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 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.
Iliades
P
P
CSIRO, Division of Molecular Science, Parkville, Victoria, Australia.
Dougan
D A
DA
Oddie
G W
GW
Metzger
D W
DW
Hudson
P J
PJ
Kortt
A A
AA
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D000888:Antibodies, Anti-Idiotypic; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins