Optimization of a time-resolved immunofluorometric assay for tumor-associated trypsin inhibitor (TATI) using the streptavidin-biotin system.

ABSTRACT

We have developed two 'sandwich'-type time-resolved immunofluorometric assays (IFMA) for tumor-associated trypsin inhibitor (TATI) using monoclonal and polyclonal antibodies. In the standard assay the monoclonal antibody was immobilized onto the walls of polystyrene microstrip wells and the polyclonal reagent was labeled with a europium chelate. We tested various assay conditions in order to optimize the assay for sensitivity and measuring range. Purification of the labeled antibody by hydrophobic interaction chromatography was found to be the most important single factor affecting sensitivity. Assay sensitivity and range were also improved by acid treatment of the solid phase antibody. To improve the sensitivity further the streptavidin/biotin (SAB) system was incorporated into the IFMA technique. In this simple and fast streptavidin/biotin IFMA (SAB-IFMA) we used streptavidin-coated wells to which we added biotinylated monoclonal antibody and a serum or urine sample. After incubation for 1.5 h and washing, the polyclonal europium-labeled tracer antibody was added. After incubation for 1 h the wells were washed and the Eu fluorescence measured. The assay performance of the SAB-IFMA was compared to the standard IFMA and radioimmunoassay (RIA). The detection limit was 0.05 microgram/l and the analytical range 3000-fold. The mean analytical recovery was 101%. Other advantages of the SAB-IFMA were high sensitivity and the low amounts of monoclonal antibody required, only 1/50 of that used in the standard IFMA.