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.

Magnetic Dynal beads (M270 Epoxy 142.01) were labeled using $1 mg/ml antigen (Invitrogen, Carlsbad, CA, USA). 100 ml of these beads were washed with 2Â fresh sonication buffer, added to the cell lysate described above, and mixed by rotation for at least 45 min at room temperature. Supernatant was removed and the beads were washed with 1 ml of sonication buffer followed by 1 ml wash buffer (sonication buffer without the salmon sperm DNA). The beads were centrifuged and all remaining buffer was removed. Next, the beads were mixed for 5 min with 50 ml of wash buffer plus 0.5 M NaCl (to dissociate the zinc fingerplasmid complex) and the supernatant containing the eluted plasmids was removed. The eluted plasmid DNA was purified using Roche High Pure PCR Product Purification Kit (Roche Diagnostics, Manheim, Germany) and transformed into high transformation efficiency DH10B or XL1-blue Escherichia coli (E. coli) cells by heat shock at 428C for 1 min. The transformed cells were grown on LB-Carbenicillin agar plates, harvested, and the plasmid DNA recovered using a Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA, USA). In some cases, this plasmid DNA was taken through another cycle of screening for additional enrichment.