A sensitive assay for the detection of IgE bound to the major birch pollen allergen, Bet v 1, in the form of immune complexes

Allergen-IgE immune complexes, in which the IgE paratopes are occupied by allergen, remain undetected in standard IgE-detection assays which are based on capturing specific IgE by allergens. We describe an assay for the detection of immune complexes consisting of IgE bound to one of the most frequent environmental allergens, the major birch pollen allergen, Bet v 1. This assay is based on a Bet v 1-specific monoclonal antibody, Bip 1, which binds to an epitope on Bet v 1 that is distinct from the epitopes recognized by allergic patients' IgE. IgE-immune complexes formed with sera from birch pollen-allergic patients (n = 46) were undetectable with solid phase-bound allergen but could be captured by Bip 1 immobilized to nitrocellulose membranes or ELISA plates and traced with radioactively or enzymatically labelled anti-human IgE antibodies. The levels of IgE complexed with Bet v 1 measured in our assays were highly correlated with the amounts of non-complexed allergen-specific IgE as determined by the ImmunoCAP assay which is based on solid phase-bound IgE (r = 0.95; p < 0.01). Bet v 1-specific IgE could even be detected at serum dilutions below the cut off of the ImmunoCAP system (i.e., 0.35 kUA/L). We have thus developed a robust and sensitive assay for the detection and quantification of Bet v 1-IgE immune complexes which should be useful to measure allergen-bound IgE in human body fluids and in in vitro experiments.