Basophil Activation Test

To determine the triggering capacity of allergen or anti-IgE Abs, BAT was performed using heparinized whole blood (100 μl/test) within 4 hours of blood draw, with the stimulating time at 15 minutes 37°C for peanut or cat allergens, and 30 minutes for anti-IgE mAbs E4.15, E7.12, C6, E5.1, p6.2 and E2.18. For therapeutic effect determination using BAT based approach, the allergic (peanut and cat) subjects’ heparinized whole blood (200 μl/test) was incubated with mouse anti-human IgE mAbs or mIgG₁ isotype control in the ranges of 0.01–0.1 μg/mL for the high affinity anti-IgE mAb E4.15, E7.12, C6, E5.1, and the range of 0.25–4 μg/mL for low affinity anti-IgE mAb p6.2 and E2.18. The tested blood was incubated with anti-IgE mAbs at room temperature (22–24°C) with slow agitation (100 RPM) for 24 or 48 hours prior to basophil activation by the mixed purified Ara h allergens (Ara h1, h2, and h6 combined) or cat allergen Fel d1, respectively, for 15 minutes at 37°C with shaking. The activation was stopped by EDTA at final concentration of 10 μM. The allergen and anti-IgE Ab stimulated blood samples were stained with CD123-FITC, CD63-PE, HLA-DR-PerCP and CD203c-APC, followed by erythrocyte lysis and flow cytometry acquisition and analysis (12–14). The cut-off value of the unstimulated blood basophil CD63 expression was always set as <3% as the background, and CD203c expression level <10% since unstimulated basophils constitutively express low level CD203c. Histamine release of basophils was performed with the ELISA kit from Immnuo-biological Laboratories Inc (IBL-America, Minneapolis, MN) followed the instruction manual.