Binding of a monoclonal antibody and its Fab fragment to supported phospholipid monolayers measured by total internal reflection fluorescence microscopy

Abstract

The association of an anti-dinitrophenyl monoclonal antibody and its Fab fragment with supported phospholipid monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine has been characterized with total internal reflection fluorescence microscopy. The surface densities of bound antibodies were measured as a function of the antibody and Fab solution concentrations, and as a function of the solution concentration of dinitrophenylglycine. The apparent association constant of Fab fragments with surface-associated haptens was approximately 10-fold lower than the association constant for haptens in solution, and the apparent surface association constant for intact antibodies was only approximately 10-fold higher than the constant for Fab fragments. Data analysis with simple theoretical models indicated that, at most antibody surface densities, 50-90% of membrane-associated intact antibodies were attached to the surface by two antigen binding sites.

Multivalent ligand–receptor binding on supported lipid bilayers

“…It should be noted that multivalent ligand-receptor binding interactions have been studied by a wide variety of techniques from total internal reflection fluorescence microscopy (Pisarchick and Thompson, 1990) and isothermal titration calorimetry (Goins and Freire, 1988) to surface plasmon resonance spectroscopy (Terrettaz et al, 1993) and quartz crystal microbalance analysis (Janshoff et al, 1997). Flow cytometry (Lauer et al, 2002), fluoroimmunoassays (Singh et al, 2000), fluorescence resonance energy transfer (Ma and Cheng, 2006), atomic force microscopy (Rinker et al, 2008; Sulchek et al, 2005), and a colloid particle phase transition method (Baksh et al, 2004) have also been used.…”

“…Common model haptens include dinitrophenyl (DNP), trinitrophenyl (TNP), nitroxide, dinitrophenyl nitroxide, fluorescein, and biotin. All of these moieties have been found to be active in antibody binding studies (Hafeman et al, 1981; Pisarchick and Thompson, 1990; Tamm, 1988; Tamm and Bartoldus, 1988; Wright et al, 1988). …”

“…McConnell and co-workers first claimed that IgG molecules interact with the membrane through both of their binding sites (Parce et al, 1979). Thompson and co-workers estimated that 10-50 % of the IgGs were bound monovalently in their studies (Pisarchick and Thompson, 1990). These investigators also found that the apparent equilibrium dissociation constant ( K D ) of the anti-DNP antibody was ~10 fold stronger than the corresponding Fab fragment under conditions with very high lipid-conjugated DNP concentrations.…”

“…Three common platforms are self-assembled monolayers (SAM) (Rao et al, 1999; Smith et al, 2003), phospholipid vesicles (Lee et al, 2005), and supported lipid bilayers (Doyle et al, 2003; Pisarchick and Thompson, 1990; Yang et al, 2003). Both vesicles and supported bilayers are composed of the same lipid molecules found in the plasma membranes of living cells.…”

Study of the Interaction of Methylene Violet with Calf Thymus DNA Using an SPR-Based DNA Sensor

“…The steadystate response was then plotted vs. the analyte concentration (Fig. 3B), and the data were fit to a simple Langmuir isotherm to determine the bimolecular interaction (Pisarchick and Thompson 1990;Lin et al 2005):…”