The use of a membrane-based continuous flow displacement immunoassay for detection of nanomolar quantities of explosives is demonstrated, and the kinetics of this system are characterized through experimentation. Antibodies of 2,4,6-trinitrotoluene (TNT) are immobilized onto a porous membrane with surface reactive sites designed to facilitate the covalent binding of the antibody. After saturating the immobilized antibody binding sites with labeled antigen, target analyte is introduced in flow, and the displacement reactions are monitored using a fluorometer. The displaced labeled antigen detected is proportional to the concentration of the analyte introduced to the antibody-labeled antigen complex. Multiple assays were performed at flow rates of 2.0, 1.0, 0.50, and 0.25 mL/min using membranes saturated with varying TNT antibody concentrations. The signal intensity (i.e. the concentration of displaced labeled antigen) was independent of antibody concentration at 1.0 mL/min, but proportional to antibody concentration at 0.25 mL/min. Our data suggests that the lower flow rate created a longer interaction time between the injected analyte and the antibody-labeled antigen complex, resulting in greater displacement of the labeled antigen and higher signal intensities than seen at higher flow rates.
