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 (original) (raw)

B cell epitopes of the major house dust mite allergen Der f 2 from Dermatophagoides ,jarinae were analysed using deletion mutants of Der f 2 expressed as fusion proteins in Escherichia co/i. 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, l-120 and 3G129, 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 l-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, l-90 and 67-l 29, 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 pferonyssinus. These findings are important for the understanding of the antigenic structure of Der f 2 and for the manipulation of the allergen for immunotherapy.