Development of fluorescence based flow immunoassays utilising restricted access columns

The development of high-speed flow immunoassay techniques is described. The principles are based on heterogeneous flow immunoassay interactions. High sample throughput can be used for screening small analytes in a number of biological matrices originating from samples of water from environmentally polluted areas, or biological fluids such as urine and plasma. The immunochemical detection principle is based on chromatographic separation of the immunocomplex formed (AbAg or AbAg^*) and the free antigen (Ag) by a restricted access (RA) column, utilising size-exclusion and reversed phase mechanisms. A fluorescein-labelled analyte (Ag^*) was used in the competitive assay format with fluorescence detection. Sample throughput was 80 h^-1 and detection limits 1.4 nM (300pgml^-1) for atrazine and 2.3 nM (500 pg ml^-1) for the sum of triazines. Analyses could be performed at a sample throughput of 400 6 h^-1 shift. Basic immunoaffinity interactions of a number of immunoreagents, using fluorescence polarisation were studied and outlined both for triazines and for 2,4-D. Structural variations in tracer synthesis confirmed that this is an important part in the design and optimisation of flow immuno methodologies.