Dataset Information

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

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.

SUBMITTER: McKenzie CI

PROVIDER: S-EPMC8518718 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Publications

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

McKenzie Craig I CI Varese Nirupama N Aui Pei M PM Wines Bruce D BD Hogarth Philip Mark PM Thien Francis F Hew Mark M Rolland Jennifer M JM O'Hehir Robyn E RE van Zelm Menno C MC

Allergy 20210505 10

<h4>Background</h4>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.<h4>Objective</h4>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.<h4>Methods</h4>Recombinant forms of Lol p 1 and Lol p 5 protein ...[more]

PMID: 33772805

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Project description:Background: Manifestation of respiratory allergy to American cockroach (Periplaneta americana) is prominent in the subtropical and tropical areas. However, co-existing perennial indoor inhalant allergies frequently compromise clinical diagnosis of cockroach allergy, and the analysis of sensitization pattern is limited by the lack of Periplaneta allergens widely available for component-resolved diagnostics (CRD). Objective: To evaluate a collection of previously described recombinant Periplaneta allergens for CRD in cockroach allergy. Methods: A panel of nine recombinant Periplaneta allergens (Per a 1-5, 7-10) was generated, purified, and subjected to physicochemical characterization by applying circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), amino acid (AA) analysis, and mass spectrometry (MS). Patients (n = 117) from India, Korea, Venezuela, and Iran, reporting perennial respiratory indoor allergies with IgE sensitization to cockroach (P. americana and/or Blattella germanica), were included. The sensitization profile was monitored by the experimental ImmunoCAP testing. Results: ImmunoCAP testing confirmed IgE sensitization to Periplaneta and/or Blattella extract in 98 of 117 patients (r = 0.95). Five out of 117 patients were sensitized to only one of the two cockroach species. Within the whole study group, the prevalence of sensitization to individual allergens varied from 4% (Per a 2) to 50% (Per a 9), with the highest IgE values to Per a 9. Patients from four countries displayed different sensitization profiles at which Per a 3 and Per a 9 were identified as major allergens in India and Korea. Periplaneta-derived lipocalin and myosin light chain were characterized as new minor allergens, designated as Per a 4 and Per a 8. Periplaneta extract showed higher diagnostic sensitivity than all individual components combined, suggesting the existence of allergens yet to be discovered. Conclusion: Utilization of a panel of purified Periplaneta allergens revealed highly heterogeneous sensitization patterns and allowed the classification of lipocalin and myosin light chain from Periplaneta as new minor allergens.

Dataset Information

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

Background

Objective

Methods

Results

Conclusion

Publications

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

Similar Datasets

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