Identification and characterization of epitopes on Cyn d I, the major allergen of Bermuda grass pollen

Background: We identified three epitopes on Cyn d I by using four anti-Cyn d I monoclonal antibodies (MoAbs). Methods: In a cross-inhibition binding assay, the binding of MoAbs 1-61 and 10-7 to Cyn d I was completely blocked by each other but not by MoAbs 4-37 and 11-7; the binding of MoAb 4-37 and MoAb 11-7 to Cyn d I was inhibited by themselves but not by other MoAbs. The epitope recognized by MoAbs 1-61 and 10-7 is designated as El, and those recognized by MoAbs 4-37 and 11-7 are designated as E2 and E3, respectively. Results: In a radioallergosorbent inhibition assay, we found that MoAbs 1-61 and 4-37 (1:50 diluted) can inhibit the binding of human Immunoglobulin Es to Cyn d I by more than 30%, whereas MoAb I1-7 was less efficient (reduced by only 6%). These results suggest that both El and E2 are major allergenic epitopes but that E3 is only a minor one. Further characterization of E1 and E2 reveals that they are labile in alkaline but resistant to acid and sodium periodate treatments. Moreover, E1 is heat-labile, but guanidine- and urea-sensitive, whereas E2 is not. Both E1 and E2 lost their antigenicity after reduction and alkylation. Conclusions: Results of the present study provide important information on the physicochemical properties of major allergenic epitopes on Cyn d 1, which may be useful for future development of therapeutic peptides for patients allergic to Bermuda grass pollen.