Determination of prostaglandin metabolites in biological samples by competitive time-resolved fluoroimmunoassay

We describe a time-resolved fluoroimmunoassay method for the determination of 13,14-dihydro-15-ketoprostaglandin E2 (PGEM) and 13,14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) in femtomolar ranges. Polyclonal antibodies were raised in rabbits and the antigen was labelled with europium by the avidin-biotin technique. A second antibody, directed to rabbit IgG, was coated onto the solid phase. The IgG-PG-europium complex was bound by the second antibody, giving a rapid and complete separation of antibody-bound and free antigen. The detection limit was 0.2 pg/assay for PGFM and 1.2 pg/assay for PGEM. The intra-assay CV ranged from 4.6% to 9.3% and the interassay CV from 6.7% to 11.4%. A good correlation was obtained between the results from TR-FIA and RIA when the method was applied to the investigation of tissues from breast cancer. We conclude that the TR-FIA is more sensitive and much faster than the RIA and avoids the use of radioactive material.