Project completed 2015
Dr Kai-Michael Toellner, Institute of Immunology and Immunotherapy
Dr Hamid Dehghani, School of Computer Science
Dr James Bowen, School of Chemical Engineering
Dr Alex Robinson, School of Chemical Engineering
Germinal Centres (GCs) play a central role in adaptive immunity; involving processes of cell migration, clonal expansion, hypermutation, and selection. To elucidate the role of affinity in regulating these processes, a technique for measuring B cell receptor affinity maturation in GCs in situ was developed. To facilitate interrogation of individual antibody-antigen interactions, atomic force microscopy (AFM) was chosen, offering nanometre positional resolution, and pico-Newton force sensitivity. Specificity of gold-coated AFM cantilevers towards the targeted receptors was achieved via a bespoke modification scheme, using self-assembled amine terminated alkanethiol to facilitate attachment of the receptor specific antigen NP ((4- hydroxy-3-nitrophenyl)acetyl). Influences on molecule deposition and subsequent NP addition were investigated, as were control measures facilitating identification of successful modifications. Effects of sample preparation techniques on AFM adhesion measurements were investigated. Subsequently, the developed AFM technique was applied in interrogation of B cells and hybridomas – expressing receptors of varying affinity towards NP – and two GCs in tissue sections. For the automated and unbiased evaluation of large amounts of varying AFM data, bespoke data analysis methods were developed. The project finds that AFM is capable of quantifying specific antibody-antigen interactions, but was unable to measure these in tissue sections.
Link to ethesis: http://etheses.bham.ac.uk/7130/