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.