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CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers
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  • Craig McKenzie,
  • Nirupama Varese,
  • Pei Aui,
  • Bruce Wines,
  • P Hogarth,
  • Francis Thien,
  • Mark Hew,
  • Jennifer Rolland,
  • Robyn O'Hehir,
  • Menno van Zelm
Craig McKenzie
Monash Univeristy
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Nirupama Varese
Alfred Health
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Pei Aui
Monash Univeristy
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Bruce Wines
Burnet Institute
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P Hogarth
Burnet Institute
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Francis Thien
Eastern Health and Monash University, Box Hill Hospital
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Mark Hew
Alfred Hospital
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Jennifer Rolland
The Alfred Hospital
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Robyn O'Hehir
Alfred Hospital
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Menno van Zelm
Monash University
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Abstract

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.

Peer review status:UNDER REVIEW

20 Nov 2020Submitted to Allergy
20 Nov 2020Assigned to Editor
20 Nov 2020Submission Checks Completed
20 Nov 2020Reviewer(s) Assigned