To gain an understanding as to why Hev b 5 is such a potent allergen, we characterized the epitopes for three monoclonal antibodies to Hev b 5.

The monoclonal antibodies included 2 IgG1 (6A10, 3G3) and 1 IgG 2b (6F6) isotypes. We used SPOTscan analysis with overlapping peptides to identify the binding regions for the monoclonals and then alanine substitution analysis to further define the critical amino acids in each epitope. Site directed mutagenesis was used to selectively eliminate the IgG binding for each epitope and single and multiple mutations were expressed as recombinant GST fusion proteins. Antibody recognition of the fusion proteins was determined by inhibition ELISA assay.

All three monoclonal antibodies recognized the same AA sequence by SPOTs analysis and slight variations of this epitope are repeated 3 times within the Hev b 5 sequence; ⁶³APETEK⁶⁸, ¹²⁰PAEGEK¹²⁵, and ¹²⁶PAEEEK¹³¹. Sequential alanine substitution by SPOTsalogue identified K⁶⁸, E¹²², K¹³¹ as critical AAs in each epitope and they were changed to alanine by site directed mutagenesis. Inhibition ELISA with the mutant proteins indicated that the AA^126–131 epitope was the dominant epitope but mutation of the AA^120–125 epitope was required to eliminate MAb reactivity to Hev b 5. The antibodies did not appear to recognize the AA^63–68 epitope in the recombinant fusion protein.

We have identified an immunodominant epitope in Hev b 5 that is repeated 3 times within the sequence making Hev b 5 a multivalent protein. Multivalent epitopes may help to explain the potent nature of this allergen.