The photophysical characterisation of a fluorescence-based immunoassay for the detection of gonadotropin-releasing hormone, type-1 (GnRH-I)

• Main Author: Dowd, Peter Dylan
• Published: University of Strathclyde 2013
• Subjects: 610.28
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.632676

The homogeneous assay format has been identified as having the potential to make an effective impact in the field of 'point-of-care or near patient testing. Homogeneous assays have the advantage that, by eliminating the need for multiple preparation steps, they can be rapid and easy to use in comparison with most solid-phase assay formats. However homogeneous assays tend to be generally less sensitive than their heterogeneous counterparts, giving results that are qualitative or at best semi-quantitative. This work presents a 'model' fluorescence-based homogeneous immunoassay for the detection of gonadotropin-releasing hormone, type-1, (GnRH-I) described by fluorescence spectroscopy and in particular time-resolved fluorescence techniques. In the model assay a new synthetic labelled 9-amino acid peptide, [des-pGlup1s]-LH-RH-Acp-FITC, is introduced to compete with GnRH-I for the two binding sites on the antibody 7B10.1D10. The core results demonstrate a photophysical characterisation of the binding of [des-pGlup1s]-LH-RH-Acp-FITC and 7B10.1D10 in homogeneous solution based on time-resolved fluorescence techniques. Specifically, values extracted from the plateau region of the time-resolved anisotropy decay curves are used to estimate the amount of free and bound [des-pGlup1s]-LH-RH-Acp-FITC and comment on the presence of interference processes in the assay. Furthermore, disruption to a system of [des-pGlup1s]-LH-RH-Acp-FITC bound to 7B10.1D10 by the addition of GnRH-I is described.