scFv/Nb-Fc antibody ELISA

The CD30 or CD5 antigen proteins were coated onto 96-well microtiter plates in coating buffer overnight at 4°C and blocked with 3% BSA for 1 h at 37°C. The coated plates were incubated with Nb-Fc or scFv-Fc for 1 h at 37°C and, subsequently, with goat anti-human IgG-Fc secondary antibody (HRP) (Sino Biological) for 1 h at 37°C. Each incubation was followed by four washes with PBS-T. Finally, TMB substrate (Invitrogen, Thermo Fisher Scientific) was added, and absorption was measured at 450 nm.