Fig. 1.

Scheme for the unbiased selection of active antibodies against cellular targets from combinatorial libraries. (A) Lentiviral antibody libraries were constructed from naïve human combinatorial antibody libraries. Plasmids of lentiviral antibody libraries and virus packaging were used to transiently transfect HEK293T cells. TF-1 erythroblast cells were infected with lentiviral antibody libraries. The resulting cells were grown on methylcellulose agar for morphogenic screening. Thirty colonies that showed unusual and different morphologies were picked after 2 wk and antibody genes were recovered by PCR. After verification of the activity of the antibodies by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay, each antibody was incubated with cell lysates followed by collection of the complexes on protein A/G beads. The beads were collected and washed, and the bound proteins were eluted by acid (pH 2.8) and separated on SDS/PAGE gels that were silver-stained. The bands of gel were sliced and analyzed by nano-LC-MS/MS after trypsinization. (B) Representative morphologies of colonies. (C) All possible combinations of antibody genes isolated from the same colonies were transfected into HEK293T cells. TF-1 cell proliferation was tested using the conditioned medium from HEK293T cells 48 h after transfection of antibodies.