Production and use of multi-species scFv-Fc antibodies. A: Schematic map of the generic pFuse-xIgG-Fc2 used to express scFvs (adapted from Invivogen). Three vectors are available bearing either a human (pFuse-hIgG-Fc2), mouse (pFuse-mIgG-Fc2) or rabbit IgG2 (pFuse-rIgG-Fc2). The scFv is inserted (NcoI/NotI) inframe between the IL2 secretion signal and the Fc portion. The vector can be selected using Zeocin both in E. coli and in mammalian cells. B: Western Blot analysis of the scFv F2C fused to Fc domains from 3 different species. hF2C, rF2C and mF2C secreted by CHO cells were purified on protein A and analyzed by western blot using either species-specific anti-Fc antibody, or using a polyclonal anti-scFv. Each F2C fusion antibody is detected in CHO supernatants and is respectively seen as a human, rabbit or mouse antibody.C: scFv-hFc are expressed as dimers. hF2C proteins were separated by SDS-PAGE either in reducing (left) or in non-reducing conditions (right) and analyzed by western blot. This confirmed that hF2C behaves as a dimer in non-reducing conditions. D: Using multi-species scFv-Fc antibodies for Western blot immuno-labeling. A HeLa cell protein lysate was separated by SDS-PAGE and transferred on a nitrocellulose membrane. The membrane was probed using anti-MyosinII SF9 (lane 1, 3 and 5) or anti-Tubulin F2C (2, 4, 6) antibodies fused to either human (h; 1,2), rabbit (r; 3,4) or mouse (m; 5,6) Fc. This experiment shows that each scFv kept its specificity when used as a human, mouse or rabbit antibody.