Pregnancy tests and related immunoassays are heavily dependent on specific and non-specific protein adsorption. These interfacial processes are affected by many factors that influence the in situ conformations of interfacially immobilised antibodies. This thesis examines a number of representative features with dual polarisation interferometry (DPI) and neutron reflection (NR), thus combining real-time dynamic monitoring with high interfacial structural resolution. Bovine serum albumin (BSA) was initially used as a model system to compare the surface coverage and thickness measurements of DPI and NR. The results show that DPI and NR provided similar surface coverage data but the measured thicknesses differed at BSA concentrations above 0.1 mg/ml. This discrepancy arose from the adoption of the uniform-layer model used by DPI for data analysis and the greater thickness sensitivity of NR. A model pregnancy immunoassay was built in steps on a silica surface so that the adsorption of each protein could be accurately monitored. Both DPI and NR provided evidence of BSA insertion into the gaps on the surface between the antibody molecules. This suggests that BSA adsorption is an excellent method to block the non-specific adsorption of target antigens to the immunoassay test surface. A magnetic tweezer system was designed and built in order to measure the specific antibody/antigen binding force. The antibodies and antigens were used to immuno-link magnetic beads to the experimental surface before the immuno-links were broken by increasing the attractive force between the magnetic tweezers and beads. The force per antibody/antigen immuno-link was estimated to lie between the values of 13.6 pN and 43.8 pN.Immuno-link detachment as a function of time was investigated. It was found that the immuno-link comprised both a strong and a weak interaction. The dissociation constant of the strong antibody/antigen interaction was found to equal 3E-4 /s and had an interaction length of 0.06 nm. The low population of beads bound by the second, weaker interaction meant that it was not possible to obtain accurate values of the dissociation constant and bond length of the second weaker interaction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:558050 |
| Date | January 2012 |
| Creators | Cowsill, Benjamin James |
| Contributors | Waigh, Thomas |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-physics-of-pregnancy-tests-a-biophysical-study-of-interfacial-protein-adsorption(538b8e9c-9111-4eb9-ac7d-e5e75110e315).html |
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