Figure 2. Characterization of selected anti-CAIX scFvFc antibodies.

A. Different anti-CAIX scFvFcs were expressed and purified from 293FT cells as discussed in Material and Methods. A total of 2 µg purified each anti-CAIX scFvFc were treated with 5XSDS-PAGE loading buffer (Pierce) in the presence (lower panel) and absence (upper panel) of reducing reagent DTT. The antibodies were separated by SDS- 10% Tris-Glycine PAGE gel electrophoresis and visualized by Coomassie Blue staining. B. Flow cytometric analysis of the anti-CAIX scFvFc binding activity. One microgram (1 µg) of each anti-CAIX scFvFc was incubated with 5×10⁵ sk-rc-52 (CAIX positive) or sk-rc-59 (CAIX negative) cells in 50 µl FACS buffer (1% BSA/PBS), followed by staining with PE-conjugated goat anti-human IgG. Binding activity of each antibody was examined by flow cytometric analysis and data is presented as overlapping histograms of the CAIX positive and negative cells as well as the secondary antibody only controls for each cell line. Similar findings were found in two independent experiments. C & D. Dosage-dependent binding of the anti-CAIX scFvFcs to 293T-CAIX cells. Anti-CAIX scFvFcs or a control of irrelevant anti-CXCR4 scFvFc (X33) at the concentrations indicated on the X-axis were incubated with 5×10⁵ 293T-CAIX cells followed by staining with FITC conjugated goat anti-human IgG. Flow cytometric analysis was performed and binding activity of each antibody is presented as both percentage of positive cells (C) and absolute geometric mean fluorescence intensity (GMFI) (D). A summary of the calculated EC₅₀ for half-maximal percent binding (C) and GMFI (D), along with maximal percent positive cells and GMFI reached, are presented in Table 1.