Confirmation of immuno‐reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity‐CIEF

Affinity‐CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno‐chemically. For this purpose mAbs of the IgG‐type have been produced in‐lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS‐PAGE, MALDI‐TOF‐MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering p I ranges of 6.98–7.09 and 6.78–7.03 for clones 2 and 5.1 with major peaks at p I 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (p I 4.95) were incubated with 2.0 μmol/L mIgG, novel peaks were progressively induced in a p I range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All p I values were calculated using two p I marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno‐chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag–antibody complexes and Ag variants coexisting in one sample.