title: The influence of binding capacity and affinity on the improved performance of N-terminally extended hCG peptides, determined by ELISA-based procedures.
abstract: The improvement of peptide-ELISA responses by the use of small synthetic peptides elongated at the N-terminus with an Ata-group or a (Lys)7 extension has been analyzed. For this purpose, binding capacity and affinity were evaluated by specific ELISA procedures. The ELISA experiments on binding capacity, performed with saturating antibody concentrations, revealed a difference of more than three orders of magnitude in binding capacity between the parent peptides and the N-terminally linked peptides, in favor of the latter peptides. Antibody affinity values were determined by a liquid-phase equilibrium method as well as by a solid-phase equilibrium method. N-terminal extension of the peptides had almost no effect on the affinity when equilibrium between the peptide and the antibody was reached in solution. In contrast, solid-phase affinity was greatly enhanced when the N-terminally linked peptides were adsorbed to the polystyrene surface. This enhancement was determined by the N-terminal extension and the peptide amino acid sequence (40 to 600 times higher). Thus, the use of N-terminally extended peptides can greatly increase the performance of a peptide-ELISA through improved surface effects, resulting in higher binding capacity and functional affinity.
authors.affiliation: ATO-DLO, Department of Industrial Proteins, Wageningen, The Netherlands.
authors.forename: E E
authors.initials: EE
authors.lastname: Loomans
authors.forename: T C
authors.initials: TC
authors.lastname: Gribnau
authors.forename: H P
authors.initials: HP
authors.lastname: Bloemers
authors.forename: W J
authors.initials: WJ
authors.lastname: Schielen
mesh_terms: D001667:Binding, Competitive; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000378:metabolism; D007700:Kinetics; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*
chemical_list: D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments