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.