ScFv fragments were purified via His-tag affinity chromatography. The scFv encoding bacterial XL1-Blue MRF culture was induced with 50 µM IPTG for 3 h at 250 rpm at 30°C. The bacterial pellet was resuspended in ice-cold PE-Buffer (pH 8, 20% (w/v) sucrose, 50 mM Tris, 1 mM EDTA) and centrifuged at 20,000×g for 30 min. The supernatant was dialyzed against PBS (Servapor dialysing tube, 12–14). Chelating Sepharose Fast Flow (Amersham) was incubated with nickel sulfate solution (0.1 M NiSO4) and washed with PBS. Afterwards, the sepharose was incubated with the dialyzed scFv supernatant at 4°C for 30 min. After several washing steps, scFv were eluted with 0.1 M EDTA and dialyzed against PBS at 4°C o.n.
ScFv were cloned into the pCMV-hIgG1-Fc-XP vector [53] (link) to express scFv-Fc antibodies and were analyzed by PCR with primers pCMVfor (5′-CGC AAA TGG GCG GTA GGC GTG-3) and pCMVrev (5′-CCA GGA GTT CAG GTG CTG-3′ ). ScFv-Fc were expressed in 293T cells after transient transfection with 10 µg/plate scFv-Fc encoding vectors. 293T cell supernatants were incubated with protein A agarose (Pierce), eluted in several fractions with elution buffer (0.1 M citric acid, 0.1 M Tri-sodium citrate, pH 2.5) and neutralized with 2 M Tris, pH 9. Pooled fractions were centrifuged and concentrated with 30 kDa cut-off amicons (Millipore).
ScFv were cloned into the pCMV-hIgG1-Fc-XP vector [53] (link) to express scFv-Fc antibodies and were analyzed by PCR with primers pCMVfor (5′-CGC AAA TGG GCG GTA GGC GTG-3) and pCMVrev (