Investigations of Bivalent Antibody Binding on Fluid-Supported Phospholipid Membranes:  The Effect of Hapten Density

Abstract

Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.

Molecular Dynamics of POPC Phospholipid Bilayers through the Gel to Fluid Phase Transition: An Incoherent Quasi-Elastic Neutron Scattering Study

“…It is also the structural host for functional proteins that control molecular traffic and cellular communication across the membrane. Membrane phospholipid bilayers are extensively used as model membranes for investigating the biological processes that occur at the cellular level [1]. In the last years, they received an increasing interest owing to a growing number of applications in biophysical and biomedical research, which include the passive transport through biomembranes [2], the pharmacokinetics of drugs [3,4] or anaesthetics [5][6][7][8], and the lodging of membrane proteins [9].…”

Making Lipid Membranes Rough, Tough, and Ready to Hit the Road

“…[21][22][23] Such microfluidic devices employ solid-supported lipid membranes intermingled with antigens as coatings on the channel walls. The channels are made from polydimethylsiloxane and glass and are fully enclosed to maintain an aqueous environment above the bilayers.…”

Integration of a Reconstituted Cell-free Protein-synthesis System on a Glass Microchip

“…11,12 Therefore, methods to modify the surfaces of PDMS microchips have been developed to prevent these problems. [13][14][15][16] Although a hydrophilic coating can prevent adsorption, the surface becomes more likely to absorb many biomolecules. On the other hand, although a hydrophobic coating will prevent absorption, it can make the surface more likely to adsorb many biomolecules.…”