Measurement of two-dimensional binding constants between cell-bound major histocompatibility complex and immobilized antibodies with an acoustic biosensor.
Gaining insights into the dynamic processes of molecular interactions that mediate cell-substrate and cell-cell adhesion is of great significance in the understanding of numerous physiological processes driven by intercellular communication. Here, an acoustic-wave biosensor is used to study and characterize specific interactions between cell-bound membrane proteins and surface-immobilized ligands, using as a model system the binding of major histocompatibility complex class I HLA-A2 proteins to anti-HLA-A2 monoclonal antibodies. The energy of the acoustic signal, measured as amplitude change, was found to depend directly on the number of HLA-A2/antibody complexes formed on the device surface. Real-time acoustic data were used to monitor the surface binding of cell suspensions at a range of 6.0 x 10(4) to 6.0 x 10(5) cells mL(-1). Membrane interactions are governed by two-dimensional chemistry because of the molecules' confinement to the lipid bilayer. The two-dimensional kinetics and affinity constant of the HLA-A2/antibody interaction were calculated (k(a) = 1.15 x 10(-5) mum(2) s(-1) per molecule, k(d) = 2.07 x 10(-5) s(-1), and K(A) = 0.556 mum(2) per molecule, at 25 degrees C), based on a detailed acoustic data analysis. Results indicate that acoustic biosensors can emerge as a significant tool for probing and characterizing cell-membrane interactions in the immune system, and for fast and label-free screening of membrane molecules using whole cells.
Saitakis
Michael
M
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Vassilika Vouton, 71110 Heraklion, Crete, Greece.
Dellaporta
Anastasia
A
Gizeli
Electra
E
D000162:Acoustics / Q000295:instrumentation*; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D020014:K562 Cells; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation*
D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I