The influence of binding capacity and affinity on the improved performance of N-terminally extended hCG peptides, determined by ELISA-based procedures

• Published in: Journal of immunological methods Vol. 221; no. 1; pp. 119 - 130
• Main Authors: Loomans, Elma E.M.G., Gribnau, Tom C.J., Bloemers, Henri P.J., Schielen, Wim J.G.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-12-1998; Elsevier
• Subjects: Agrotechnological Research Institute; Binding capacity; Binding, Competitive; Biological and medical sciences; Chorionic Gonadotropin, beta Subunit, Human - immunology; Chorionic Gonadotropin, beta Subunit, Human - metabolism; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Epitopes - metabolism; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Instituut voor Agrotechnologisch Onderzoek; Kinetics; Liquid-phase affinity; Molecular immunology; Monoclonal antibody; Oligopeptides - immunology; Oligopeptides - metabolism; Peptide; Peptide Fragments - immunology; Peptide Fragments - metabolism; Solid-phase affinity; Techniques; K aff, affinity constant; BSA, bovine serum albumin; Ata, acetyl-thio-acetyl; Solid-phase affinity; Mab, monoclonal antibody; OD, optical density; EC 50, peptide concentration in the coat solution at 50% of the maximum ELISA signal; Monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; Liquid-phase affinity; Binding capacity; hCG, human chorionic gonadotropin; Peptide; ELISA; Human chorionic gonadotrophin; Microtiter plate; Molecular model; Property structure relationship; Gonadotropin; Glycoprotein hormone; Enzyme immunoassay; N terminal peptide; ELISA assay; Placental hormone; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(98)00173-2

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.