Determination of the N- and C-terminal sequences required to bind human IgE of the major house dust mite allergen Der f 2 and epitope mapping for monoclonal antibodies

Abstract

B cell epitopes of the major house dust mite allergen Der f 2 from Dermatophagoides farinae were analysed using deletion mutants of Der f 2 expressed as fusion proteins in Escherichia coli. The reactivities of these partial Der f 2 molecules to human anti-mite IgE antibodies in atopic patients and to murine anti-Der f 2 monoclonal antibodies (mAbs) were examined by immunoblotting. A C-terminal deletion mutant of Der f 2, 1–123, had almost the same reactivity to human IgE as the whole Der f 2 (1–129) and an N-terminal deletion mutant of Der f 2 (25–129) still had weak reactivity. On the other hand, in two deleted Der f 2 molecules, 1–120 and 30–129, reactivity was lost in spite of long overlapping sequences. These results suggest that the human IgE antibodies to Der f 2 in atopic patient sera recognize the conformational structures dependent on the tertiary structure of Der f 2, including disulfide bond formations, rather than the contiguous sequences of amino acids. The sequences 1–24, 25–29 and 121–123 were revealed as the minimum N-and C- terminal amino acid sequences required for IgE binding. Contrastingly, all three murine mAbs bound to the smaller deletion mutants, 1–90 and 67–129, suggesting that the cores of the epitopes for these mAbs exist in the 24 amino acid sequence of Der f 2, 67–90 overlapping the sequential human IgE epitope on Der p 2, the equivalent allergen from Dermatophagoides pteronyssinus. These findings are important for the understanding of the antigenic structure of Der f 2 and for the manipulation of the allergen for immunotherapy.