Food allergic reactions are an increasing cause of morbidity and mortality worldwide, and are triggered by allergen-specific IgE antibodies bound to mast cell and basophil effector cells. The molecular features that make some human IgE antibodies pathogenic have been difficult to study from polyclonal sera that contain mixtures of unknown numbers and proportions of antibody types. Similarly, the mechanisms by which IgG4 and IgG1 antibodies elicited by oral immunotherapy (OIT) can protect patients from allergic reactions would be clarified by studying defined antibodies of known sequence from patients following treatment. We will study the functional activity of defined peanut allergen-specific human IgE, IgG4 and IgG1 monoclonal antibodies (mAbs) isolated from symptomatic allergic patients compared to sensitized but non-allergic controls, and from patients post-treatment who vary in their response to OIT. Cultured primary human mast cells will be used as effector cells, and will be studied with three assays extending from early cell activation (Calcium influx), through effector release, to later cytokine secretion. IgE clones will be evaluated for their allergen specificity, binding affinity, and epitope recognition, and tested in combinations to determine which species can sensitize mast cells. Blocking of mast cell activation by mAb IgG4 and IgG1 will be tested, and Fc mutations used to determine whether Fcγ receptors contribute to decreases in mast cell effector functions. Finally, IgE and other antibody isotypes isolated from patient gastrointestinal tract biopsies will be compared to antibodies isolated from the blood, to determine whether there is enrichment for potentially pathogenic IgE in the mucosal sites where allergic reactions occur. These studies are likely to have a significant impact on basic and translational human allergy research, as they will use a fully human, but well-defined experimental system to identify the molecular features of pathogenic IgE clones in the blood and GI tracts of allergic patients, and evaluate potential IgG4 and IgG1- mediated mechanisms of protection following OIT. Improved understanding of IgE clone affinities, epitope reactivities, and combinations that result in mast cell sensitization could enable better diagnostic and prognostic testing in allergic patients, while more knowledge about the criteria leading to protective IgG4 and IgG1 could guide therapeutic trials and the development of improved immunotherapy regimens.