Biacore amine coupling kit

Surface plasmon resonance experiments were performed on a Biacore 8K instrument (Cytiva) using HBS-EP+ (Teknova) at pH 7.4 as a running buffer. For the SPR chip, Protein A (Pierce) was immobilized on a CM5 chip (Cytiva) using a Biacore amine coupling kit (Cytiva) according to the manufacturer’s instructions. Multi-cycle kinetics experiments were run as follows. Antibodies were diluted in running buffer to 0.5μg/ml and captured on the sample flow cells of all channels at a flow rate of 10 μL/min for 60s. Antigen was flowed over both flow cells of all channels at a flow rate of 30μl/min for 180s followed by a 600s dissociation time. The chip surface was regenerated between cycles using 50mM NaOH for 30s at a flow rate of 30μL/min. Antigen concentrations used were 0, 0.39, 1.56, 6.25 and 25nM. The data was analyzed in the Biacore analysis software to a 1:1 binding model using local Rmax and double referencing. Off-rates fitted as slower than 1x10^-5 1/s and too slow to be fitted reliably were manually adjusted to 1x10^-5 1/s. Antigens used were human complement C7 (Complement Tech) and cyno complement C7 purified from cynomolgus serum (Seralab).