Abstract. Due to problems of immobilizing functional tumor antigens in their natural conformation on surfaces for immunoassays, it is often difficult to evaluate the binding of antibodies derived from phage display libraries depleted and selected by panning on cell lines and living tumor cells. Performing cell membrane based ELISA methods does not reveal any up front kinetic binding information and depends on the performance of secondary antibodies and substrates. To overcome these limitations, we developed a new method to visualize direct antibody-cell membrane interactions by surface plasmon resonance using the Biacore 3000 and on-line signal subtraction on antigen-negative cell membrane vesicles. Conditions for the coating of cell membrane preparations to a carboxymethyl dextran hydrogel surface of a commercially available chip and the proof of concept for this application by the analysis of different formats of anti- CD30 and anti-carcinoembryonic antigen (CEA) antibodies interacting with coated membrane vesicles of CD30-positive/ CEA-negative and CD30–negative and CEA-positive cell lines are described. Introduction One method for generating recombinant antibodies or other ligands against novel tumor antigens is to use phage display antibody libraries, in which every single antibody is connected to its encoding gene in one particle. For this purpose the immunoglobulin V-genes of immunized donors, e.g. tumor patients or immunized mice, or the V-genes of B-lymphocytes of non-immunized donors are amplified in standardized polymerase chain reactions (PCR). The PCR products are cloned into especially developed expression vectors and transformed into E. coli bacteria, which produce individual bacteriophage. Each bacterial clone produces one type of bacteriophage, which displays one individual antibody- fragment on its surface and carries the genetic information of this antibody-fragment within this phage particle (1). This provides the researcher with an immunized or naïve antibody library, respectively, which reflects the whole diversity of the provided antibody repertoires. The library of phage particles displaying their individual antibody fragment on their surface are incubated with a source of antigen e.g. purified and immobilized protein or a cell line. Only phage binding to a target protein via their antibody fragment are propagated and enriched in successive rounds of this selection procedure. This will result in several, individual antibody fragments, depending on the number of provided target proteins, the selection strategy and the diversity of the generated antibody repertoires (2). Selecting them on tumor cells, including subtraction of unspecific binders by depletion against other cell lines, can reveal a huge number of so far uncharacterized specific tumor antigens and their targeting antibodies at the same time (3). The resulting binders have to be screened for their specificity against unknown and presumably unstable antigens under natural conditions. Performing whole cell or cell membrane based ELISA methods (4,5) are established tools to investigate, if the selected antibodies bind to the target cells or not but do not provide analogue cross reactivity analysis in one tube, kinetic binding information and depend on the performance of secondary antibodies and substrates. The surface plasmon resonance (SPR) can serve as a useful alternative to overcome these limitations since a commercially available chip with a modified carboxymethylated dextran matrix enables the capturing of membrane vesicles (6). The real-time measurement of interactions with antibodies, peptides or receptor ligands in a flow-through mode using very small amounts of protein became feasible with this technology. We developed a new method to visualize direct antibody-cell membrane interactions by SPR Here we describe a fast and simple method of coating a SPR L1-chip (Biacore, Uppsala, Sweden) with predominantly right-side out membrane vesicles and the determination of specific membrane-antibody interactions using the Biacore 3000 and on-line signal subtraction on antigen-negative cell membrane vesicles. INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 14: 765-768, 2004 765 Measurement of antibody-membrane interactions by surface plasmon resonance ALEXANDER KLIMKA 1 , MEHMET K. TUR 1 , MICHAEL HUHN 1 , UWE BIERFREUND 3 , ESTELA TERRADA 2 , RAINER FISCHER 1,2 , RICARDA FINNERN 1 and STEFAN BARTH 1 1 Fraunhofer IME and 2 University Aachen, Worringerweg 1, 52074 Aachen; 3 Biacore AB, Jechtinger Strasse 8, 79111 Freiburg, Germany Received April 22, 2004; Accepted July 7, 2004 _________________________________________ Correspondence to: Dr Stefan Barth, Fraunhofer IME, Dept. of Pharmaceutical Product Development, Worringerweg 1, 52074 Aachen, Germany E-mail: barth@molbiotech.rwth-aachen.de Key words: surface plasmon resonance, cell membrane, Biacore L1-sensor chip, tumor-specific antibodies