Human ige

Venous blood was collected from normal donors following guidelines from the ethics committee of the Leuven University Hospital (number B322201111373/S53239). Platelet-rich plasma was prepared, and platelet aggregation was monitored as described (8 (link)). Platelets (4 × 10⁵/μl) were preincubated with either soluble recombinant purified pentameric proteins extensively dialyzed into PBS (PEAR1 (s5-PEAR1), FcεR1α (s5-FcεR1α), and control rat Cd200 all used at 10 μg/ml) or the commercially available antibodies human IgE (10 μg/ml (53 nm); Abcam), anti-PEAR1 (3 μg/ml; R&D Systems), and omalizumab (10 μg/ml (67 nm); Xolair®, Novartis) at 37 °C as appropriate. The IgE fragments were used at an equimolar concentration of 67 nm. Platelet aggregation was triggered by collagen with the concentration adjusted to reach 40–70% platelet aggregation for each donor and measured as the percentage of change in light transmission relative to a blank (buffer without platelets) set to 100%.

To assess the ability of IgE_TRAP and omalizumab to disrupt IgE-FcεRI complexes, His-tagged human FcεRI (SinoBiological, China; 5 μg mL⁻¹) was immobilized on Ni-NTA biosensors (Pall ForteBio, USA) for 600 s before human IgE (Abcam, UK; 100 nM) was allowed to bind sensor-immobilized FcεRI for 120 s. IgE_TRAP- and omalizumab-mediated dissociation of the IgE-FcεRI complexes was then measured for 600 s. To determine the binding potential of IgE_TRAP and omalizumab to IgG receptors, recombinant human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB (R&D Systems, USA; 5 μg mL⁻¹) in 10 mM acetate buffer (pH 5) was immobilized on activated AR2G biosensors for 300 s. The binding kinetics of IgE_TRAP and omalizumab to the sensor-immobilized IgG receptors were measured for 300 s. To assess Interaction of IgE_TRAP and omaizumab to C1q protein, biotinylated IgE_TRAP and omalizumab (10 μg mL⁻¹) were immobilized on streptavidin biosensors (Pall ForteBio, USA) and binding kinetics of C1q protein were analyzed^{45 (link)}. All experiments were performed at 30 °C with the sample plate shaker speed set at 1000 rpm using an Octet RED384 or Octet K2 system (Pall ForteBio, USA).