Binding analysis of adenovirus fibers

Binding analysis was performed using a BIAcore 3000™ equipped with a CM5 sensor chip. Approximately 5000 RU of CD46, CAR, and DSG2 was attached to the CM5 sensor chip, using amine coupling, at a slow flow-rate of 10 μl/min to ensure uniform distribution on the chip surface. A blank flow cell was used as a negative control surface on flow cell 1. All measurements were performed at 25 °C in PBS buffer (Sigma, UK) at a flow rate of 30 µl/min. For equilibrium binding analysis, the HAdV-D26K and HAdV-B3K fiber knob proteins were purified and concentrated to 367 and 3 μM respectively. 5× 1:3 serial dilutions were prepared for each sample and injected over the relevant sensor chip. The equilibrium binding constant (K_D) values were calculated assuming a 1:1 interaction by plotting specific equilibrium-binding responses against protein concentrations followed by non-linear least squares fitting of the Langmuir binding equation. For single cycle kinetic analysis, HAdV-D26K, HAdV-D48K, HAdV-B35K, HAdV-C5K, and HAdV-B3K were injected at a top concentration of around 200 µM, followed by four injections using serial 1:3 dilutions. The K_D values were calculated assuming Langmuir binding (AB = B×ABmax/(KD + B)) and the data were analysed using the kinetic titration algorithm (BIAevaluationTM 3.1). Receptor proteins were obtained commercially, as follows: Recombinant Human Desmoglein-2 Fc Chimera Protein, R&D Systems, Catalogue number 947-DM-100. Recombinant Human CXADR Fc Chimera Protein (CAR), R&D Systems, Catalogue number 3336-CX-050. Recombinant Human CD46 Protein (His Tag), Sino Biological, Catalogue number 12239-H08H.