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Development of an Immunoassay For Addressing The Question 'Do 15,16-Dihydrocyclopenta[a]Phenanthren-17-one Analogues Exist in Man?'.

Bartlet-Jones, Michael. (1994) Development of an Immunoassay For Addressing The Question 'Do 15,16-Dihydrocyclopenta[a]Phenanthren-17-one Analogues Exist in Man?'. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

Five derivatives of 15,16-dihydrocyclopenta[a]phenanthren-17-one were synthesised bearing 5-6 atom-long linker arms terminating with carboxyl groups. Linkage at C-3 and C-11 was through O-butyryl ethers, at C-15 by way of an S-ether with thiopropionic acid, at C-16 via a hemisuccinate ester, and at C-17 through a carboxymethyloxime. These hapten acids were allowed to react, as their active N-hydroxysuccinimide esters, with the lysyl e-amino groups present on BSA to form physiologically stable conjugates suitable for antibody production. These conjugates were independently used as antigens for the production of polyclonal antibodies. Rabbits were inoculated and serum collected at monthly intervals; the resultant sera were assayed by a direct ELISA assay procedure described, and the immunisation profile per animal, per antigen are presented. A competition assay designed to probe human biofluids directly was developed and up to 60 compound analytes tested against each of the 5 polyclonal sera generated, with the view to characterising the respective antibody specificity. The data was normalised between assay plates and polyclonal sera, and a direct comparison of the D-ring linked antigen derived polyclonal sera carried out. The polyclonal (17-N-linked serum) was shown to recognise most of the carcinogens tested, these included 11-methyl- and 7-methyl-15,16-dihydrocyclopenta[a]phenanthren-17-one,3-methylcholanthrene, and benzo[a]pyrene, in addition to other closely related compounds. Both the 15-Sand the 16-O-linked derived antibodies demonstrated specificity toward cyclopenta[a]phenanthrene analogues they were designed to recognise. The sera raised against antigens derived from conjugates linked directly through the aromatic rings via the 3- and 11-positions, showed remarkable specificity toward their target antigens. A collaboration was set up with Mr John. H. Davies (surgeon) at the Royal Surrey County Hospital and two affinity-purified anti-sera raised against C-11 (11-O-linked) and C-17(17-N-linked) were employed in a competitive ELISA assay on human urine and blood samples. Having carefully eliminated recognition of human serum albumins closely homologous with the antigen BSA carrier protein, one of the sera gave positive results with 10 out of 46 urine samples tested. When a minimum 10% cross-reaction threshold was applied to the analysis of data, only two of these urine samples were positive, one by a margin of 2% and the second patient displayed an activity of about 100 times that of the other positives. Collection of further samples from this patient confirmed the ELISA activity. It was demonstrated that the activity was partially extracted into organic solvent (ethyl acetate) suggesting it to be a small hydrophobic molecule. HPLC analysis of the ethyl acetate extract revealed a peak having a U. V spectrum consistent with that expected for a cyclopenta[a]phenanthrene. This peak has not been identified or shown to be responsible for the ELISA activity.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Bartlet-Jones, Michael.
Date : 1994
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1994.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 11:56
Last Modified : 06 May 2020 12:02
URI: http://epubs.surrey.ac.uk/id/eprint/855633

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