Comparative study of fluorescent ternary terbium complexes. Application in enzyme amplified fluorimetric immunoassay for alpha-fetoprotein

A systematic spectrofluorimetric study of the formation of ternary complexes of terbium ions (Tb3+) with ethylenediamine tetraacetic acid (EDTA) and a variety of bidentate organic ligands has been performed. Fourteen ligands (salicylic acid, quinolones and hydroxybenzene sulfonic acid derivatives) were examined as efficient energy donating chelators for Tb3+. The fluorescence properties of the ligands, alone as well as of the ternary complexes with Tb3+ and EDTA have been examined. Optimization studies, including the effect of pH, buffer systems and the use of EDTA analogs, were performed. A possible relationship between the structure and the ability of the ligand to act as an efficient light absorbing and energy donating chelator for Tb3+ is discussed. The phosphate ester of diflunisal, one of the most sensitive ligands for Tb3+, was used as an alkaline-phosphatase substrate for the determination of a-fetoprotein (AFP) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay by means of second derivative synchronous fluorescence spectroscopy as a detection technique. By using this substrate, AFP was determined in serum with a limit of detection (3xs.d. blank) of 5 pg ml(-1), coefficients of variation in the 2.4-5.3% range and mean recovery from four pooled serum samples at four concentration levels (5, 40, 100 and 250 ng ml(-1)) equal to (99+/-3)%.