3.2 Recombinant Api m 1 tetramer staining detects functional
allergic sensitization to bee venom
To determine whether positive staining of basophils with [Api m
1]4 indicates functional allergic sensitization to BV,
blood samples from 41 BV-allergic patients and 24 controls were
incubated for 20 min at 37oC with increasing amounts
of [Api m 1]4-APC or, as a control, 1 µg/ml
streptavidin-APC. Samples were then stained with mAbs to detect
basophils, and within this population, expression of CD63 was assessed
as a marker of activation and degranulation. There was a dose-dependent
increase in staining of basophils following incubation with [Api m
1]4-APC in all 41 patients with BV allergy
(Figure 2A ). In contrast, there was no increase in staining
with [Api m 1]4-APC over the streptavidin-APC
reagent on basophils from the control cohort (Figure 2A ). Thus,
the specific binding of allergen tetramer to basophils of BV-allergic
patient was accompanied by a dose-dependent increase in the frequency of
basophils with surface expression of CD63 (Figure 2B ). The
frequency of CD63+ basophils following incubation with
[Api m 1]4-APC was negligible for control subjects.
Similarly, parallel incubation of blood samples with BV extract resulted
in CD63 expression only on basophils of BV-allergic patients and not
controls (Figure 2C ). Basophils from all BV-allergic and
control subjects expressed surface CD63 following incubation with
positive controls anti-IgE and/or fMLP, indicating that basophils in all
blood samples were functionally capable of degranulation (Suppl.
Figure 2A, B ). Thus, the CytoBas approach with [Api m
1]4 has the capacity to detect relevant and functional
allergic sensitization to BV.