Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent
Fig 4
Interaction of Bet v 1 isoforms with serum IgE in the absence and presence of Q3OS.
A, Binding of serial dilutions of pool serum IgE to equimolar amounts of surface-coated Bet v 1a, Bet v 1d, and Bet v 1m. Allergen-specific human IgE was detected with a horseradish peroxidase-conjugated mouse anti-human IgE antibody. As substrate 3,3′,5,5′-tetramethylbenzidine was used and the absorbance at 450 nm was measured after stopping the reaction with 25% H2SO4. B, Mediator release induced by recombinant Bet v 1 isoforms. Humanized rat basophil leukemia cells were sensitized with a pool of human birch-specific sera. Cross-linking of membrane-bound human IgE by IgE-Bet v 1 isoform interaction and subsequent release of β-hexosaminidase was determined with serial dilutions of Bet v 1 a, d and m. The β-hexosaminidase activity in the culture supernatants was quantified by photometric measurements. The percentage of β-hexosaminidase activity relative to cells lysed with Triton X-100 was calculated and corrected for spontaneous release. C, Binding of serial dilutions of pool serum IgE to equimolar amounts of surface-coated Bet v 1a, Bet v 1d, and Bet v 1m (as described in A) and D, mediator release (as described in B) in the presence of a 5-molar excess of Q3OS.