Abstract
BackgroundBlomia tropicalis is a dust mite and an important source of allergens in tropical regions. Up to now, the assays to diagnose atopy to this mite use whole body extract as antigens. However, anti-B. tropicalis IgE antibodies cross-react with Ascaris lumbricoides antigens, hindering the diagnosis of allergy to this mite. In this study, B. tropicalis recombinant allergens were evaluated with the purpose of developing an immunodiagnostic assay for allergy to this mite with greater specificity than those commercially available.MethodsTwo B. tropicalis allergens (Blo t 5 and Blo t 21) were cloned into a plasmidial expression vector, expressed in Escherichia coli and purified by affinity chromatography. Sixty-three sera containing anti-B. tropicalis extract (BtE) IgE antibodies were used to investigate IgE reactivity to the recombinant Blot 5 and 21 allergens. Inhibition assays with 20 sera pre-adsorbed with A. lumbricoides extract were performed using rBlo t 5, rBlo t 21, and BtE as antigens. All the assays were carried using indirect ELISA.ResultsEighty-two point nine percent and 80.0% of the sera with anti-BtE antibodies from 35 children reacted with rBlo t 5 and rBlo t 21, respectively, whereas 92.8% and 89.3% of the 28 sera with anti-BtE antibodies from adult asthma patients reacted with the same allergens, and 96.4% of these sera reacted with a mixture of rBlo t 5 and rBlo t 21. In an inhibition ELISA, the absorption of sera by A. lumbricoides extract affected less the reaction with rBlo t 5 and rBlo t 21 than with BtE.ConclusionsThe rBlo t 5 and rBlo t 21 allergens contain important epitopes recognized by IgE antibodies of individuals allergic to B. tropicalis antigens. Moreover, the assays using the recombinant allergens had lower IgE cross-reactivity with A. lumbricoides antigens, a fact which would confers higher specificity to serodiagnostic assays than the crude mite extract. However, additional recombinant allergens should be evaluated in order to reach the same sensitivity of the commercially available assays based on mite extract.
Highlights
Blomia tropicalis is a dust mite and an important source of allergens in tropical regions
Several individuals had a higher IgE antibody reactivity to rBlo t 5 and rBlo t 21 than to B. tropicalis extract (BtE), there is a small percentage of sera (7.9%, corresponding to 5 out of 63 sera) that does not recognize any of the recombinant allergens, a fact that would limit the sensitivity of assays using these allergens
We demonstrated that rBlo t 5 and rBlo t 21 were antigenic for the B. tropicalis-sensitized population evaluated in this work
Summary
Blomia tropicalis is a dust mite and an important source of allergens in tropical regions. The majority of the methods for detecting anti-allergen IgE antibodies is based on the use of natural allergenic extracts, a fact that may compromise the interpretation of their results. Around 12 recombinant allergens have been molecularly and immunologically characterized and deposited into the allergen database, according to International Union of Immunological Societies (IUIS) Allergen Nomenclature (www.allergen.org) Among these allergens, it includes Blo t 1 (cysteine protease, 26 kDa), Blo t 3 (trypsin protease, 25 kDa), Blo t 5 (unknown molecular function, 14 kDa), Blo t 11 (paramyosin, 110 kDa), Blo t 12 (unknown molecular function, 14 kDa), and Blo t 21 (unknown molecular function, 13.2 kDa), which are present in 50% or more of the sera from B. tropicalis-sensitized individuals [3,4]. Despite the fact that some putative IgE epitope residues are conserved in both Blo t 5 and Blo t 21 three-dimensional structures, these allergens present a low to moderate cross-reactivity [5,8]
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