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

A novel simultaneous measurement method for cw-fetoprotein (AFP) and carcin oembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay ( TR-FIA) is described. The proposed approach combines the use of europium-la beled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody c omplexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microti ter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibod ies 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-Eu3+ and bi otinylated anti-CEA antibody was added. The AFP concentration was determine d by measuring the solid-phase fluorescence of the europium-labeled anti-AF P antibody at 615 nm. Then a BHHCT-Sm3+-labeled streptavidin-bovine serum a lbumin 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 t o dissociate the samarium-labeled SA-BSA in the immune complex on the surfa ce of the well into the solution. The CEA concentration was determined by m easuring the solution fluorescence of 643 nm from the samarium-labeled SA-B SA. 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 a nd CEA concentrations in 27 human serum samples were determined by the pres ent method as well as by single assay for comparison. A good correlation wa s obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA. (C) 1999 Academic Press.