Surface plasmon resonance: multi-cycle kinetics, C5

Multi-cycle kinetics experiments were performed using Biacore 8 K and 8 K+ instruments (GE Healthcare). Following normalization of a Biacore sensor chip CM5, human C5 protein (purified from serum, as described)²⁹ was amine coupled as follows: flow cells 1 and 2 were activated using a 1:2 molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (flow rate 10 µL/minute; contact time, 30s). A 5 µg/mL solution of human C5 in 10 mM sodium acetate at pH 4.5 was immobilized in flow cell two only and, finally, both flow cells were blocked with 1 M ethanolamine-HCl, pH 8 (flow rate 10 µL/minute; contact time, 420 s). This typically resulted in immobilization of 230–730 response units (RU). To derive kinetics, five point, three-fold serial dilutions of analyte (range of 100–0.4 nM) were prepared in HBS-EP+ buffer. For each injection, a flow rate of 40 µL/minute, a contact time of 300 s and dissociation time of 5400 s was used. After each injection, regeneration of the surface was performed with sequential injections of 2 M MgCl₂ (flow rate 30 µL/min; contact time 30s). The data was fitted with the reference surface subtracted using a Biacore evaluation software 1:1 binding model to determine the binding kinetics.