2.5 IgG1 and IgG4 kinetics by Bio-Layer interferometry (BLI)
The analysis of binding kinetics of anti-Fel d 1 IgG1 and IgG4 monoclonal antibodies (F127, A044 and G078) to FcγRIIb was analyzed by Bio-Layer Interferometry (BLI) using an Octet RED96e (Sartorius) instrument. Briefly, high precision Streptavidin (SAX, Sartorius, Fremont, CA, USA) biosensors were saturated with 2 µg/ml of human recombinant biotinylated FcγRIIb (CD32b) (Sino Biological, Cat No 10259_H27H-BI, Beijing, China) in kinetics assay buffer (PBS, 0.1% BSA, 0.02% Tween 20) for 10 min. Association was carried out in 300 s, with the IgG serially diluted from 100 to 6.3 nM in 1:2 steps. Finally, dissociation was also performed in 300 s. All proteins were diluted in kinetics buffer (KB), a loaded sensor dipped in BLI assay buffer served as drift control. The resulting curves were aligned to the beginning of association step and a 1:1 global model was applied on the fitting.