Key Points
Four human Der p 2–specific IgE mAbs were identified by a new hybridoma technology.
The human IgE mAbs recognize two nonoverlapping epitopes on Der p 2.
NMR identified residues in the epitopes of human IgE and murine IgG mAbs.
Abstract
IgE Abs drive the symptoms of allergic disease upon cross-linking allergens on mast cells or basophils. If the IgE binding sites on the allergens could be identified, it may be useful for creating new forms of immunotherapy. However, direct knowledge of the human IgE (hIgE) epitopes is limited because of the very low frequency of IgE-producing B cells in blood. A new hybridoma technology using human B cells from house dust mite–allergic patients was used to identify four Der p 2–specific hIgE mAbs. Their relative binding sites were assessed and compared by immunoassays with three previously studied murine IgG mAbs. Immunoassays showed that the recognition of Der p 2 by the first three hIgE was inhibited by a single murine IgG, but the fourth hIgE recognized a different epitope from all the other mAbs. The functional ability of the hIgE that bind different epitopes to cross-link Der p 2 was demonstrated in a mouse model of passive systemic anaphylaxis. Nuclear magnetic resonance analyses of Der p 2 in complex with IgG and IgE Abs were used to identify specific residues in the epitopes. To our knowledge, the combination of immunoassays to distinguish overlapping epitopes and nuclear magnetic resonance analyses to identify specific residues involved in Ab binding provided the first epitope mapping of hIgE mAbs to an allergen. The technologies developed in this study will be useful in high-resolution mapping of human epitopes on other Ags and the design of improved therapeutics.
Footnotes
This work was supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (Project Z01-ES102906 to G.A.M.). This work was supported in part by the NIH, National Institute of Allergy and Infectious Diseases under Award R01AI077653 (to A.P. and M.D.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- hIgE
- human IgE
- HMQC
- heteronuclear multiple-quantum coherence
- mIgG
- murine IgG
- nDer p 2
- natural Der p 2
- NMR
- nuclear magnetic resonance.
- Received March 17, 2020.
- Accepted August 5, 2020.
- Copyright © 2020 by The American Association of Immunologists, Inc.
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