Single-chain Fv of anti-idiotype 11-1G10 antibody interacts with antibody NC41 single-chain Fv with a higher affinity than the affinity for the interaction of the parent Fab fragments

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining V(H) and V(L) domains with a (Gly(4)Ser)(3) linker, with a pelB leader sequence, and two C-terminal FLAG(TM) tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 mu g/ml) was stable for 7 days at 21 degrees C and 30 days at 4 degrees C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer-dimer equilibrium mixture after 30 days at 4 degrees C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of M(r) approximate to 156,000. Binding affinities, determined in solution using a BIAcore(TM) biosensor, showed that the affinity for the interaction of 11-1G10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.