Non-protease native allergens partially purified from bodies of eight domestic mites using p-aminobenzamidine ligand

Main Article Content

T. Erban
R. Klubal

Keywords

Allergen, Tropomyosin, Paramyosin, Native antigen, Actin, House dust mite, Stored-product mite, Affinity chromatography

Abstract

Background: Optimised purification steps for concentrating trace target native antigens are needed. Combining the p-aminobenzamidine ligand with protease inactivation enables partial purification of mite non-protease allergens lacking proteases.


Objective: We sought to analyse in detail proteins obtained using this method from eight species of synanthropic acaridid mites and tested IgE reactivity using pooled human sera.


Materials and methods: Proteins affinity bound to p-aminobenzamidine as a ligand were identified by MALDI TOF/TOF. After electroblotting, the proteins were visualised using the fluorescent SYPRO-Ruby protein blot stain, and IgE reactivity was further analysed using pooled human sera collected from patients allergic to house dust mites.


Results: MS/MS identification confirmed previous results that no proteases were purified. Protein patterns corresponding to the allergens Der f 7, Der f 30 and actins indicated that these proteins are purified using p-aminobenzamidine and are present across a wide spectrum of acaridid mites. When using Dermatophagoides farinae, apolipophorins (Der f 14), chitinase-like Der f 15 and 18, 70-kDa heat shock protein, and a Der f Alt a10 allergen homolog (gi|37958173) were also detected. The target antigens tropomyosins and paramyosins showed similar IgE binding among the mite species tested. IgE reactivity with miscellaneous D. farinae antigen was also observed.


Conclusions: Partial purification of mite non-protease antigens using a strategy combining paminobenzamidine with protease inactivation was verified by 1D-E and 2D-E analyses. IgE binding to p-aminobenzamidine-purified native non-protease mite antigens was tested using pooled sera. This preliminary study allows for further work on individual serum samples, allowing confirmation of immunoreactivity.

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