Design of a heterotetravalent synthetic allergen that reflects epitope heterogeneity and IgE antibody variability to study mast cell degranulation

The present paper describes the design of a HtTA (heterotetravalent allergen) as a multi-component experimental system that enables an integrative approach to study mast cell degranulation. The HtTA design allows presentation of two distinct haptens, each with a valency of 2, thereby better reflecting the complexity of natural allergens by displaying epitope heterogeneity and IgE antibody variability. Using the HtTA design, synthetic allergens HtTA-1 and HtTA-2 were synthesized to model a combination of epitope/IgE affinities. HtTA-1 presented DNP (2,4-dinitrophenyl) and dansyl haptens (K_d=22 and 54 nM for IgE^DNP and IgE^dansyl respectively) and HtTA-2 presented dansyl and the weak-affinity DNP-Pro (DNP-proline) haptens (K_d=550 nM for IgE^DNP). Both HtTAs effectively induced degranulation when mast cells were primed with both IgE^DNP and IgE^dansyl antibodies. Interestingly tetravalent DNP-Pro or bivalent dansyl were insufficient in stimulating a degranulation response, illustrating the significance of valency, affinity and synergy in allergen–IgE interactions. Importantly, maximum degranulation with both HtTA-1 and HtTA-2 was observed when only 50% of the mast cell-bound IgEs were hapten-specific (25% IgE^dansyl and 25% IgE^DNP). Taken together, results of the present study establish the HtTA system as a physiologically relevant experimental model and demonstrates its utility in elucidating critical mechanisms of mast cell degranulation.