Allergenicity of native and recombinant major allergen groups 1 and 2 of Dermatophagoides mites in mite sensitive Thai patients.

BACKGROUND AND OBJECTIVE: Natural allergenic extracts using for diagnosis and immunotherapy may have batch-to-batch variations and contaminations with unrefined allergens or non-allergenic components. Thus, recombinant allergen is believed to overcome these shortcomings. In this study, native and recombinant allergens of group 1 and 2 of Dermatophagoides mites were produced and their allergenicities were compared. METHODS: Native allergens were prepared by MAb affinity chromatography. All recombinant allergens were produced in E. coli expression system. IgE reactivities of these allergens were determined by IgE-ELISA. RESULTS: The native and recombinant Der p 1, Der p 2, Der f 1, Der f 2 had molecular weights of approximately 25, 15, 25 and 15 kDa, respectively. IgE reactivities of nDer p 1, nDer f 1, rDer p 1 and rDer f 1 were 96.67%, 90%, 43.33% and 46.67%, respectively. Allergenicities of nDer p 2, nDer f 2, rDer p 2 and rDer f 2 were 86.67%, 96.43%, 76.67% and 89.29%, respectively. The findings indicated that recombinant group-1 products were minor allergens which revealed no correlation with their native forms. In contrast, recombinant group-2 allergens were major allergens and showed a significant correlation to their native allergens. CONCLUSION: We successfully produced native and recombinant group-1 and group-2 allergens. According to their allergenicities, recombinant Der p 2 and rDer f 2 have potential to replace native allergen in diagnostic and therapeutic extracts. Moreover, they can employ as a standard reagent to measure the amount of group 2 allergen in the environment by sandwich-ELISA and utilise this as an immunogen for MAb production.

Proteome, Allergenome, and Novel Allergens of House Dust Mite, Dermatophagoides farinae.

Dermatophagoides farinae mite is a predominant source of indoor allergens causing high incidence of allergy worldwide. People with different genetic background respond differently to the mite components, and thus the component-resolved diagnosis (CRD) is preferred to the conventional allergy test based on crude mite extract. In this study, proteome and culprit components in the D. farinae whole body extract that sensitized the allergic patients were studied by using SDS-PAGE (1DE) and 2DE-IgE immunoblotting followed by LC-MS/MS and database search for protein identification. From the 1DE, the mite extract revealed 105 proteins that could be classified into seven functionally different groups: allergens, structural components, enzymes, enzyme inhibitor, receptor proteins, transporters, and binding/regulatory/cell signaling proteins. From the 2DE, the mite extract produced 94 spots; 63 were bound by IgE in sera of 20 D. farinae allergic patients. One more protein that was not revealed by the 2DE and protein staining reacted with IgE in 2 allergic patients. Proteins in 40 spots could be identified as 35 different types. Three of them reacted to IgE of >50% of the allergic patients, and hence they are major allergens: tropomyosin or Der f 10 (75%), aconitate hydratase (70%), and one uncharacterized protein (55%). Aconitate hydratase is a novel D. farinae major allergen unraveled in this study. Several mite minor allergens that have never been previously reported are also identified. The data have clinical applications in the component-resolved diagnosis for tailor-designed allergen-specific immunotherapy.