'Real-time' biomolecular interaction analysis: novel applications and developments

The work described in this thesis involves the application of direct 'real-time' biomolecular interaction analysis using existing SPR-based biosensors and the development of novel alternative SPR-based technologies. These systems were applied to the investigation of whole cell binding to an immobilised ligand. Solid-phase blood typing was demonstrated by employing monoclonal anti- A IgG and anti-B IgM as affinity ligands for the capture of washed whole red blood cells (RBC). Protein A affinity-capture of anti-A IgG was used as an alternative to direct immobilisation thereby facilitating surface regeneration. A new non-'real-time' fibre optic-based system (BIACORE Probe) was evaluated using the same affinity-capture format and was successfully applied to direct whole blood typing. Non-'real time' analysis limited the data quality. Therefore, a similar 'real-time' fibre optic device was constructed. In addition, a miniature SPR-based biosensor employing a single platform optical configuration was developed with a view towards the production of a smaller, cheaper and portable alternative to the existing range of instruments. Determination of kinetic rate constants using direct 'real-time' biosensors has proved more complex than suggested by early investigators and has lead to misleading conclusions from artifactual data. A model study, employing the interaction of CD4 and glutathione-s-transferase with specific monoclonal antibodies, investigated experimental and data analysis methods for the determination of reliable rate constants. In addition, novel assays for determination of the active analyte concentration without the requirement for a standard curve were examined