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.

The antigen-coated 96-well Nunc MaxiSorp plate (i.e. spike protein at 1 mg/ml, infected cell lysate, or whole SARS virus) was prepared by incubating 100 ml antigen/well at 48C overnight, and then washed with 200 ml/well of PBS with 0.05% Tween 20 (PBS-T). In some cases, half of the plate was coated with BSA to determine non-specific binding. The plate was blocked overnight at 48C with 200 ml/well blocking buffer (PBS-T with 5% dry milk powder or 1% BSA). The plate was then washed four times (4Â) with PBS-T and 100 ml/well induced supernatant added for 1 h at room temperature. Following 4Â washes with PBS-T, 100 ml/well rat anti-mouse kappa light chain antibody conjugated to peroxidase (Invitrogen, Carlsbad, CA, USA) was added and incubated for 1 h, and washed again 4Â with PBS-T. Subsequently, 100 ml of SureBlue TM TMB (KPL, Gaithersburg, MD, USA) was incubated for 30 min at room temperature, 1 M phosphoric acid was added to stop the reaction, and absorbance at OD 450 nm measured using a SpectroMax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).