Fig 1: Ad5 is inactivated by binding to human erythrocytes via the CAR. (A) Fresh washed human erythrocytes were incubated with Ad5 in the presence or absence of fiber or hexon. Ad5 erythrocyte binding was assessed by separating the liquid (□) and erythrocyte (■) fractions by centrifugation and performing quantitative PCR specific for the Ad5 genome on each fraction. Data are represented as the percentage of the total input dose recovered; n = 4, SEM shown. **P < .005. (B) Human erythrocytes from 4 donors (i) or erythrocytes from C57BL/6 mice (ii) or rhesus macaques (iii) were analyzed for CAR presentation using the anti-CAR primary antibody RmcB,8 secondary antibody R0480 (Dako Denmark), and flow cytometric analysis. (i) Filled peak represents isotype control (antibody W6/32); empty peak, donors 1 to 4 with anti-CAR. (ii,iii) Filled peak represents isotype control; empty peak, + anti-CAR (data are superimposed). (C) Human erythrocytes from 4 donors (D1-D4) were treated to produce hemoglobin-free ghosts and then analyzed by SDS-PAGE/Western blotting using 100 ng/lane, primary antibody 15405 (Santa Cruz Biotechnology), and secondary antibody W4011 (Promega). Positive control (+ve) indicates mouse liver lysate (10 ng); negative control, mouse A9 cells (∼ 100 ng). (D) Human erythrocytes in PBS were incubated with anti-CAR antibody (RmcB) before Ad5 addition. After separation of the liquid (□) and cell fraction (■) by centrifugation, quantification of each fraction was achieved by quantitative PCR; n = 4, SEM shown. **P < .005. (E) A549 cells were incubated with Ad5 in the presence of serially diluted erythrocytes for 90 minutes at 37°C; after thorough washing in PBS and the addition of fresh media, the A549 cells were returned to the incubator and luciferase expression was measured 24 hours later; N = 4, SEM shown. **P < .005. (F) SKOV-3 cells were incubated with Ad5 in the presence or absence of physiologic FX levels (8 μg/mL) and serially diluted erythrocytes in PBS, for 90 minutes at 37°C; after thorough washing in PBS and the addition of fresh media, cells were returned to the incubator and luciferase expression was measured 24 hours later. The fold increase on FX addition was calculated by dividing the value achieved with FX by that achieved without FX, and plotted for each erythrocyte dilution; N = 4, SEM shown. **P < .005.