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.

RNA was extracted using TRIzol LS (Invitrogen, Carlsbad, CA, USA) from the frozen splenocytes and cDNA libraries were constructed using the BD SMART TM kit (BD Biosciences, San Jose, CA, USA). From the isolated cDNA, light chain antibody domains were amplified using 11 V L forward primers and a kappa reverse primer from oligonucleotide sequences described in Essono et al. (2003) representing 75% of the light chains. cDNA encoding heavy chain antibody domains was amplified using 11 V H forward primers and six reverse primers to complement IgG1-1, IgG1-2, IgG2a, IgG2c, IgG2c-2 and IgG3, representing 71% of the heavy chains. A subset of the possible forward primers was used during construction of the libraries simply to limit the number of primers required for library construction.