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.

Following sequence analysis of the human framework reassembly (HuFR TM ) and GSSM TM libraries, the appropriate clones were identified and arrayed into deep 96-well plates. The cultures were grown for 36 -48 h at 308C with shaking and plasmid DNA isolated using the PerfectPrep 96-well plasmid isolation kit (Eppendorf, Hamburg, Germany). Using an automated robotics system, 96-well plates containing 293-C18 cells (50 -80% confluent) were transfected with the plasmids and grown for five days at 378C in 8% CO 2 . Supernatants were harvested at five days and two ELISAs were performed on each plate using a robotics system. One ELISA was a functional ELISA using the spike protein as the antigen following the protocol described above except that the secondary antibody was an anti-human kappa antibody conjugated to peroxidase (Sigma, St Louis, MO, USA). The second ELISA measured the relative amount of antibody expressed. This ELISA was similar to the functional assay with the exception that streptavidin-coated plates (Sigma, St Louis, MO, USA) were used with a biotinylated anti-human IgG (Sigma, St Louis, MO, USA) to capture the expressed antibody on the plates.