Abstract

BACKGROUND: Diagnostic tests for allergyrely on detecting allergen-specific IgE.Component-resolved diagnostics incorporatemultipledefined allergen components to improve the quality of diagnosis and patient care. OBJECTIVE: To develop a new approach for determining sensitization to specific allergen componentsthat utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flowcytometry. METHODS: Recombinant forms ofLol p 1 and Lol p 5 proteins from ryegrass pollen (RGP) and Api m 1 from honeybee venom (BV) were produced, biotinylated and tetramerized with streptavidin-fluorochromeconjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation ofbasophil allergen binding and activation. RESULTS: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves.Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single tubeassay enabled rapid detection of sensitization to both Lol p 1 and Lol p 5 in RGP-allergic patients and discriminated between controls, BV-allergic and RGP-allergic patients. CONCLUSION: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.

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