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.
INTRODUCTION
Diagnosis of IgE-mediated allergy is based on the patient’s clinical symptoms and medical history, confirmed with laboratory tests.1 The latter are becoming increasingly important for patient management, as well as for selection for and monitoring of allergen-specific immunotherapy (AIT). Currently, skin prick testing (SPT) and detection of serum specific IgE using allergen extracts are standard methods to differentiate sensitized from non-sensitized individuals.2 While serum specific IgE detection is highly sensitive, the use of allergen extracts can yield positive results due to recognition by cross-reactive antibodies of components that are not the drivers of disease.3-8
Basophil activation tests (BAT), i.e. in vitro exposure of blood basophils to increasing doses of allergen,9 have the advantage of detecting functional IgE that on binding allergen, can directly result in degranulation and development of allergic symptoms.10 IgE bound to FcεRI is cross-linked by allergen and triggers signaling and basophil degranulation. Resultant surface expression of CD63 is a reliable indicator of the sensitivity and reactivity of basophils to an allergen.9 BAT is a functional assay with advantages over serology for specific IgE in that it can more accurately diagnose allergies and monitor responses to immunotherapy.10 However, BAT requires in vitrostimulation of multiple blood samples with serial dilutions of allergen as well as positive and negative controls, which has thus far hampered large-scale diagnostic implementation.8
A different approach for detection of relevant allergen sensitization is the use of molecular allergen components that are either purified from allergen extracts or produced recombinantly. Detection of IgE reactivity to major allergen components has the potential to be highly sensitive with high specificity through omission of cross-reactive components that are less relevant for disease.11-13 For example sensitization to Der p 1 and/or Der p 2, the major house dust mite (HDM) allergens, is a better positive predictor for HDM-driven asthma than whole HDM extract.14,15 Furthermore, sensitization to Ara h 2, a major peanut allergen, is more specific for systemic allergic responses than whole peanut extract.16,17Consequently, sensitization patterns to molecular allergen components can be relevant for patient management and treatment.18,19 The major ryegrass pollen (RGP) allergens Lol p 1 and Lol p 5 are each detected by serum IgE of 80-90% of RGP-allergic individuals.20-23 Importantly, Lol p 5 sensitization is associated with a higher risk of thunderstorm asthma,24,25 and would be a potential indicator for AIT.26
The major allergen component of BV is Api m 1, whereby 78-90% of people allergic to BV have Api m 1-specific IgE in serum.27,28 BV extracts used in AIT for BV allergy typically contain Api m 1 but may lack another key major allergen associated with systemic reactions, Api m 10. Patients with high sensitization to Api m 10 will not benefit from treatment with such currently-available SCIT preparations, highlighting the importance of resolving both disease and treatment at the component level for accurate prescription of AIT.5,19,29
Despite our understanding of these allergen components, the use of component-resolved diagnostics has not become routine or standard practice. At present, these may be applied as follow-up tests to confirm or refute an initial test using whole allergen extract.17 Ideally, a single laboratory test would enable differential detection of allergen sensitization and stratification of patients into clinical risk groups. This would constitute a multiplex assay that can incorporate a relevant set of allergen components for e.g. aeroallergens, food allergens or stinging insect allergens.
We have generated fluorescently-labelled tetramers of the major allergen components Api m 1, Lol p 1 and Lol p 5 to explore component-resolved diagnostics using direct staining of blood basophils in a single test - CytoBas. Application of these reagents in flow cytometry showed specific binding to basophils of sensitized patients, with a higher positive predictive value for allergy than BAT. Fluorescence intensity of allergen tetramers on the surface of basophils correlated with titers of allergen-specific IgE in serum. Importantly, by using different fluorochromes, binding of multiple allergen components can be assessed simultaneously, enabling a differential and component-resolved allergy diagnostic in a single flow cytometric assay and without the need for cell stimulation.
METHODS