Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen

Background The knowledge of IgE-binding epitopes on allergen molecules is i mportant for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy. Objective Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen. Methods We performed an IgE-binding epitope mapping of rHol 1 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragment s (length between 15 and 186 amino acids), which were expressed in E. coli as MBP fusion proteins. Using sera of 65 grass pollen allergic patients, th e fragments were analysed by immunoblotting for IgE reactivity. Specificity of antibody binding was confirmed by competitive blot inhibition assays. Results At least four different continuous IgE-binding epitopes were identi fied on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroy ed by further fragmentation. The fragments were differentially recognized b y individual patients' sera. By investigating IgE- binding to one of the sm all fragments in more detail, we found further epitope regions on this frag ment. It was noteworthy that IgE reactivity to small fragments was weak com pared to large fragments or to the complete molecule. Competitive blot inhi bition experiments showed that binding of IgE antibodies to the small fragm ents was specific but with lower avidity than to the complete rHol 1 5. Conclusion rHol 1 5 harbours multiple discontinuous as well as continuous I gE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients. Low avidity of IgE a ntibodies to small fragments suggests that the continuous epitope regions d o not represent the complete epitope and are most probably parts of discont inuous epitopes.