Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.592246
Title: Clinical applications of Goodpasture antigen
Author: Chopra, S.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1999
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Abstract:
Goodpasture's disease is a well described autoimmune disorder characterised by rapidly progressive glomerulonephritis and pulmonary haemorrhage. The antigen had been identified as the carboxyl terminal of the non-collagenous domain of the α3 chain of type IV collagen, α3(IV)NC1. This has been successfully expressed as a recombinant minichain construct in insect cells. This project investigates ways in improving the production and purity of the recombinant antigen as well as probing possible clinical uses for the recombinant product, rAg. A minichain construct expressing α4(IV)NC1 was developed. α3(IV), α4(IV) and α5(IV) chains are normally found together and it was possible that co-expression of these constructs enhance production. Attempts to improve purity of the rAg produced in insect cells concentrated on forming an antibody to the 7S region of the minichain constructs. Proteolytic digestion of the rAg and subsequent separation of the fractions by reverse phase HPLC was unsuccessful in isolating this region. Therefore, constructs were developed which encoded the 7S region alone allowing expression in insect, mammalian and bacterial cells. Polyclonal antibodies raised against these constructs were found not to recognise the minichain constructs. α3(IV)NC1, α4(IV)NC1 and α5(IV)NC1 were successfully produced in a drosophila expression system. This system was found to support co-expression of α3/α4; α3/α5 and α3/α4/α5 NC1 domains. Bacterial rAg and an on-column refolding strategy were used to develop an immunoadsorption column. This column was found to recognise antibody in patient serum. An ELISA technique using rAg produced in mammalian cells was developed to identify the presence of Goodpasture antibodies in serum.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.592246  DOI: Not available
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