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Title: Effect of VH and VL consensus sequence-specific primers on the binding and neutralizing potential of a single-chain FV directed towards HuIFN-gamma
Authors: Froyen, Guido
Hendrix, Diane
Ronsse, I
Fiten, P
Martens, E
Billiau, A #
Issue Date: Jul-1995
Series Title: Molecular immunology. vol:32 issue:7 pages:515-21
Abstract: We have previously reported on the cloning and bacterial expression of a biologically active scFv antibody fragment (scFv-D9D10) derived from the mouse anti-human interferon-gamma (HuIFN-gamma) antibody, D9D10. Since the variable (V) regions were isolated by means of VH and VL consensus sequence-specific PCR primers and cloned in an expression vector relying on primer-incorporated restriction sites, some amino acids (aa) at the N- and C-terminal ends of the cloned V domains were expected to differ from the corresponding ones in the natural D9D10 antibody. Therefore, the naturally occurring sequences of both V domains were isolated by means of traditional cDNA synthesis procedures. In comparison with scFv-D9D10, the "natural" V sequences differed in three aa in VH and three in VL. The V domains of scFv-D9D10 were adapted to their natural sequence by means of PCR-directed mutagenesis to yield scFv-D9D10N. Comparison of the binding and neutralizing potentials of both antibody fragments did not reveal differences in either of both activities. In addition, their affinities for HuIFN-gamma were found to be equal. These results show that murine VH and VL consensus-specific primers can yield antibody fragments having functional properties equivalent to those of the natural scFv. Information on the impact of the use of V-specific primers on kinetics of interaction between the recombinant antibody and the corresponding antigen is important for the development of most engineered antibodies or their fragments.
ISSN: 0161-5890
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Faculty of Medicine - miscellaneous
Molecular Genetics Section (-)
Faculty of Pharmaceutical Sciences - miscellaneous
Human Genome Laboratory
Department of Human Genetics - miscellaneous
# (joint) last author

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