Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases

Rogers, J.; Schoepp, R.J.; Schröder, O.; Clements, T.L.; Holland, T.F.; Li, J.Q.; Li, J.; Lewis, L.M.; Dirmeier, R.P.; Frey, G.J.; Tan, X.; Wong, K.; Woodnutt, G.; Keller, M.; Reed, D.S.; Kimmel, B.E.; Tozer, E.C.

Vero E6 cells (ATCC, Manassas, VA, USA) were grown in minimal essential medium containing penicillin (200 IU/ml) and streptomycin (200 mg/ml), 2 mM L-Glutamine, 1 mM pyruvic acid, and fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 378C and 5% CO 2 until confluent. Following trypsinization, Vero E6 cells were harvested by centrifugation at 1000 g for 5 min, washed with PBS, and resuspended in 250 ml PBS ($4 Â 10 6 cells/ml). The Vero E6 cell suspension was added to 250 ml antibody-spike solution and incubated at room temperature for 1 h. The samples were washed three times by centrifugation with 500 ml PBS, resuspended in 500 ml PBS containing 200 mg/ml streptavidin-phycoerythrin (Invitrogen, Carlsbad, CA, USA), and incubated for 1 h at room temperature. After incubation, the cells were washed 3Â (PBS), resuspended in 500 ml PBS, and analyzed on a Dako MoFlo flow cytometer (Dako, Fort Collins, CO, USA).