An understanding of the impact of processing on food allergenicity is critical for allergen risk assessment. In vitro cell-based assays have... Show moreAn understanding of the impact of processing on food allergenicity is critical for allergen risk assessment. In vitro cell-based assays have been developed to assess the allergenicity of food proteins by measuring the capacity of these proteins to induce degranulation in effector cells. This study evaluated the use of sensitized rat basophilic leukemia (RBL) cells for assessing the allergenic potential of ovalbumin (OVA), ovomucoid (OVM) and NIST whole egg standard reference material (NIST egg) by measuring the proteins’ ability to induce the release of β-hexosaminidase and histamine. RBL-2H3 cells at a concentration of ~105 cells/mL were seeded in six-well plates and incubated for 24 hours. After washing with PBS and resuspension in Eagle’s minimal essential medium, cells were sensitized by incubation for 24 h with various dilutions of anti-OVA, anti-OVM or anti-egg antisera derived from Brown Norway or Wistar rats. The sensitized cells were then challenged with different concentrations of unheated or boiled (for 10 min) OVA, OVM or NIST egg. After 45 min, cell degranulation was determined by measuring the increase in β-hexosaminidase and histamine in culture supernatant. The release of β-hexosaminidase was observed after the cells were challenged with OVA, OVM or NIST egg. Assay optimization studies using sensitized cells challenged with OVA indicated that the greatest degree of release occurred when RBL-2H3 cells were sensitized with 1:10 dilution of anti-sera and challenged with 0.01mg/mL of OVA. Histamine release was not detected under any serum or OVA concentration. Boiling caused a decrease in the recovery of OVA and NIST egg (70.34±3.61% and 73.69±7.66% respectively), but did not affect the recovery of OVM (96.28±2.95%). Under the optimal assay conditions, boiled OVA and boiled NIST egg x triggered the release of more β-hexosaminidase than the unheated forms of these proteins in most cases. However, boiling did not significantly affect the ability of OVM to induce the release of β-hexosaminidase. Due to the large variation in % release of β-hexosaminidase obtained from different trials, the RBL mediator release assay may not be an accurate method to evaluate the impact of thermal processing on the allergenic potential of egg allergens. M.S. in Food Safety and Technology, May 2013 Show less
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