Equilibrium and kinetic parameters for the interaction of a monoclonal antibody with liposomes bearing fluorescent haptens.
We have obtained equilibrium and rate constants for the interaction of monoclonal IgG and its monovalent Fab fragment with a hapten (fluorescein) attached to the surface of a liposome. Binding was detected at nanomolar hapten concentrations by the quenching of the hapten's fluorescence on antibody binding. The binding parameters were computed from nonlinear least squares fits, using mass-action models. Crypticity of the hapten was observed and interpreted as an equilibrium between two states, extended and sequestered, the latter representing haptens associated with the membrane surface. Depending on the lipid composition of the liposomes, the fraction of sequestered hapten ranged from 0.25 to 0.975; transitions between the two states took place on the time scale of minutes. Fab interactions with extended hapten on the membrane were similar to interactions with water-soluble hapten. The ability of IgG to bind bivalently to membrane gave it an avidity two to six times the affinity for purely monovalent binding. However, the equilibrium constant for the monovalent-bivalent binding equilibrium was effectively four to five orders of magnitude less than that for the initial binding step. This probably reflects steric penalties for the simultaneous binding of two haptens on a membrane.
Petrossian
A
A
XOMA Corporation, Santa Monica, CA 90404.
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity / Q000276:immunology; D000937:Antigen-Antibody Reactions; D005452:Fluoresceins / Q000737:chemistry; D005470:Fluorometry; D006241:Haptens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008081:Liposomes; D010743:Phospholipids