B-067 Human IgE Monoclonal Antibodies as Unique Tools for Allergy Diagnostics

Abstract Background Current blood-based tests for allergy diagnosis rely on human sera or plasma as the source of IgE antibody for method validation and calibration. Obtaining human samples with sufficiently high levels of IgE presents challenges for researchers and diagnostic companies. Here, we present a unique panel of allergen-specific human IgE monoclonal antibodies (mAb) that target inhalant, food and alpha-gal allergens and offer novel approaches for investigation of IgE responses and standardization of allergy diagnostic reagents and methods. Methods Human IgE mAb (n=30) targeting inhalant allergens from dust mite, cat and dog; food allergens from peanut, egg, milk, walnut and cashew, and galactose-α-1,3-galactose, were generated from B cells from symptomatic allergic patients with rhinitis, asthma, atopic dermatitis or red meat allergy using hybridoma technology. Total and allergen-specific IgE were quantified by ImmunoCAP (whole allergen ImmunoCAP were also performed). Allergen binding profiles and mAb titers were determined by two-site immunoassay (ELISA). Antibody purity was assessed using SDS-PAGE and Western Blot. Results SDS-PAGE showed single heavy and light chain bands under reducing conditions. Antibody recognition of target allergens was confirmed by Western Blot. Allergen-specific IgE mAb concentrations for inhalant allergens ranged: 9,000-37,000 kU/L for dust mite (n=3), 2,100-53,000 kU/L for cat (n=4), 5,300-21,000 for dog (n=2). Food allergens ranged: 16,300-49,400 kU/L for peanut (n=14), 15,700-81,000 for egg (n=2), 41,600 for milk (n=1), 7,100 for walnut (n=1) and 37,000 for cashew (n=1). Alpha-gal IgE monoclonals (n=2) ranged in concentration from 15,700-81,000 kU/L. The ratios of specific IgE to total IgE measured by ImmunoCAP vary by allergen and by individual human IgE mAb. Ratios for the inhalant mAb ranged from 0.04-1.05, for the food mAb the ratios ranged from 0.33-0.99. The two alpha-gal mAb showed relatively lower specific IgE levels (average ratio was 0.14; range=0.08-0.20). Conclusions Monoclonal IgE antibodies derived from patients presenting with allergic symptoms may provide a reliable replacement for human sera to investigate allergic disease. IgE mAb to inhalant and food allergens, and to the alpha-gal carbohydrate epitope, provide unique diagnostic markers and will offer improved accuracy for assessing patient allergy by standardizing diagnostic reagents and methods. Crystal structures of several of the human IgE mAb epitopes have recently been reported (Pomés et al, J Allergy Clin Immunol, 2024). The current panel of human IgE mAb can be expanded to include additional allergen targets, which may lead to the development of improved allergy therapeutics.