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 b iological matrices originating from samples of water from environmentally p olluted areas, or biological fluids such as urine and plasma. The immunoche mical detection principle is based on chromatographic separation of the imm unocomplex 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 fo rmat with fluorescence detection. Sample throughput was 80 h(-1) and detect ion limits 1.4 nM (300 pg ml(-1)) for atrazine and 2.3 nM (500 pg ml(-1)) f or the sum of triazines. Analyses could be performed at: a sample throughpu t of 400 6 h(-1) shift. Basic immunoaffinity interactions of a number of immunoreagents, using fluo rescence 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 immune methodologies.