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Comparative interaction kinetics of two recombinant fabs and of the corresponding antibodies directed to the coat protein of tobacco mosaic virus
Author(s) -
Chatellier Jean,
RaufferBruyère Nathalie,
Van Regenmortel Marc H. V.,
Altschuh Danièle,
Weiss Etienne
Publication year - 1996
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/(sici)1099-1352(199601)9:1<39::aid-jmr239>3.0.co;2-v
Subject(s) - recombinant dna , monoclonal antibody , antibody , chemistry , peptide , tobacco mosaic virus , microbiology and biotechnology , biochemistry , escherichia coli , biology , virus , virology , gene , genetics
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 BIAcore TM 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.