Mapping Human Monoclonal IgE Epitopes on the Major Dust Mite Allergen Der p 2 (1)
Both subjects had nearly identical IgE B cell frequencies; subject P1 had 10 and P2 showed 12 IgE B cells for every 10 million PBMCs cultured.
These three IgG mAbs, 7A1, 1D8, and aDpX inhibited binding of IgE Abs from sera/plasma to Der p 2 up to 10.3, 23.2, and 36.8%, respectively. Three hIgE mAbs (2G1, 5D10, and 1B8) bound Der p 2 presented by mAb 1D8 and 7A1 but not aDpX, which indicates that aDpX and the three IgE mAbs 2G1, 5D10, and 1B8 bind to overlapping epitopes. However, the IgE mAb 2F10 bound Der p 2 presented by each of the three coating mIgG mAbs (1D8, 7A1, and aDpX).
The aDpX at 100 ug/ml inhibited 50–71% of the binding of IgE mAbs 2G1, 5D10, and 1B8 but not 2F10 to Der p 2. The murine mAbs 7A1 and 1D8 did not inhibit binding of these IgE mAbs in a similar way as the negative control the anti–Der p1 mAb 4C1.
The three biotinylated IgE mAbs 2G1, 5D10, and 1B8 detected the Der p 2 presented by coating IgE mAb 2F10; and vice versa, biotinylated 2F10 detected Der p 2 presented by either the IgE mAb 2G1 or 1B8. Recognition of Der p 2 by three hIgE (5D10, 1B8, and 2G1) was inhibited by aDpX, but IgE mAb 2F10 binds to a different site than the epitopes for all the other six mAbs.
The methyls of residues L61, V63, and V105 have broadened below detection, whereas I97 has shifted. These residues are in close proximity on Der p 2. Using the knowledge that I97 is buried in the 7A1 epitope, the shift of I97 is suggested to be a distal conformational change because of the complex formation. Therefore, the 2F10 epitope is proposed to center around V105.
V81 and V40 showed shift changes, but they were smaller and in different directions. The shifts of V116 were so far novel to the 5D10 family of Abs. There are possibly new shifts for the adjacent L117 that are subtly different for these three Abs.
Dashed lines highlight the proposed hIgE epitopes for 5D10/1B8/2G1 and 2F10. The methodology described in this study can be directly applied to other allergen epitopes and more generally to other Ag–Ab interactions.
IgG Abs generated during peanut oral immunotherapy demonstrated that three different patients generated Abs against Ara h 2 with very similar sequences.