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 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.
Kortt
A A
AA
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Lah
M
M
Oddie
G W
GW
Gruen
C L
CL
Burns
J E
JE
Pearce
L A
LA
Atwell
J L
JL
McCoy
A J
AJ
Howlett
G J
GJ
Metzger
D W
DW
Webster
R G
RG
Hudson
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015374:Biosensing Techniques; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA; D019281:Dimerization; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007130:Immunoglobulin Idiotypes / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009439:Neuraminidase / Q000276:immunology*; D017434:Protein Structure, Tertiary
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D004247:DNA; D009439:Neuraminidase