Synthesis of a terbium fluorescent chelate and its application to time-resolved fluoroimmunoassay

A new nonadentate ligand, N, N, N-1, N-1-[2,6-bis(3'-aminomethyl-1 1'-pyraz olyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA) for a Tb3+ fluorescent complex was synthesized. The Tb3+ complex is strongly fluorescent, having a large fluorescence quantum yield of 1.00 and very long fluorescence lifeti me of 2.681 ms in 0.05 M berate buffer of pH 9.1. Streptavidin (SA) was lab eled with SPTA by using its succinimidyl monoester, and the BPTA-Tb3+-label ed SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of alpha -fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb3+-labeled SA was also used in competitive type TR-FIA of bensulfuro n- methyl (BSM) in water. The detection limits of these assays are 42 pg/mL for AFP, 70 pg/mL for CEA, and 0.4 ng/mL for BSM. In addition, a new simul taneous measurement method for AFP and CEA in a human serum sample was deve loped by using 4,4'-bis(1 " ,1 " ,1 " ,2 " ,2 " ,3 " ,3 " -heptafluoro-4 " ,6 " -hexanedion-6 " -yl)chlorosulfo-o-terphenyl ((BHHCT)-Eu3+-labeled anti -AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb3+-labeled SA. Th e concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AF P and CEA by TR-FIA with a single-label method. A good correlation was obta ined with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.