Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero- residue linker a trimer

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10were constructed by joining the VH and VL domains with 10-residue(Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFvwas constructed by joining the C-terminal residue of the VH domain to theN-terminus of the VL domain. The scFv with the 10- and five- residuelinkers exclusively formed dimeric antibody fragments (M(r) 52000). Thesewere shown to be bivalent and were able to cross-link two neuraminidasetetramers to form a 'sandwich' type complex; each antigen combining sitecould also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r)70000) was shown to form a trimer with three active antigen combiningsites, each binding an anti-idiotype Fab' to yield a complex of M(r)212000. The orientation of the combining sites in the zero-residue linkerscFv, however, was such that it could not cross- link tetramers ofneuraminidase. BIAcore biosensor experiments showed that the affinity ofeach individual antigen combining site in both the 10- and five-residuelinker scFv dimers and zero-residue linker scFv trimer was essentially thesame when the scFvs were immobilized onto the sensor surface. However, whenthe scFvs were used as the analyte, the dimeric and trimeric scFvs showedan apparent increase in binding affinity due to the avidity of binding themultivalent scFvs.