Panel A. ScFv sequence alignment and mutagenesis design. The scFv 32, 43 and 51 aminoacidic sequences were aligned to the consensus sequences of the respective germ-line genes, namely DP47 for the VH region of the three antibodies, DPL16 for the VL of scFv32 and 51 and DPK22 for the VL of scFv43. The positions of the mismatching amino acids are indicated for the three scFvs; the numbering is according to Kabat and Wu . The amino acids mutated in the scFv43M1 and M2 sequences with respect to that of scFv43 are in bold. Panel B. Comparison of the scFv43, 43M1 and 43M2 reactivity against E7 protein. The purified scFvs have been utilized at a concentration of 0.7 μg/ml in ELISA (left hand) and of 1 μg/ml in Western blotting (right hand). The E7 is indicated by an arrow. The immunocomplexes were revealed by anti-FLAG tag mAb followed by GAM-HRP incubation.