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.
Matsumoto
K
K
Department of Chemistry, Waseda University, Shinjuku-ku, Japan Science and Technology Corporation, Tokyo, 169-8555, Japan. kmatsu@mn.waseda.ac.jp
Yuan
J
J
Wang
G
G
Kimura
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D001774:Blood Chemical Analysis / Q000379:methods*; D002272:Carcinoembryonic Antigen / Q000097:blood* / Q000276:immunology; D005063:Europium; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000379:methods*; D006588:Hexanones; D006801:Humans; D012493:Samarium; D013730:Terphenyl Compounds; D000509:alpha-Fetoproteins / Q000032:analysis* / Q000276:immunology; D005466:Fluorocarbons
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D005063:Europium; D005456:Fluorescent Dyes; D006588:Hexanones; D012493:Samarium; D013730:Terphenyl Compounds; D000509:alpha-Fetoproteins; C110071:4,4'-bis(1'',1'',1'',2'',2'',3'',3''-heptafluoro-4'',6''-hexanedione-6''-yl)chlorosulfo-o-terphenyl; D005466:Fluorocarbons