A new tetradentate beta-diketonate--europium chelate that can be covalently bound to proteins for time-resolved fluoroimmunoassay.

A new chlorosulfonylated tetradentate beta-diketone, 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6 "-yl) chlorosulfo-o-terphenyl (BHHCT), was synthesized as a chelating label for Eu3+. BHHCT can be covalently bound to proteins under mild conditions and forms a strongly fluorescent chelate with Eu3+. Bovine serum albumin (BSA) and streptavidin (SA) were labeled with BHHCH-Eu3+, and the latter was used for time-resolved fluoroimmunoassay of alpha-fetoprotein (AFP) in human sera. A remarkably high sensitivity was obtained, with a detection limit of 4.1 x 10(-3) pg/mL, which corresponds to an improvement of about 4-5 orders of magnitude, compared to those of all conventional immunoassays including fluoroimmunoassay, enzyme immunoassay, and radioimmunoassay. The high sensitivity has been attained both by strong fluorescence of the present label and by the extremely suppressed background level brought about by the direct labeling of proteins with the beta-diketone-Eu3+ complex. A general consideration and ideas are given for designing ideal label ligands for strongly fluorescent Eu3+ complexes.