Kemp, Philip Arthur; (1998) The development of novel antibodies and antibody fragments for use in pharmaceutical biodetection systems. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Antibodies and antibody fragments are widely used as reagents for a variety of detection systems (e.g. ELISA, Western blotting, in situ hybridization, receptor binding and cell bioassays) but there is also a growing use for these molecules as drug discovery leads or indeed as therapeutic compounds. Two different approaches to the preparation of antibodies and antibody fragments were performed and the use of one of them in an assay was investigated. Desferal is an iron chelating compound (Novartis Pharmaceuticals) used to treat transfusion dependent anaemias. Although this is a marketed compound it would greatly benefit from better analysis systems to improve delivery and open up opportunities for new indications in iron chelation therapy. To aid the development of a more patient compliant dosage form, a polyclonal antibody that specifically recognises the iron bound form of desferrioxamine was produced and an ELISA assay using this antibody developed. This assay was used to quantify the amount of drug in samples from animal and human pharmacokinetic studies to determine the bioavailability of new forms and formulations of Desferal and new routes of administration. Antibody fragments generated by phage display offer several advantages over conventional antibodies in that they are rapidly generated, generally smaller in size and may not possess the normally associated effector functions. As a result they may have a selected advantage in that they won't suffer from steric hindrance and may not stimulate immune systems when used in vivo. The human clotting cascade protein Factor Vila (activated form of Factor VII) was used as a model protein to determine whether specific phage displayed single chain antibodies (ScFv) could be generated. ScFv's were constructed using the variable heavy and light chain genes from an antigen stimulated mouse immunoglobulin repertoire linked together by a flexible hydrophilic peptide and subsequently expressed or "displayed" on the outside of a bacteriophage. A single anti-Factor Vila ScFv phage clone was generated and it's binding constant was determined using surface plasmon resonance. The nucleic acid sequence was determined and the CDR regions assigned. In conclusion these studies demonstrate that the development of novel antibodies or antibody fragments can be utilised as valuable tools in the pharmaceutical development process.

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