Measurement of Two-Dimensional Binding Constants between Cell-Bound Major Histocompatibility Complex and Immobilized Antibodies with an Acoustic Biosensor

Abstract

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 × 10 4 to 6.0 × 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 × 10 −5 μ m 2 s −1 per molecule, k d = 2.07 × 10 −5 s −1, and K A = 0.556 μm 2 per molecule, at 25°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.