Antibody binding to rBla g 2 presented by mAb 7C11 or 4C3 using multiplex array.

<p>A) Anti-Bla g 2 polyclonal antibody bound to rBla g 2 (mean ± standard deviation, n = 5 experiments, p = 0.059). B) IgE from sera of cockroach allergic patients bound to rBla g 2. The allergen was presented by either mAb 7C11 (gray) or 4C3 (black). The first and second row of numbers under the X-axis are ratio of IgE bound to Bla g 2 presented by mAb 7C11 versus mAb 4C3 (100%) and serum number, respectively. MFI are Median Fluorescence Intensity Units.</p

Fab 8066 and 8062 titrations.

<p>(A) Sedimentation <i>c(s)</i> distributions for samples containing 2.4 µM of the 3-H and 2.1 (red), 4.3 (green), 6.4 (purple) and 8.5 µM (blue) of added Fab 8066. (B) Sedimentation <i>c(s)</i> distributions for samples containing 2.0 µM of the 3-H and 1.2 (red), 2.4 (green), 4.8 (purple), 6.0 (blue) and 7.2 µM (gray) of added Fab 8062. In both cases the actual amount of CCIZN36 was estimated from the integral contribution of the complex at saturation. Profiles presented are based on sedimentation velocity data collected at 50 krpm and 25.0°C using the absorbance optical system at 280 nm. Similar profiles were observed using the interference optics.</p

Characterization of the 3-H, the Fab 8066 and Fab 8062 and their complexes by analytical ultracentrifugation.

<p>(A) Sedimentation <i>c(s)</i> distributions for the 3-H at 6 µM (green), the Fab 8062 at 1.4 µM (blue) and the Fab 8066 at 1.7 µM (red). (B) Sedimentation <i>c(s)</i> distributions for a purified (Fab 8062)₃/3-H complex at loading concentrations of 0.66 (blue), 0.33 (red) and 0.15 µM (green). (C) Sedimentation <i>c(s)</i> distributions for a purified (Fab 8066)₃/3-H complex at loading concentrations of 0.4 (blue), 0.2 (red) and 0.1 µM (green). All data are based on sedimentation velocity data collected at 50 krpm and 25.0°C using the absorbance optical system at 230 nm.</p

Superposition of EcTI in free and bound state.

<p>(a) Interface between EcTI (yellow ribbon) and trypsin (green space filling representation) in the inhibitor-trypsin complex. The side chains of Arg64 and Leu115 of EcTI are shown as sticks. A superimposed tracing of molecule B of free EcTI is shown in red, clearly indicating that the conformational changes in the reactive loop around Arg64 are needed in order to avoid clashes and allow the proper fit of its side chain into the S1 pocket of the enzyme. (b) Steroview of the superposition of trypsin-bound EcTI and STI (PDB ID: 1AVW). Color highlights are for the loops which are most different between these two proteins, blue for EcTI and red for STI, whereas other parts of the chain are gray.</p

Superposition of the corresponding Fab 8066 and 8062 complexes with 3-H and 5-Helix based on the Cα coordinates of three N-HR helices.

<p>(A) The (Fab 8066)₃/3-H (red) and the Fab 8066/5-Helix (green) complexes. (B) The (Fab 8062)₃/3-H (blue) and Fab 8062/5-Helix (yellow) complexes.</p

Dose-response curves of Bla g 2 and epitope mutants by ELISA.

<p>A) Binding of the anti-Bla g 2 polyclonal antibody to mAb 7C11 epitope mutants. B) Binding of the anti-Bla g 2 polyclonal antibody to mAb 4C3 epitope mutants. C) Binding of mAb 7C11 to mAb 7C11 epitope mutants, D) Binding of mAb 4C3 to mAb 4C3 epitope mutants. Data are mean ± standard deviation of duplicates from one representative experiment out of two performed for each panel. Legends in panels C and D also apply to panels A and B, respectively.</p

Inhibition of biotinylated mAb binding to rBla g 2 by allergen mutants using ELISA.

<p>A) Inhibition of binding of biotinylated-mAb 7C11 to rBla g 2-N93Q by mAb 7C11 epitope mutants. B) Inhibition of binding of biotinylated-mAb 4C3 to rBla g 2-N93Q by mAb 4C3 epitope mutants. Data are mean ± standard deviation of duplicates from one representative experiment out of two performed for each panel.</p

Rational design for mutagenesis.

<p>A) Principal residues involved in the the mAb 7C11 epitope on the N-terminal lobe of Bla g 2. The longest consecutive part of the epitope (shown in yellow) interacts with all three CDR of the light chain (surface in green) and has 9 residues (starting at 60) from which two were mutated to alanine (K65 and D68a). K65, R83 and K132 form cation-π interactions with tyrosines Y53, Y92 and Y33 from the mAb 7C11, respectively. The heavy chain of the antibody is shown in lilac. B) Main residues involved in the mAb 4C3 epitope on the C-terminal lobe of Bla g 2. The surface of the allergen, with the main residues involved in antibody binding is shown in gold. The third loop of the heavy chain of the antibody (in blue) includes: a) R103 attracted to the negatively charged E233, and b) W105 that docks into a hydrophobic pocked formed by A234, I199 and P256 in Bla g 2. Residues K251 and Y32 involved in a cation-π interaction are also shown.</p

Electron density for the fragments of the structure comprising CDRs H2 of the Fabs and N helices of 3-H.

<p>The 2Fo-Fc maps are contoured at 1.0 σ and are shown for the fragments of N helices comprising residues 571–575, and for CDRs H2 (residues 51–57). A) (Fab 8066)₃/3-H. Fab is green and 3-H is blue. B) (Fab 8062)₃/3-H. Fab is yellow and 3-H is orange.</p

Comparison of the antigen-antibody interactions of single Fabs 8066 and 8062 in the complexes with 3-H and 5-Helix.

<p>Fabs were superimposed using the Cα traces of the β-sheet framework of the variable domains. CDRs and N-HR helices of the complexes of Fab 8066 with 3-H and 5-Helix are shown in red and green, respectively, while the corresponding fragments of the complexes of Fab 8062 are blue and yellow. (A) Superposition of all CDRs in two complexes with 3-H. (B) Contacts between CDRs H2 and two 2 N-HR helices in the four complexes. (C) A similar conformation of CDR H3 in the four complexes is stabilized by a cluster of aromatic residues from CDRs H3, L1 and L3. Residues F96, Y100B, and F100D of CDR H3 are displayed, but, for clarity, are not labeled.</p