CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers

Craig I McKenzie; Nirupama Varese; Pei M Aui; Bruce D Wines; Philip Mark Hogarth; Francis Thien; Mark Hew; Jennifer M Rolland; Robyn E O'Hehir; Menno C van Zelm

doi:10.1111/all.14832

CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers

Allergy. 2021 Oct;76(10):3028-3040. doi: 10.1111/all.14832. Epub 2021 May 5.

Authors

Craig I McKenzie¹, Nirupama Varese^{1

2}, Pei M Aui¹, Bruce D Wines^{1

3

4}, Philip Mark Hogarth^{1

3

4}, Francis Thien⁵, Mark Hew^{6

7}, Jennifer M Rolland^{1

2}, Robyn E O'Hehir^{1

2

7}, Menno C van Zelm^{1

7}

Affiliations

¹ Department of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, VIC, Australia.
² Department of Allergy, Immunology and Respiratory Medicine, Central Clinical School, Monash University, Melbourne, VIC, Australia.
³ Immune Therapies Group, Burnet Institute, Melbourne, Australia.
⁴ Department of Pathology, The University of Melbourne, Parkville, VIC, Australia.
⁵ Respiratory Medicine, Eastern Health, Box Hill and Monash University, Melbourne, VIC, Australia.
⁶ School of Public Health and Preventive Medicine, Monash University, Melbourne, VIC, Australia.
⁷ Allergy, Asthma and Clinical Immunology, Alfred Health, Melbourne, VIC, Australia.

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-fluorochrome 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.

Keywords: allergen tetramers; basophils; component-resolved diagnostics; flow cytometry; surface IgE.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Allergens
Basophils*
Flow Cytometry
Humans
Hypersensitivity* / diagnosis
Staining and Labeling

Substances

Allergens