ISSN:
1573-4943
Keywords:
Antibody
;
anti-idiotype
;
single-chain Fvs
;
solution properties
;
complexes with antigens
;
binding affinities
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract 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 μg/ml) was stable for 7 days at 21°C and 30 days at 4°C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer–dimer equilibrium mixture after 30 days at 4°C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of M r ≍ 156,000. Binding affinities, determined in solution using a BIAcore™ biosensor, showed that the affinity for the interaction of 11-1G10 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1022584702108
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