Kinetic Analysis of Antibody–Antigen Interactions at a Supported Lipid Monolayer

✍ Scribed by Matthew A. Cooper; Dudley H. Williams

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 153 KB
• Volume: 276
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4333

✦ Synopsis

Modified phospholipids possessing carboxyl head groups synthesized from phosphatidylethanolamine were incorporated into supported lipid monolayers on top of a thin gold film. A monoclonal antibody was chemically coupled to the modified lipids in these monolayers and the kinetics of antigen binding were determined by surface plasmon resonance. The binding could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity constants were almost identical to those reported for the same interaction on a dextran hydrogel-based sensor chip. When an antigen was chemically coupled to a modified lipid monolayer, the binding of a monoclonal antibody to this surface was biphasic. A two-step algorithm describing the formation of a 1:2 antibody:antigen complex was developed which accurately described the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the density of antigen on the surface. The affinities determined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA.