Simultaneous Determination of α-Fetoprotein and Carcinoembryonic Antigen in Human Serum by Time-Resolved Fluoroimmunoassay

Abstract

A novel simultaneous measurement method for α-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–Eu3+ 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–Sm3+-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 × 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.