Comparative interaction kinetics of two recombinant Fabs and of the corresponding antibodies directed to the coat protein of tobacco mosaic virus.
Two recombinant Fab fragments, 57P and 174P, recognizing peptide 134-146 of the coat protein of tobacco mosaic virus have been cloned, sequenced and expressed in Escherichia coli. They differ by 15 amino acid changes in the sequence of their variable region. The interaction kinetics of the Fabs with the wild-type and four mutant peptides have been compared using a BIAcoreTM biosensor instrument. The recombinant Fab 174P had the same reactivity as the Fab fragment obtained by enzymatic cleavage of monoclonal antibody 174P. The two recombinant Fabs recognized the various peptides in the same ranking order but Fab 174P consistently dissociated somewhat faster from the peptides compared to Fab 57P. The two whole antibodies showed the same relative differences in reactivity as the two recombinant Fabs. The location of amino acid changes was visualized on a model structure of the Fab. Differences in dissociation rates of the two antibodies are most likely due to changes located at the periphery of the antigen-combining site and/or at the interface between the light and heavy chain domains. Our results demonstrate the feasibility of detecting very small differences in binding affinity by the biosensor technology, which is a prerequisite for assessing the functional effect of limited structural changes.
Chatellier
J
J
Institut de Biologie Moléculaire et Cellulaire du CNRS, Laboratoire d'Immunochimie, Strasbourg, France.
Rauffer-Bruyère
N
N
Van Regenmortel
M H
MH
Altschuh
D
D
Weiss
E
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017951:Antigen Presentation; D015374:Biosensing Techniques; D002213:Capsid / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011994:Recombinant Proteins