbern et al, 2003;Kaul et al, 2007;Untersmayr et al, 2005;Vogel et al, 2006). Humanized RBL cells, in combination with human serum pool, might then represent a preferred test system.…”

“…Therefore, RBL cell activation mediated through serum IgE is a result of complex relationships not only dependent on allergenspecific IgE content, but also the capacity to efficiently sensitize cells and trigger signaling responses that lead to degranulation. Further, some serum factors may hamper binding of IgE to F ce RI expressed on the cell membrane and adverse effects on the cells occur (Dibbern et al, 2003;Marchand et al, 2003;Vogel et al, 2006). Those factors and how they may act on cells to interfere with the assay have not yet been specifically identified.…”

“…Despite the risk of severe allergic reactions and even death, there is no curative treatment for food allergy, and strict avoidance of the offending food remains the only effective measure to prevent allergic reactions. This strategy is not optimal as there is still the risk of unintentional ingestion due to mislabeling of pre-packaged foods, allergen cross-contact during manufacture, or consuming foods in restaurants and catering (Vogel et al, 2006). Besides, with an increasing interest in genetically engineered foods, the potential allergenicity of newly introduced proteins has become an important safety evaluation issue.…”