Figure 1

CellectAb antibody screening methodology and antigen identification. (A) Schematic of phage-displayed synthetic antibody selections using FACS sorted AC133^high CICs as positive selection, and AC133^low bulk cells as negative selection (see Methods for details). (B) Histograms from a representative flow cytometry experiment showing binding of AN01, AN02 and AN03 to POP92 cells. (C) Flow cytometry analysis from a representative experiment of POP92 co-stained with CD133-FITC and AN0#-APC (top). Histograms comparing the AN0# binding on the CD133^high to CD133^low populations gated above (bottom). (D) Bar chart illustration of the mean fold increase in AN01, AN02 or AN03 mean fluorescent intensity (MFI) on CD133^high cells compared to CD133^low (normalized to 1 in each experiment). Error bars are standard error of the mean (SEM). For AN01 N = 7, for AN02 N = 6, and for AN03 N = 7 biological replicates. (E) Bar chart illustration of percent maximum AN01 and AN03 binding on HEK293T cells co-transfected with various integrin subunit pairs relative to in-tube control (see Methods for details). Error bars are SEM from N = 3 separate transfections. Significance was calculated for each transfection compared to control transfection by unpaired student’s t-test. (F) Bar chart illustrating AN02 binding to POP92 cells stably transduced with shRNA against HLA-A or shLacZ negative control. Data is represented as % shLacZ MFI, error bars are SEM, N = 3 independent transductions. Significance was calculated using two-tailed paired t-test. *p < 0.05, **p < 0.01, ***p < 0.001.