The Use of Fab' Enzyme Conjugates for the Measurement of Urinary Growth Hormone

Couper, James S (1994) The Use of Fab' Enzyme Conjugates for the Measurement of Urinary Growth Hormone. MSc(R) thesis, University of Glasgow.

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Abstract

This project was initiated following work performed within this Institute to produce antibodies against human growth hormone for use in a serum immunoradiometric assay (IRMA) and some work to investigate the use of insulin like growth factor I (IGF-I) as a diagnostic and management tool for patients with particular growth disorders. Following initial attempts by other workers within the Institute to measure urinary growth hormone, it was concluded that an IRMA was unlikely to be capable of providing adequate sensitivity for this application. This project was intended to investigate the possibility of using the available antibodies in a fluorimetric assay, using similar methodologies to those of Ishikawa et. al. (1987), (1988), to provide a sensitive assay for measuring urinary growth hormone on a routine basis. The production of a suitable antibody fragment from sheep polyclonal IgG was the first major hurdle in the project and it was not until this problem was successfully traversed, by the use of an alternative method to that of Ishikawa et. al. (1988), that it was possible to progress onto production of a peroxidase conjugate or to develop a working assay. In the resulting assay, microtitration wells were used as an alternative separation system to polystyrene beads, for reasons of practicability. Initially a number of colourimetric peroxidase substrates were used in the optimization of the assay, and later when appropriate measuring equipment became available, fluorimetric and luminometric substrates were used in the hope of achieving enhanced sensitivity. The luminometric substrate,which was only investigated briefly at the last stage of the project, provided an assay with sensitivity in the range necessary for a urine growth hormone assay. A number of problems were encountered when attempting to measure growth hormone in urine due to the presence of matrix effects, and attempts were made to eliminate these as far as possible from the assay. Hence a significant proportion of the work carried out involved investigating these matrix effects and showed that dialysis of the urine samples produced unsatisfactory results. An assay was developed with the sensitivity required to measure growth hormone in urine but the matrix effects of urine samples on the assay could not be overcome adequately. Perhaps the realistic conclusion to be drawn is that a successful assay of this type requires investment in state of the art microtitre plate equipment, detection systems and the best available antibodies and enzyme labels. These are most likely to be provided by a commercial company with an international market for the product and the necessary financial backing.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: Graham Beastall
Keywords: Biochemistry, Endocrinology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-74911
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:13
Last Modified: 27 Sep 2019 15:13
URI: http://theses.gla.ac.uk/id/eprint/74911

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