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.

Antibodies were diluted 2-fold in Earle's minimum essential medium (EMEM) containing 10% fetal bovine serum and antibiotics [penicillin (200 IU/ml) and streptomycin (200 mg/ml)]. A polyclonal rabbit serum was used as a positive control and diluent as a negative control with each set of samples tested. Approximately 100 pfu SARS-CoV in 100 ml of EMEM media was added to an equal volume of diluted antibodies and incubated overnight at 48C. After incubation, the virus-antibody mixtures were applied to confluent monolayers of Vero cells grown in six-well tissue culture plates, and adsorbed for 1 h at 378C in a CO 2 incubator. Plates were overlaid with EMEM 0.6% agarose medium containing 10% fetal bovine serum and antibiotics and incubated for 24-48 h at 378C in a CO 2 incubator. Plaques were visualized by staining with a second EMEM agarose overlay containing 5% fetal bovine serum, 5% neutral red, and antibiotics. Plaques were counted 24 h after staining and 50 and 80% plaque reduction neutralization titers were calculated relative to the negative controls.