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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay.

A novel simultaneous measurement method for alpha-fetoprotein ( AFP) and carcinoembryonic antigen ( CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti- AFP antibody for AFP TR-FIA and biotinylated anti- CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti- AFP and anti- CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti- AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti- CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti- AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti- CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.[1]


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