Binding assessment of single phage antibodies to BECs and LECs in cell ELISA and FACs.
A: Representative whole cell ELISA of phage antibodies prepared from 166 unique scFv clones derived from panning rounds #5 and #6 on BECs (red) and LECs (green). Values represent mean ± SD (n = 3). Negative controls were: WT-phage, 2xTY medium without phage, 2nd antibody only. Phage antibody binding was visualized via peroxidase-conjugated anti-M13 phage antibody. OD value represents absorbance at 450nm. B: Phage amount ELISA of single phage antibodies. To allow for normalization of cell ELISA signal intensities, phage antibody concentrations were determined in a second ELISA format, where anti-fd bacteriophage antibody (S1 Table) was coated, and phage containing supernatants were added. Bound phage particles were detected by peroxidase-conjugated anti-M13 phage antibody (S1 Table). C: Effect of decreasing numbers of phage antibody concentrations on binding to BECs and LECs by cell-ELISA. Phage antibody dilution series in cell ELISA assessing specific binding to BECs and LECs. Control: same dilutions of WT phage. D: Binding of three selected phage antibodies to BECs and LECs analyzed by flow cytometry. Shown are histograms of BECs and LECs stained with the three phage antibodies (black lines). Grey-filled graphs show VCS-M13 wildtype phage binding as negative control. Cell binding was detected by sequential incubation with anti-M13 and FITC-conjugated anti-rabbit antibodies. The percentage of BECs and LECs bound to phages was evaluated by setting a marker (M1) indicated by the blue bar and is depicted in the graph.
