Altered antigenicity of M-177, a 177-kDa allergen from the house dust mite Dermatophagoides farinae, in stored extract

Background A high molecular weight allergen, M‐177 (177 kDa) was isolated from Dermatophagoides farinae using a specific antibody raised to an allergenic clone Mag 3, which was obtained by immunoscreening a mite cDNA library. The potent IgE reactivity of M‐177 is comparable with that of Der f 2. Objective The aim of this study was to analyse the molecular characteristics and the allergenic activity of M‐177 in stored mite extracts. Methods Antigens were analysed by immunoblotting and enzyme‐linked immunosorbent assay (ELISA; inhibition). Allergenic activity was estimated from IgE reactivity and the results of a histamine release assay. Results The intact M‐177 molecule was present in high concentrations in fresh extract obtained from purified mite bodies, but was only detected in small amounts in stored extracts. Instead of the intact molecule, anti‐Mag 3 antibody detected various cross‐reactive antigens in the stored preparations. Studies of a stored liquid extract showed that these cross‐reactive antigens were produced by the degradation of M‐177, and that this change was suppressed by the addition of protease inhibitors. Interestingly, the allergenic activity of the fragmented M‐177 (sM‐177) isolated from the stored extracts was greater than that of the intact antigen. Specific IgE reacted with sM‐177 in 84.2% of 38 sera samples from patients allergic to mites, while 65.8% were positive for M‐177‐specific IgE. Similarly, the histamine release test showed that sM‐177 had greater allergenic activity in vitro. ELISA inhibition indicated that the increased allergenic activity resulted from alteration of the antigenicity with the degradation of M‐177. Conclusions M‐177 is a protease‐sensitive allergen. The breakdown products of M‐177 provoked higher allergenic activity than the intact allergen.