Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross-link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.

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Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab′)2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti‐neuraminidase NC10 scFv‐streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcoreTM biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti‐ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.

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 VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG 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 microg/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 monomerdimer 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 Mr approximately 156,000. Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-IG10 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.