Fig 1.
Structural integrity of recombinant Bet v 1a and Bet v 1aS112P/R145P.
A) Secondary structure topology of Bet v 1a (pdb: 1BV1). The amino acids S112 (purple) of β-strand 7 and R145 (cyan) of α-helix 3 were exchanged for proline. B) Circular dichroism of rBet v 1a and rBet v 1aS112P/R145P. C) 1H-NMR spectra of 30 μM of rBet v 1a (upper panel), 30 μM rBet v 1aS112P/R145P (middle panel), and the overlay rBet v 1a in red and rBet v 1aS112P/R145P in black (lower panel). 3D-model of Bet v 1a was visualized with PyMOL [54].
Fig 2.
Immunoglobulin binding of rBet v 1a and rBet v 1aS112P/R145P.
Binding of IgE from sera of subjects allergic to birch pollen bound to purified (lane 14, Coomassie stain) rBet v 1a (upper panel) and rBet v 1aS112P/R145P (lower panel) (lanes 1–10). Binding of rabbit anti-Bet v 1 IgG to the rBet v 1a proteins is shown (lane 13). A minor degradation product of rBet v 1aS112P/R145P detected by IgG is shown (*). As controls, serum of non-allergic subject (lane 11) and buffer control (HRP-conjugated anti human IgE antibody only) (lane 12) were used.
Fig 3.
Circular dichroism of rBet v 1a/rBet v 1aS112P/R145P combinations.
A) Circular dichroism of defined molar ratios of rBet v 1a/rBet v 1aS112P/R145P. 5 μM total rBet v 1a was measured with increasing fractions of rBet v 1aS112P/R145P from 0% to 100%. Mean residual ellipticities with 95% confidence interval and statistical evaluations at wave lengths 193 nm (B), 195 nm (C), 200 nm (D), 218 nm (E), and 222 nm (F) are shown.
Fig 4.
IgE Immunoblot of rBet v 1a/rBet v 1aS112P/R145P mixtures.
A) 1 μg of total rBet v 1a with increasing fractions of rBet v 1aS112P/R145P 0% to 100% were transferred onto nitrocellulose and stained with Ponceau S. Binding of sera pool IgE to rBet v 1a combinations was determined by chemiluminescence after 100 milliseconds. To visualize IgE signals with molar ratios of rBet v 1aS112P/R145P from 99% to 100% (lanes 7–10) a 2 min exposure is shown (left). B) Chemiluminescence was quantified densitometrically and plotted against the molar fraction of rBet v 1aS112P/R145P (right).
Fig 5.
Inhibition of IgE binding to immobilized rBet v 1a.
Dose-dependent inhibition of IgE binding to surface-coated rBet v 1a by rBet v 1a/rBet v 1aS112P/R145P mixtures in the presence of increasing molar ratios of rBet v 1aS112P/R145P in ELISA. Curves were fitted in parallel with a 4-parameter, logistic sigmoidal curve fit with same slope, lower and upper asymptote for all curves.
Fig 6.
Mediator release of humanized rat basophils.
β-hexosaminidase release of humanized rat basophil leukemia cells sensitized with a pool of human sera of donors allergic to birch pollen. Cross-linking of membrane-bound human IgE by IgE-Bet v 1 interaction and subsequent release of β-hexosaminidase was determined in the presence of defined molar ratios of rBet v 1a/rBet v 1aS112P/R145P. The legend shows molar ratios (in %) of rBet v 1aS112P/R145P in the rBet v 1a/rBet v 1aS112P/R145P combinations analyzed.
Fig 7.
Experimental/theoretical comparison of CD, IB, ELISA and RBL mediator release.
The experimental/theoretical comparisons of the individual experiments shown in Figs 3–6 for molar fractions of rBet v 1aS112P/R145P from 0% to 90% in rBet v 1a/rBet v 1aS112P/R145P combinations analyzed are shown.
Table 1.
Accuracy, precision and resolution of methods to distinguish rBet v 1a conformation-dependent IgE binding and secondary structure of rBet v 1a/rBet v 1aS112P/R145P mixtures.