| Abstract |
Molecular interactions that govern cell‐cell interactions are mediated by molecules embedded in the cell membrane, are very common in the immune system and are distinct from interactions between soluble molecules. Assays that can detect these interactions in a non‐invasive way are particularly useful. Work was focused towards the development of such assays using biosensors. A well‐defined cellular model system was used to study the responses of biosensor systems during the interaction of cell membrane receptors, class I MHC HLA‐A2 molecules on the surface of cells, bound to ligands, anti‐HLA‐A2 monoclonal antibodies, immobilized on the sensor devices. The acoustic biosensor systems used, a Love‐wave sensor and a quartz crystal microbalance (QCM) sensor detected the molecular interaction quantitatively but showed different sensitivity to different cell treatments: the QCM sensor detected cytoskeleton perturbations by cytochalasin whereas the Love‐wave sensor distinguished cell types on the basis of cell surface HLA densities. For the Love‐wave sensor, acoustic energy dissipation was found to correlate directly with the number of cell‐membrane receptors specifically attached to immobilized antibodies, which enabled the characterization of the interaction via the calculation of the 2D kinetic and affinity parameters. Also, the dependence of cell adhesion on glycocalyx condition was demonstrated for HLA/anti‐HLA binding. In order to move towards the characterization of a more biologically significant interaction pair, membrane models on biosensor surfaces were developed for the attachment of proteins of interest. Two types of lipid membranes were prepared, lipid monolayers on self‐assembled thiol layers and lipid bilayers on hydrophilic silica surfaces. A real‐time biosensing technique to study the binding of T cells to a pMHC‐modified surface was proposed. As a TCR system, the JM22 TCR, which recognizes the influenza matrix peptide MP(58‐66) presented on HLA‐A2, was selected. The biosensor surface was prepared using a lipid monolayer presenting HLA‐A2/MP(58‐66) complexes. Two T cell systems were prepared for the biosensor technique, a cell line transfected with JM22 TCR and primary T cell clones isolated from peripheral blood. These cells were characterized for TCR expression and cytotoxicity. The long‐term goal is to measure the 2D binding parameters of the TCR‐pMHC interaction and correlate these with the physiological response of cells to antigen recognition.
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Χαρακτηρισμός μοριακών ανοσολογικών αλληλεπιδράσεων χρησιμοποιώντας βιοαισθητήρες και ολόκληρα κύτταρα
