Measurement of antibody-membrane interactions by surface plasmon resonance.
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.
Klimka
Alexander
A
Department of Pharmaceutical Product Development, Fraunhofer IME, Worringerweg 1, 52074 Aachen, Germany.
Tur
Mehmet K
MK
Huhn
Michael
M
Bierfreund
Uwe
U
Terrada
Estela
E
Fischer
Rainer
R
Finnern
Ricarda
R
Barth
Stefan
S
D000906:Antibodies / Q000276:immunology* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D017730:Ki-1 Antigen / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D000906:Antibodies; D017730:Ki-1 Antigen