Goat anti human igg fc fragment antibody

TfRMAb-TNFR fusion protein binding to TNF-α (Additional file 2: Fig. S1B), and TfRMAb-TNFR and etanercept plasma concentrations were quantified by a sandwich ELISA. The human TNF-α (hTNF-α) (PeproTech, Rocky Hill, NJ, USA) was the capture agent and 2 µg/mL of hTNF-α was plated in 96-well plates, and incubated overnight at 4 ºC. The wells were blocked with TBS containing 1% bovine serum albumin (TBSB) (0.01 M Tris/0.15 M NaCl/1% BSA/pH7.4) for 30 min at room temperature. Plasma samples (diluted 1:10 in TBSB) were added to the wells and incubated for 1 h at room temperature followed by washing with TBST. Alkaline phosphatase-conjugated detector agents, goat anti-human IgG-Fc fragment antibody (Bethyl, TX, USA) that binds to the human Fc domain of etanercept and goat anti-mouse light chain (kappa) antibody (Bethyl Laboratories, Inc., TX, USA) that binds to the TfRMAb domain of the TfRMAb-TNFR, were added to the wells for 1 h at room temperature followed by washing with TBST. Wells were then incubated with P-nitrophenyl phosphate solution (Sigma Aldrich, St. Louis, MO, USA) for 15 min in the dark at room temperature and the reaction was stopped by adding 1.2 M NaOH. Blank corrected absorbance measured at 405 nm was used to calculate plasma concentrations using a standard curve.