Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline...

Off- and on-line strategies for a non-competitive heterogeneous flow immunoassay were developed comparing three different labels. The samples, containing the model compounds digoxin or digoxigenin, were either pre-incubated off-line or on-line in a mixing coil with excess of labelled anti-digoxigenin Fab-fragments. The excess of Fab-fragments was then separated from the digoxin bound Fab-fragments by passing the sample through a column with immobilised digoxin. The off-line immunochemical detection system is suitable for sensitive high through-put screening of the analytes, whereas the on-line system is more suitable for coupling as a post-column detection unit to liquid chromatography. The digoxin and digoxigenin content in the sample were quantified using fluorescein (F) and enzyme (peroxidase (POD), alkaline phosphatase (AP)) labelled Fab-fragments. The fluorescein label was directly measured with the fluorescence detector, whereas a fluorescent enzyme product was measured in the two enzyme based systems, using 3-(p-hydroxyphenyl)-propionic acid (HPPA) and hydrogen peroxide for POD and, and 4-methylumbelliferyl phosphate (4-MUP) for AP. The highest sensitivity and lowest limit of detection (LOD) was obtained with the Fab-POD system with LODs for digoxin and digoxigenin in the off- and on-line configurations of 0.025 and 0.01 nM, respectively. The sample through-put for the off- and on-line systems were 43 and 32 samples per hour, respectively.