Figure 2.
Rendomab-B4 specifically recognizes with high affinity human ETB overexpressed in CHO cell line. To check human ETB expression on CHO-ETB surface, cells were incubated with increasing concentrations of ET-1-FAM (A) and ET-3-FAM (B) for 24 h at 4°C in the dark. To determine rendomab-B4 affinity, increasing concentrations of the monoclonal antibody were incubated with CHO-ETB cells for 24 h at 4°C (C). Rendomab-B4 binding was revealed using R-PE-labeled anti-mouse antibody. The binding of labeled-peptides and monoclonal antibody were measured on a GUAVA flow cytometer and resulting curves (A, B and C), which correspond to mean fluorescence intensity (MFI) as a function of ligand concentrations, were plotted and fitted using GraphPad Prism software. Kd or apparent Kd (Kd*) corresponding to half maximal effective concentration values correspond to the mean ± s.d. of three (A and B) or 5 (C) independent experiments. (D) CHO and CHO-ETB cells were fixed and incubated with 100 nM of rendomab-B4, followed by Cy3-conjugated anti-mouse IgG antibody incubation.