Development of immunoassay screening methods using long wavelength fluorescence

The developments of immunoassay methods for the early stage diagnosis of tuberculosis (TB) are described. These went through two different routes, one through flow injection analysis (FIA), and the other using immunochromatography methodology. The design of a simple longwavelength fluorescence detector to serve the above purposes has also been described. The FIA immunoassay methods involve immobilising antibodies on to beads, either directly or through protein A based solid phases. The beads are then packed into a micro-column reactor for incorporation into the FIA system. In this case reactor-bound molecules are eluted from the system by a change of pH, thus limiting the available fluorophores to those that are reasonably fluorescent in acid solution. Sandwich (reagent excess) assays have been investigated. A couple of long wavelength (600-800) fluorophores have been studied. The bead injection option has also been investigated. The immunochromatographic method uses a lateral flow system and a sandwich (two-site) immunometric assay. Capture antibodies are immobilised on a coated membrane matrix at a pre-determined position and the antigen is analysed after binding to a fluorescence-labelled antibody. Both fluorescent latex preparations and conventional fluorescent labels have been used and compared. The strips are simply immersed in a small volume of sample to start the analysis. The chromatographic step is rapid and extremely simple. The fluorescence detector is fitted with a motor-driven sample holder to allow the length of the immunochromatographic strip to be scanned. The detector utilises a diode laser light source, optical filters in the emission beam and a miniaturised photomultiplier. It can be easily modified for the FIA, and can readily be adapted to operate from batteries, so is suitable for field use