Epitope mapping of major insect allergens (chironomid hemoglobins) with monoclonal antibodies

Monoclonal antibodies (m ABs) were raised against the physically and chemically well-defined insect allergen, component CTT III from Chironomus thummi thummi (CTT) hemoglobin. With these m ABs, we observed epitopes present in as many different hemoglobin (Hb) molecules from the widespread insect family Chironomidae as possible. These Hbs were identified as important allergens in previous studies. Twenty m ABs from seven different clones were tested for specificity against the CTT Hbs III, IIIa, IV, and I to X, as well as against Hbs from 14 other chironomid species; m ABs 1 to 3 were found to be specific for an epitope that was expressed on the CTT III component as well as in Hbs from three other species. m ABs 5 to 7 and 15 recognized an epitope expressed in CTT III, CTT IIIa, CTT IV, and in Hbs from 13 other species. m ABs 8 to 14 and 16 to 20 demonstrated heteroclitic behavior, that is, they bound CTT IIIa better than the immunogen CTT III. Reactivity of m AB 6 with a clinically relevant epitope was demonstrated by up to 15% inhibition of antibody binding by human IgE from sensitized patients. Our results demonstrate that clinically relevant allergenic epitopes can be localized on different molecules with m ABs. This could be important in the development of immunotherapy protocols with highly purified or fragmented allergens.