title: Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal.
abstract: A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA.
authors.affiliation: School of Life Sciences, University of Warwick, Warwick, CV4 7AL, United Kingdom.
authors.forename: George O
authors.initials: GO
authors.lastname: Badescu
authors.affiliation: Department of Chemistry, University of Warwick, Warwick, CV4 7AL, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Marsh
authors.affiliation: Department of Chemistry, University of Warwick, Warwick, CV4 7AL, United Kingdom.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Smith
authors.affiliation: School of Life Sciences, University of Warwick, Warwick, CV4 7AL, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.affiliation: School of Life Sciences, University of Warwick, Warwick, CV4 7AL, United Kingdom.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Napier
mesh_terms: D000040:Abscisic Acid / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D058619:Maltose-Binding Proteins / Q000378:metabolism; D010937:Plant Growth Regulators / Q000494:pharmacology*; D057127:Single-Chain Antibodies / Q000276:immunology*; D012996:Solutions; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D058619:Maltose-Binding Proteins; D010937:Plant Growth Regulators; D057127:Single-Chain Antibodies; D012996:Solutions; D000040:Abscisic Acid