OPTIMIZATION OF A TIME-RESOLVED IMMUNOFLUOROMETRIC ASSAY FOR TUMOR-ASSOCIATED TRYPSIN-INHIBITOR (TATI) USING THE STREPTAVIDIN-BIOTIN SYSTEM

We have developed two 'sandwich'-type time-resolved immunofluorometric assays (IFMA) for tumor-associated trypsin inhibitor (TATI) using mon oclonal and polyclonal antibodies. In the standard assay the monoclona l antibody was immobilized onto the walls of polystyrene microstrip we lls and the polyclonal reagent was labeled with a europium chelate. We tested various assay conditions in order to optimize the assay for se nsitivity and measuring range. Purification of the labeled antibody by hydrophobic interaction chromatography was found to be the most impor tant single factor affecting sensitivity. Assay sensitivity and range were also improved by acid treatment of the solid phase antibody. To i mprove the sensitivity further the streptavidin/biotin (SAB) system wa s incorporated into the IFMA technique. In this simple and fast strept avidin/biotin IFMA (SAB-IFMA) we used streptavidin-coated wells to whi ch we added biotinylated monoclonal antibody and a serum or urine samp le. After incubation for 1.5 h and washing, the polyclonal europium-la beled tracer antibody was added. After incubation for 1 h the wells we re washed and the Eu fluorescence measured. The assay performance of t he SAB-IFMA was compared to the standard IFMA and radioimmunoassay (RI A). The detection limit was 0.05 mug/l and the analytical range 3000-f old. The mean analytical recovery was 101%. Other advantages of the SA B-IFMA were high sensitivity and the low amounts of monoclonal antibod y required, only 1/50 of that used in the standard IFMA.