COMPARATIVE-STUDY OF FLUORESCENT TERNARY TERBIUM COMPLEXES - APPLICATION IN ENZYME-AMPLIFIED FLUOROMETRIC IMMUNOASSAY FOR ALPHA-FETOPROTEIN

A systematic spectrofluorimetric study of the formation of ternary com plexes of terbium ions (Tb3+) with ethylenediamine tetraacetic acid (E DTA) and a variety of bidentate organic ligands has been performed. Fo urteen ligands (salicylic acid, quinolones and hydroxybenzene sulfonic acid derivatives) were examined as efficient energy donating chelator s for Tb3+. The fluorescence properties of the ligands, alone as well as of the ternary complexes with Tb3+ and EDTA have been examined. Opt imization studies, including the effect of pH, buffer systems and the use of EDTA analogs, were performed. A possible relationship between t he structure and the ability of the ligand to act as an efficient ligh t absorbing and energy donating chelator for Tb3+ is discussed. The ph osphate 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 amplifie d lanthanide luminescence immunoassay by means of second derivative sy nchronous 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 con centration levels (5, 40, 100 and 250 ng ml(-1)) equal to (99+/-3)%.