3.3 Recombinant Lol p 1 tetramer staining detects functional allergic sensitization to ryegrass pollen
The ability of [Lol p 1]4 to detect functional allergen sensitization was examined using blood samples from 50 RGP-allergic patients and 20 controls. Following incubation with increasing amounts of [Lol p 1]4-APC or control streptavidin-APC, basophils of patients and controls were examined for allergen binding (Figure 3A ) and activation (CD63 positivity;Figure 3B ). Incubation with [Lol p 1]4-APC resulted in a dose-dependent increase in signal observed in all 50 patients with RGP allergy (Figure 3A ). The increase in staining intensity indicated functional allergen binding, as this was accompanied by increased frequencies of CD63+ basophils in the patient samples (Figure 3B ). In contrast, basophils in the control cohort did not show any increase of allergen staining over the strep-APC reagent (Figure 3A ). There was negligible CD63 expression on basophils of controls. Similarly, parallel incubations of blood with RGP extract only resulted in significant CD63 expression on basophils of patients and not controls (Figure 3C ). Activation by anti-IgE and/or fMLP resulted in increased CD63 expression on basophils from all patients and controls, indicating that these cells were functionally capable of degranulation (Suppl. Figure 3A, B ). Thus, as for BV allergy, the CytoBas approach can be utilized for RGP allergy with a major recombinant allergen component, Lol p 1, to detect relevant and functional allergic sensitization.