Horseradish peroxidase hrp conjugated goat anti human fc antibody

100 ng HGF (R&D Systems) was immobilized on a Maxisorp microtiter plate (MTP) in 100 μl PBS overnight at 4°C. The remaining binding sites of the MTP surface were blocked with 200 μl PBS / BSA 3% (w/v). c-Met-Fc 250 ng was pre-incubated with various concentrations of aptamer in a final volume of 100 μl PBS / BSA 1% (w/v). The pre-incubated solutions were then added to the pre-treated wells. c-Met-Fc was detected with a horseradish peroxidase (HRP)-conjugated goat anti-human Fc antibody (1:5,000) (Jackson ImmunoResearch). For detection 100 μl 1step Ultra TMB ELISA reagent (Pierce/Thermo Scientific) was added and the chromogenic reaction was stopped with the addition of 100 μl 2 M sulphuric acid. The spectrometric detection at 450 nm was carried out on a Synergy4 reader with the Gen5 software (BioMek/BeckmanCoulter). The values were analyzed by non-linear regression with the log(inhibitor) vs. response—variable slope equation using GraphPad Prism software.

Virus samples were pre-incubated with Fab fragments of antibodies for the noted time periods before preparation for BN-PAGE. BN-PAGE analysis was performed using the NativePAGE bis-Tris gel system (Invitrogen), according to the manufacturer's instructions. Briefly, samples were solubilized with 1% DDM in the supplied sample buffer and run on a 3–12% gradient bis-Tris NativePAGE gel at 150 V at 4 °C for 3 h. Proteins in the gel were transferred to a PVDF membrane; membranes were blocked in 5% (w/v) non-fat dry milk dissolved in PBS, 0.05% Tween-20, and blotted overnight at 4 °C using a cocktail of antibodies to gp120 (2 μg ml⁻¹ each of b12, 2G12 and 447-52D) and to gp41 (1 μg ml⁻¹ each of 2F5, 4E10 and Z13e1) combined. Membranes were washed, probed for 30 min at RT with an horseradish peroxidase (HRP)-conjugated goat anti-human Fc antibody (Jackson) and peroxidase activity was assayed using SuperSignal West Pico Chemiluminescence (Pierce).