The development of simultaneous multi-analyte fluorescence immunoassays

Fluorescence immunoassays have been established for a number of years as valuable methods of analysis in clinical chemistry and other fields, being sensitive, safe, easy to use and available in a variety formats. Those in common use are normally single analyte assays. But in many cases (e.g. forensic drug screening, therapeutic drug monitoring, screening for cancer markers, monitoring of thyroid function, or the analysis of environmental pollutants) dual- or multi-analyte assays would be much more valuable, with the advantages of increased information content, savings in time and costs, and the elimination of some sources of sampling variance. Amongst all the labels used in immunoassays, only fluorescent groups offer realistic prospects of practicable multi-analyte assays. This project has investigated single-, dual- and multi-analyte fluorescence immunoassays using several spectroscopic and software methods to resolve multicomponent fluorescence emission or synchronous spectra. The assays have been based on flow injection analysis methodology, with solid phase reactors to effect the separation of antibody-bound and unbound labelled analytes. The use of solid phase reactors incorporating thiophiIic gels to bind antibodies has also been investigated: these stationary phases have the advantage that bound antibodies can be eluted by changes of ionic strength, rather than changes of pH. This allows the use of a much wider range of fluorescence labels, clearly important in multi-analyte assays, and it has thus proved possible to develop successful dual and triple analyte assays, with results that compare well with other independent methods.