Preparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarization

• Published in: Analytical biochemistry Vol. 196; no. 2; p. 421
• Main Authors: Jiskoot, W, Hoogerhout, P, Beuvery, E C, Herron, J N, Crommelin, D J
• Format: Journal Article
• Language: English
• Published: United States 01-08-1991
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - immunology; Antibody Affinity - immunology; Antigens, Bacterial - immunology; Bacterial Outer Membrane Proteins - immunology; Binding Sites; Fluorescein; Fluoresceins; Fluorescence Polarization; Haptens - immunology; Molecular Sequence Data; Neisseria meningitidis; Peptides - chemical synthesis; Peptides - immunology
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(91)90488-F

A simple and rapid method for determining the affinity constant of a monoclonal antibody-peptide complex under equilibrium conditions is presented. A peptide corresponding to sequence 178-185 of meningococcal strain MC50 class 1 outer membrane protein, which is recognized by monoclonal antibody MN12 (mouse IgG2a), was synthesized. After fluorescein was coupled to the peptide, the peptide-fluorescein conjugate was used for binding studies with MN12, employing fluorescence polarization of the fluorescein label to probe the bound fraction of the peptide. Scatchard analysis showed that the affinity constant was pH dependent. Storage of MN12 under alkaline conditions resulted in a loss of antigen-binding sites, but did not alter the affinity constant. Sips plots showed a homogeneity index of unity.