Investigations of bivalent antibody binding on fluid-supported phospholipid membranes: the effect of hapten density.
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.
Yang
Tinglu
T
Department of Chemistry, Texas A&M University, College Station, Texas 77843-3012, USA.
Baryshnikova
Olga K
OK
Mao
Hanbin
H
Holden
Matthew A
MA
Cremer
Paul S
PS
D019297:2,4-Dinitrophenol / Q000276:immunology; D018033:Antibodies, Bispecific / Q000276:immunology / Q000378:metabolism*; D006241:Haptens / Q000276:immunology / Q000378:metabolism*; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000276:immunology / Q000378:metabolism*; D008560:Membrane Fluidity / Q000276:immunology; D008856:Microscopy, Fluorescence; D010743:Phospholipids / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
D018033:Antibodies, Bispecific; D006241:Haptens; D007074:Immunoglobulin G; D008051:Lipid Bilayers; D010743:Phospholipids; D019297:2,4-Dinitrophenol