Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas)

Abstract

House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). Apositive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for invitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.