Construction of a novel antibody derived from a positive phage clone against Bcl-2 with the TAT sequence. (A) Monoclonal ELISA: binding and specificity of phage clones to GST-Bcl-2. Isolated individual phage clones, after the fourth round of biopanning, were assayed for binding to either GST-Bcl-2 (▦)or GST (□), by phage monoclonal ELISA as described in “Materials and methods.” Three positive clones (H1, G2, and A9) were identified as showing specific binding to GST-Bcl-2. (B) Molecular structure and sequence of the anti-Bcl-2-scFv-TAT. The light and the heavy variable chains were linked by a flexible linker, which confers the possibility of conformational change. (C) Western blot analysis of the purified scFvs-TAT. After purification, the recombinant antibody anti-Bcl-2-scFv-TAT (lane 1) and nonspecific scFv-TAT (lane 2) were electroporated under denaturating conditions, transferred onto nitrocellulose paper and the 30-kDa bands were visualized using protein A-HRP.