Direct time-resolved fluorescence immunoassay of progesterone in serum involving the biotin-streptavidin system and the immobilized-antibody approach.
We developed a direct competitive-type immunoassay for progesterone in serum that combines the advantages of the biotin-streptavidin system with the antibody-immunobilization approach. We synthesized biotinylated progesterone derivatives of five different proteins and, after initial evaluation of the conjugates, selected biotinylated bovine IgG-progesterone as the most suitable tracer. Progesterone released from binding proteins with danazol competes with the biotinylated tracer conjugate for binding to a limited amount of a mouse anti-progesterone monoclonal antibody in microtitration wells coated with a goat anti-mouse IgG antibody. The binding of the biotinylated tracer is then monitored by reaction with a streptavidin-based universal detection reagent developed for time-resolved fluorometry. The assay demonstrated typical performance characteristics with respect to the dynamic range, detection limit, and precision. Recovery averaged 99.3% (SD 8.3%) and dilution experiments showed good linearity. Measurements correlated well with those from three commercially available direct immunoassays for progesterone.
Kakabakos
S E
SE
Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
Khosravi
M J
MJ
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001667:Binding, Competitive; D001710:Biotin; D002417:Cattle; D005455:Fluorescent Antibody Technique / Q000592:standards / Q000706:statistics & numerical data; D006801:Humans; D007074:Immunoglobulin G; D051379:Mice; D011374:Progesterone / Q000097:blood*; D011786:Quality Control; D019809:Streptavidin
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D011374:Progesterone; D001710:Biotin; D019809:Streptavidin