Goat anti human igg fc hrp antibody

Each converted full-length IgG antibody clone was diluted to 1.0 ug/mL in PBS and 100 µL was added to each well with 1 h incubation at 4 °C. Wells were then incubated directly with 100 µL of goat anti-human IgG Fc-HRP antibody (Abcam, ab98624) diluted 1:10,000 in 1X TBST for 1 h at 4 °C. After 6 more 1X TBST washes, 100 µL of TMB substrate (Biolegend, 421101) was added and allowed to develop. The reaction was quenched with 50 µL of 2 N sulfuric acid. Absorbance at 450 nm and 540 nm was measured with a Synergy H1 Multi-Mode Reader (BioTek, Winooski, VT). O.D. readings were measured as absorbance at 450 nm minus that at 540 nm.

To determine the interaction between DPP4 and RBD, 1 μg of mouse anti-His monoclonal antibody (Invitrogen) and 5 μg of purified MERS-RBD-Fc-His or HKU4-RBD-Fc-His were mixed overnight at 4 °C with HEK293T cell lysates after expression of hDPP4-HA, TpDPP4-HA, and dcDPP4-HA for 48 h, respectively. The formed protein complex was then precipitated by 25 μl (1 × 10⁷) Dynabeads goat anti-mouse IgG (Invitrogen). The bounding interaction of DPP4 and RBD were analyzed by western blot. MERS-RBD-Fc-His and HKU4-RBD-Fc-His were detected by using 1:6000 dilution of goat anti-human IgG Fc (HRP) antibody (Abcam) whereas hDPP4-HA, TpDPP4-HA, and dcDPP4-HA were detected by using 1:1000 dilution of primary mouse anti-HA monoclonal antibody [HA.C5] (Abcam) and 1: 4000 dilution of secondary goat anti-mouse HRP antibody (Invitrogen).