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
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.
A Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.