Second derivative synchronous scanning fluorescence spectrometry as a sensitive detection technique in immunoassays. Application to the determination of α-fetoprotein

Abstract

Second derivative synchronous (scanning) fluorescence spectrometry (SDSFS) has been used for the first time as an alternative to time-resolved fluorescence for the detection of Tb 3+ chelates. This approach minimizes the background signal by taking advantage of the large Stokes shift properties of Tb 3+ chelates and offers high sensitivity by narrowing the spectral bands. The analytical performance of this detection technique has been evaluated by choosing the well-defined enzyme-amplified lanthanide luminescence (EALL) immunoassay of α-fetoprotein (AFP) as a model. Monoclonal “capture” antibodies and monoclonal biotin-labelled antibodies in a “sandwich-type” assay configuration in a microwell format have been used. Alkaline phosphatase (ALP) conjugated to an antibiotin antibody was used as an enzyme label. ALP cleaves phosphate from salicylphosphate to produce salicylic acid which forms a highly fluorescent ternary complex with Tb 3+ and EDTA, which is monitored 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 concentration levels equal to 94 ± 11%.