Dark, Christopher; (1992) The production and characterisation of an antibody recognising alpha- tocopherol. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The visualisation and localisation of α-tocopherol within membranes would provide useful information regarding its role in biological membranes in general, and why in man neural membranes are particularly susceptible to a deficiency of this fat soluble vitamin. This study has investigated the possibility of using antibodies as a tool to localise the vitamin. Two approaches have been used for their production. Firstly using liposomes containing α-tocopherol as an immunogen and secondly using proteins as carriers of α-tocopherol. A sensitive enzyme linked immunosorbent assay (ELISA) was initially developed and validated to monitor antibody production. Liposomes containing lipid A (a polyclonal B cell activator) and α-tocopherol were prepared and characterised. Conditions and lipid concentrations were optimised to produce reproducible liposome preparations and a good immune response. An immunisation protocol was optimised and a weak IgM response (1:32 serum endpoint dilution titre) obtained. No IgG antibody could be detected even after boosting. Attempts were made to enhance the titre by producing a monoclonal antibody, and although a response was detected, antibody production could not be stabilised. For the second approach using proteins as carriers, ovalbumin was covalently bound to trolox c, where the phytyl side chain of tocopherol is replaced by a carboxyl group. The carboxyl group of trolox c was linked to free amino groups of the protein using N-hydroxysuccinimide and carbodiimide as coupling agents. Confirmation that trolox c had bound to the protein was provided by UV spectroscopy. Unbound trolox c and protein gave characteristic absorbance peaks at 288 nm and 278 nm respectively, whereas the conjugate had a maximum absorbance at 284 nm. Approximately 20-30 molecules of trolox c were shown to be bound to each protein molecule. An immunisation programme was established in rabbits using Freund's complete adjuvant to enhance the response. Antibody production was detected using a well validated ELISA system with trolox c bound to bovine serum albumin as the detecting antigen. An IgG antibody with a maximum 50% dilution litre of 1:50,000 was obtained. The antibody was then purified using ammonium sulphate precipitation followed by protein A isolation of IgG and affinity chromatography to isolate the antibodies recognising trolox c. The specificity of the antibody has been investigated using the ELISA assay and a range of molecules related to trolox c and α-tocopherol bound to bovine serum albumin. Studies were also carried out using a) tocopherol containing liposomes as test antigens which provided evidence that the antibody could be used to detect tocopherol in artificial membranes and b) physiological cell membranes as test antigens in which, using available techniques, tocopherol could not be detected.

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