Background: Diagnostic tests for allergy rely on detecting
allergen-specific IgE. Component-resolved diagnostics incorporate
multiple defined allergen components to improve the quality of diagnosis
and patient care. Objective: To develop a new approach for determining
sensitization to specific allergen components that utilizes fluorescent
protein tetramers for direct staining of IgE on blood basophils by flow
cytometry. Methods: Recombinant forms of Lol_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-fluorophore conjugates. Blood samples from 50 RGP-allergic,
41 BV-allergic and 26 controls were incubated with fluorescent protein
tetramers for flow cytometric evaluation of basophil 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 tube assay 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.
CytoBas: Precision component‐resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers
