Development of time-resolved immunofluorometric assays for the detection of house dust mite-allergic IgE in human sera

Dermatophagoides farinae and D. pteronyssinus are the prevalent house dust mites (HDM) in tropical countries and are associated with allergic diseases. This investigation developed a time- resolved immunofluorometric assay (TR-IFMA) for the first time to detect specific IgE antibody in patients with skin prick test positive to HDM but no detectable IgE by other means. Levels of IgE to natural and recombinant HDM allergens were measured by TR-IFMA in 50 HDM-allergic patients and 19 healthy participants compared to sandwich enzyme-linked immunosorbent assay (ELISA). A recombinant allergen, rDerf2, showed a 14 kDa band corresponding to broad range proteins of natural HDM.TR-IFMA showed sensitivity lower than 0.35 kUA/l. TR-IFMA employing three HDM antigens showed good correlations with sandwich ELISA at R2 0.93-0.96. TR-IFMA detected HDM IgE in 62, 62, 25 percent of allergic patient serum sample compared to 28, 32, and 22 percent detected by ELISA result using three HDM allergen. TR-IFMA also detected 26.3, 31.6, and 5.3 percent positive samples from 19 healthy participants while ELISA showed 0, 5.3, and 0 percents IgE positive samples. The use of rDerf2 as an HDM allergen for the assay was verified with no statistically different from other HDM allergens. TR-IFMA showed lower detection limit than ELISA and yielded higher sensitivity for serum of people with allergic symptoms with no detectable HDM IgE. It is anticipated that TR-IFMA for HDM-specific IgE detection will play an important role in future diagnosis of HDM allergy in clinical laboratories and for different research purposes.

T cell responses to der f2 mite allergens in Thai allergic patients

Dermatophagoides farinae group 2 (Der f2) is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variants on antibody and T cell responses has not been compared. The aim of the present study was to evaluate IgE binding, transcription and translations for IL-5, IFN-γ and TGF-β induced by mite allergens. Sera from 24 HDM-allergic patients and 20 non-allergic subjects were measured for IgE reactivity by ELISA. PBMC was cultured with mite allergens (Df, rDerf2) and mitogen (PHA). The supernatants and cell pellet obtained were evaluated for cytokine production by ELISA and cytokine gene expression by RT-PCR, respectively. Four patients showed IgE reactivity to both allergens. Five patients showed IgE reactivity to Df. Other allergic patients and all non-allergic subjects did not show IgE reactivity to mite allergens. Both allergens showed similar levels of IL-5 and IFN-γ transcriptions in allergic patients and non-allergic subjects. The rDer f2 induced IL-5 protein from allergic patients higher than non-allergic subjects, while Df showed IL-5 protein from allergic patients similar to non-allergic subjects. Df induced IFN-protein from allergic patients higher than non-allergic subjects whereas rDer f2 induced IFN-protein from allergic patients similar to non-allergic subjects. The ratio of IFN-γ to IL-5 production after stimulation with rDer f2 was higher in non-allergic subjects than in allergic patients. Our data demonstrated that the changes in the sequence of rDer f2 compared with native Df had effect on cytokine production in both allergic patients and non-allergic subjects.