2ND DERIVATIVE SYNCHRONOUS SCANNING FLUORESCENCE SPECTROMETRY AS A SENSITIVE DETECTION TECHNIQUE IN IMMUNOASSAYS - APPLICATION TO THE DETERMINATION OF ALPHA-FETOPROTEIN

Second derivative synchronous (scanning) fluorescence spectrometry (SD SFS) has been used for the first time as an alternative to time-resolv ed fluorescence for the detection of Tb3+ chelates. This approach mini mizes the background signal by taking advantage of the large Stokes sh ift properties of Tb3+ chelates and offers high sensitivity by narrowi ng the spectral bands. The analytical performance of this detection te chnique has been evaluated by choosing the well-defined enzyme-amplifi ed lanthanide luminescence (EALL) immunoassay of alpha-fetoprotein (AF P) as a model. Monoclonal ''capture'' antibodies and monoclonal biotin -labelled antibodies in a ''sandwich-type'' assay configuration in a m icrowell format have been used. Alkaline phosphatase (ALP) conjugated to an antibiotin antibody was used as an enzyme label. ALP cleaves pho sphate from salicylphosphate to produce salicylic acid which forms a h ighly fluorescent ternary complex with Tb3+ and EDTA, which is monitor ed by SDSFS. The method allows the measurement of AFP with a limit of detection of 2 pg ml(-1), coefficients of variation in the range 4.5-9 .9% and mean recovery from four pooled serum samples at two concentrat ion levels equal to 94 +/- 11%.