SINGLE-CHAIN FV OF ANTIIDIOTYPE 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, w hich recognizes an idiotope of anti-neuraminidase antibody NC41, was c onstructed 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 sequenc es, 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 7 5 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 dissoc iated to monomer to yield a 2:1 monomer-dimer equilibrium mixture afte r 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 11-1G1 0 scFv monomer with NC41 scFv monomer was five- to six-fold higher tha n the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity tha n its parent Fab.