Rapid fluorescence flow injection immunoassay using a novel perfusion chromatographic material

Abstract

A rapid fluorescence flow injection immunoassay has been developed for a model analyte (testosterone) using a novel perfusion chromatographic matrix (POROS) coated with protein A. The fluorophore used was Texas Red. The perfusion matrix contains two classes of pores, which radically enhances mass transport and provides a very high capacity unaffected by flow rate. For each assay the antibody–protein A reaction takes place at nearly neutral pH, and the complexes are eluted at acidic pH. Both pre-incubation and on-line assay formats were investigated. The total time for the pre-incubation and on-line assays was less than 4 and 6 min, respectively. Serum testosterone was determined by this method and good relative standard deviations and recoveries were achieved. The use of an avidin–biotin system in the preparation of the testosterone–Texas Red conjugate is also described.