Synthesis of a Terbium Fluorescent Chelate and Its Application to Time-Resolved Fluoroimmunoassay

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Abstract
A new nonadentate ligand, N, N, N1, N1-[2,6-bis(3‘-aminomethyl-1‘-pyrazolyl)-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 lifetime of 2.681 ms in 0.05 M borate buffer of pH 9.1. Streptavidin (SA) was labeled with BPTA by using its succinimidyl monoester, and the BPTA−Tb3+-labeled SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb3+-labeled SA was also used in competitive-type TR-FIA of bensulfuron-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 simultaneous measurement method for AFP and CEA in a human serum sample was developed 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. The concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AFP and CEA by TR-FIA with a single-label method. A good correlation was obtained with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.