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.

摘要

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 f2 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 121-123were 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.