Second derivative synchronous scanning fluorescence spectrometry as a sensitive detection technique in immunoassays. Application to the determination of α-fetoprotein
✍ Scribed by Evriklia S. Lianidou; Pinelopi C. Ioannou; Eftichia Sacharidou
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 594 KB
- Volume
- 290
- Category
- Article
- ISSN
- 0003-2670
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✦ Synopsis
Second derivative synchronous (scanning) fluorescence spectrometty (SDSFS) has been used for the first time as an alternative to time-resolved fluorescence for the detection of Tb 3+ chelates. This approach minimixes the background signal by taking advantage of the large Stokes shift properties of Th 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 @ALL) immunoassay of a-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 enxyme label. ALP cleaves phosphate from salicylphosphate to produce salicylic acid which forms a highly fluorescent ternary complex with Tb3+ and EDTA, which is monitored by SDSFS. The method allows the measurement of AFP with a limit of detection of 2 pg ml-', 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 f 11%.