Kinetic Analysis of Antibody–Antigen Interactions at a Supported Lipid Monolayer Matthew A. Cooper 1 and Dudley H. Williams Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom Received May 11, 1999 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 bind- ing could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity con- stants 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 algo- rithm describing the formation of a 1:2 antibody:anti- gen complex was developed which accurately de- scribed the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the den- sity of antigen on the surface. The affinities deter- mined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA. © 1999 Academic Press Key Words: surface plasmon resonance; lipid mono- layer; bivalent; antibody. Optical biosensors can be used to measure the inter- actions of macromolecules in real time providing access to rate constants without the need for chemical or radiolabeling of the reacting species (1). It is necessary to attach one of the reactants to a surface, however, which places limitations on the accuracy of rate con- stants determined for binding of massive molecules and demands careful analysis of the binding of multi- valent molecules (2– 4). Surface plasmon resonance (SPR), 2 a widely used technique for the analysis of binding kinetics, is usually employed with molecules covalently attached to a carboxymethyl dextran hydro- gel that coats the surface of a gold sensor chip (5). This hydrogel provides a hydrophilic environment that is conducive to the measurement of most binding inter- actions. It is not suitable for binding of extremely mas- sive species, however, because the binding components may not readily penetrate the hydrogel (vide infra). Multivalent binding is an important, inherent fea- ture of many biological systems. Bivalent molecules such as antibodies behave very differently in solution than at a surface. Studies of antibody binding using radiolabeled antibodies have shown the measured af- finity constants at surfaces are sensitive to numerous experimental variables, such as the volume of incuba- tion and the antigen surface density (6). Binding curves for antibody–antigen interactions on Lang- muir–Blodgett phospholipid monolayers (7) and on solid supports (8) determined using fluorescence recov- ery after photobleaching have also shown deviations from a simple bimolecular association. SPR has been employed in the analysis of antibody binding to anti- gens immobilized on a carboxymethyl dextran sensor chip. Monomeric scFv antibody fragments were shown to possess much lower affinities and much faster dis- sociation rates than bivalent Fab fragments (2). Few attempts have been made to quantitate the bivalent binding of antibodies to fixed ligands on this sensor chip surface (3, 4, 9). The quantitation of bivalent binding is extremely important for the comparison of the behavior of differ- 1 To whom correspondence should be addressed. Fax: 144 1223 336913. E-mail: mc221@cam.ac.uk. 2 Abbreviations used: SPR, surface plasmon resonance; HPA, hy- drophobic association sensor chip; RU, response unit; PBS, phos- phate-buffered saline; DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide; EDC, N-ethyl-N9-(3-diethylaminopropyl) car- bodiimide; PDEA, 2-(2-pyridinyldithio)ethanamine hydrochloride; PC, phosphatidylcholine; PE, phosphatidylethanolamine; BSA, bo- vine serum albumin; mAb, monoclonal antibody. 36 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 276, 36 – 47 (1999) Article ID abio.1999.4333, available online at http://www.idealibrary.com on