CytoBas: Precision Component-resolved Diagnostics for Allergy Using Flow Cytometric Staining of Basophils with Recombinant Allergen Tetramers

Overview

Journal Allergy

Specialty Allergy & Immunology

Date 2021 Mar 27

PMID 33772805

Citations 2

Authors

Craig I McKenzie

Nirupama Varese

Pei M Aui

Bruce D Wines

Philip Mark Hogarth

Francis Thien

Mark Hew

Jennifer M Rolland

Robyn E OHehir

Menno C van Zelm

Affiliations

Soon will be listed here.

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.

Citing Articles

Multiplex basophil activation tests for allergy diagnosis: present and future applications.

Koren A, Korosec P Front Allergy. 2025; 5:1515843.

PMID: 39877248 PMC: 11772483. DOI: 10.3389/falgy.2024.1515843.

Expansion of phenotypically modified type 2 memory B cells after allergen immunotherapy.

von Borstel A, Reinwald S, Aui P, McKenzie C, Varese N, Hogarth P Allergy. 2024; 80(3):867-869.

PMID: 39268605 PMC: 11891399. DOI: 10.1111/all.16320.

RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect.

McKenzie C, Varese N, Aui P, Reinwald S, Wines B, Hogarth P Allergy. 2022; 78(3):822-835.

PMID: 36153670 PMC: 10952829. DOI: 10.1111/all.15529.

Role of Basophils in a Broad Spectrum of Disorders.

Miyake K, Ito J, Karasuyama H Front Immunol. 2022; 13:902494.

PMID: 35693800 PMC: 9186123. DOI: 10.3389/fimmu.2022.902494.

References

Dang T, Tang M, Choo S, Licciardi P, Koplin J, Martin P . Increasing the accuracy of peanut allergy diagnosis by using Ara h 2. J Allergy Clin Immunol. 2012; 129(4):1056-63. DOI: 10.1016/j.jaci.2012.01.056. View

Heeringa J, McKenzie C, Varese N, Hew M, Bakx A, Aui P . Induction of IgG and IgG B-cell memory following sublingual immunotherapy for ryegrass pollen allergy. Allergy. 2019; 75(5):1121-1132. PMC: 7317934. DOI: 10.1111/all.14073. View

Niederberger V, Laffer S, Froschl R, Kraft D, Rumpold H, Kapiotis S . IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) account for a high percentage of grass pollen-specific IgE. J Allergy Clin Immunol. 1998; 101(2 Pt 1):258-64. DOI: 10.1016/s0091-6749(98)70391-4. View

Whitrow M, Moore V, Rumbold A, Davies M . Effect of supplemental folic acid in pregnancy on childhood asthma: a prospective birth cohort study. Am J Epidemiol. 2009; 170(12):1486-93. DOI: 10.1093/aje/kwp315. View

Bilo M, Ollert M, Blank S . The role of component-resolved diagnosis in Hymenoptera venom allergy. Curr Opin Allergy Clin Immunol. 2019; 19(6):614-622. DOI: 10.1097/ACI.0000000000000574. View

Tamborini E, Faccini S, Lidholm J, Svensson M, Brandazza A, Longhi R . Biochemical and immunological characterization of recombinant allergen Lol p 1. Eur J Biochem. 1997; 249(3):886-94. DOI: 10.1111/j.1432-1033.1997.00886.x. View

Shi X, Jarvis D . Protein N-glycosylation in the baculovirus-insect cell system. Curr Drug Targets. 2007; 8(10):1116-25. PMC: 3647355. DOI: 10.2174/138945007782151360. View

Treudler R, Simon J . Overview of component resolved diagnostics. Curr Allergy Asthma Rep. 2012; 13(1):110-7. DOI: 10.1007/s11882-012-0318-8. View

Blank S, Michel Y, Seismann H, Plum M, Greunke K, Grunwald T . Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells. Protein Pept Lett. 2010; 18(4):415-22. DOI: 10.2174/092986611794653923. View

10.

Lieberman J, Glaumann S, Batelson S, Borres M, Sampson H, Nilsson C . The utility of peanut components in the diagnosis of IgE-mediated peanut allergy among distinct populations. J Allergy Clin Immunol Pract. 2013; 1(1):75-82. DOI: 10.1016/j.jaip.2012.11.002. View

11.

Vieths S, Hoffmann A, Holzhauser T, Muller U, Reindl J, Haustein D . Factors influencing the quality of food extracts for in vitro and in vivo diagnosis. Allergy. 1998; 53(46 Suppl):65-71. DOI: 10.1111/j.1398-9995.1998.tb04965.x. View

12.

Knox R . Grass pollen, thunderstorms and asthma. Clin Exp Allergy. 1993; 23(5):354-9. DOI: 10.1111/j.1365-2222.1993.tb00339.x. View

13.

Knol E, Mul F, Kuijpers T, Verhoeven A, Roos D . Intracellular events in anti-IgE nonreleasing human basophils. J Allergy Clin Immunol. 1992; 90(1):92-103. DOI: 10.1016/s0091-6749(06)80015-1. View

14.

Ong E, Knox R, Singh M . Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation. Mol Immunol. 1995; 32(4):295-302. DOI: 10.1016/0161-5890(94)00127-m. View

15.

Halim A, Carlsson M, Madsen C, Brand S, Moller S, Olsen C . Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications. Mol Cell Proteomics. 2014; 14(1):191-204. PMC: 4288254. DOI: 10.1074/mcp.M114.042614. View

16.

Kohler J, Blank S, Muller S, Bantleon F, Frick M, Huss-Marp J . Component resolution reveals additional major allergens in patients with honeybee venom allergy. J Allergy Clin Immunol. 2014; 133(5):1383-9, 1389.e1-6. DOI: 10.1016/j.jaci.2013.10.060. View

17.

Huang Y, Wang C, Lin X, Lou H, Cao F, Li W . Association between component-resolved diagnosis of house dust mite and efficacy of allergen immunotherapy in allergic rhinitis patients. Clin Transl Allergy. 2020; 9:64. PMC: 6921530. DOI: 10.1186/s13601-019-0305-4. View

18.

Chan S, Pomes A, Hilger C, Davies J, Mueller G, Kuehn A . Keeping Allergen Names Clear and Defined. Front Immunol. 2019; 10:2600. PMC: 6878850. DOI: 10.3389/fimmu.2019.02600. View

19.

Chirumbolo S, Vella A, Ortolani R, De Gironcoli M, Solero P, Tridente G . Differential response of human basophil activation markers: a multi-parameter flow cytometry approach. Clin Mol Allergy. 2008; 6:12. PMC: 2584049. DOI: 10.1186/1476-7961-6-12. View

20.

Macglashan Jr D . Basophil activation testing. J Allergy Clin Immunol. 2013; 132(4):777-87. DOI: 10.1016/j.jaci.2013.06.038. View