COMPETITIVE FLOW-INJECTION ENZYME-IMMUNOASSAY FOR STEROIDS USING A POSTCOLUMN REACTION TECHNIQUE

Two types of flexible, sensitive and rapid competitive flow injection enzyme immunoassay were developed and evaluated for their potential us e in the bioanalysis of steroids. Instead of the more typical approach where the signal is generated from antibody-bound hapten-enzyme conju gate, the non-bound fraction passing through the affinity column was a llowed to react with an enzyme substrate from a merging channel in a p ost-column reaction system. The enzyme product( p-aminophenol or 4-met hyl umbelliferol) was amperometrically or fluorometrically detected, S everal parameters known to affect signal generation in the immunoassay were evaluated, including flow rate through the affinity column and t hrough the reaction coil, the length of the reaction coil and of the a ffinity column. In the pre-incubation approach, where samples were mix ed with enzyme conjugate and antibodies before injection, a sample thr oughput as high as 20 h(-1) was possible, The signal precision was abo ut 1% (RSD) for cortisol (0.6-80 pmol) and 2% (RSD) for budesonide (0. 02-12.5 pmol). In the displacement assay for cortisol, enzyme-labelled analyte was displaced from the affinity column when the sample was in jected into the flow. A standard curve was obtained with a signal prec ision of 4-20% for 12.5-1250 pmol injected. The same instrumental set- up was used in both types of immunoassay, and thus a highly flexible s ystem was obtained. A simple replacement of the affinity column from p rotein G in the pre-incubation approach to a column containing primary antibodies in the displacement assay was needed.