SPR

Surface plasmon resonance (SPR)
For the immobilization onto CM5 chip, all antigens were
activated via thiol labeling kit (GE Healthcare, Buckinghamshire,
UK). A 0.5 mg of antigens in 0.5 mL were prepared in
morpholinoethanesulfonic acid (MES) buffer. Then, 0.25 mL of
2-(2-pyridinyldithio)ethaneamine (PDEA) (15 mg/mL) in 0.1 M
MES buffer was added. After adding 25 mL of 0.4 M 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDC), the mixtures were
incubated at 25uC for 10 min. The buffers were exchanged to PBS
via dialysis after the incubation. A Biacore 3000 (GE Healthcare)
and CM5 chip (GE Healthcare) was used in all SPR analysis. SPR
experiments were carried out at a flow rate of 10 mL/min using
PBS as running buffer. For the activation of the surface of CM5
chip, 0.2 M EDC and 0.5 M N-hydroxysuccinimide (NHS)
solution was injected for 2 min. And 40 mM cystamine solution
was injected for 3 min, and then, 0.1 M dithioerythritol (DTE) in
0.1 sodium borate solution was injected for 3 min. After the
activation of the chip surfaces, 100 mg/mL of activated antigens
were injected for 7 min. For deactivation of excess reactive groups
on the chip surface, 20 mM PDEA and 1 M NaCl solution in
0.1 M sodium acetate was injected for 4 min. Finally, various
concentrations of isolated scFvs were injected for 5 min and the
binding signal was detected. The KD values of scFvs were
evaluated by measuring Kon and Koff in BIA evaluation software