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