Surface plasmon resonance (SPR) analysis of scFv antibody binding affinity

Surface plasmon resonance (SPR) was performed using a Biacore 3000 instrument (GE Healthcare, Sweden). The CM5 sensor chip (GE Healthcare) was used to immobilize mAb 3912 (40 μg/mL) via amine coupling at 25 degrees Celsius with running buffer HBS-P (0.01 M HEPES, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) to a RU level of 10,000. Either GI.1 or GI.7 VLPs were injected (100 μg/mL) at 10 μL/min for 7 minutes and allowed 5 minutes to stabilize. Next, purified NJT-R3-A3 scFvs were injected at various concentrations ranging from 50 to 500 nM at 10 μL/min for 5 minutes followed by 10 minutes to allow for dissociation. Regeneration was achieved between cycles with one injection of 10 mM glycine, pH 2.0 at 20 μL/min for 40 seconds followed by 2 minutes of stabilization. Each cycle began with a fresh injection of VLPs for capture by mAb 3912. As a negative control channel, scFvs were flowed over immobilized mAb 3912 directly in a parallel flow channel without VLPs; the responses from this channel were subtracted from responses observed in the channel with captured VLPs. Kinetic parameters were determined by fitting the resulting curves using the Langmuir model for 1:1 protein binding interactions.