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Production and Characterisation of Human Thyroid Derived Cell Lines.

Overton, Paul Douglas. (1992) Production and Characterisation of Human Thyroid Derived Cell Lines. Doctoral thesis, University of Surrey (United Kingdom)..

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In the present study, human heterohybridoma techniques were employed in an endeavour to produce stable human thyroid cell lines that would express autoantigens. Human thyrocytes obtained from a Graves patient were fused with second and third generation human X mouse heterohybridomas according to standard hybridoma technologies. The resultant hybridomas were selected by their growth in HAT medium. 70 stable cell lines (termed HTH) were established, expanded, cryopreserved and characterised. Several thyroglobulin secreting fibroblastic lines were initially identified by means of a specific thyroglobulin ELISA utilising murine monoclonals. These lines however, proved unstable and were eventually lost. Screening of the cellular supernatants for human immunoglobulin revealed the presence of 5 immortalised thyroid infiltrating lymphocytes. Immuno-histochemical staining techniques failed to identify their specificity. Karyotypic analysis of selected HTH lines confirmed their human origin. SDS-PAGE analysis of the cellular proteins suggested that the HTH lines obtained had two basic phenotypes. These were fibroblastic and lymphocytic. Following extensive screening, none of the novel HTH lines were found to express the common thyroid antigens. They were therefore deemed unsuitable as thyroid cell "models". In summary this study has demonstrated the potential usefulness of human heterohybridoma technology in producing long term stable human cell lines. Two assays were developed for the detection of TSH receptor antibodies (TRAB). Both ELISA systems depended on the immobilisation of partially purified porcine TSH receptor to microtitre plates. The purification was achieved by using lectin and TSH affinity chromatography. The competitive ELISA depended on the direct inhibition of enzyme labelled TSH by sample autoantibodies. This assay failed to function in a serum matrix and was therefore terminated in favour of an indirect approach. The indirect ELISA depended on TSH receptor autoantibodies binding specifically to the TSH receptor. Bound antibodies were detected by the means of an enzyme labelled second antibody. Validation of the proposed ELISA against the existing radioreceptor assay resulted in a correlation of 0.84. The precision and recovery of the ELISA were good and no significant cross reactivity was observed with other common autoantibodies.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Overton, Paul Douglas.
Date : 1992
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1992.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:23
Last Modified : 06 May 2020 14:29

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