Two-site enzyme immunoassay for alpha-fetoprotein involving column chromatography

We developed a sensitive enzyme immunoassay for alpha-fetoprotein by use of a novel separation method. The assay system consisted of goat antibodies labeled with beta-D-galactosidase, the same antibodies labeled with F(ab')2 fragments of rabbit IgG, and a microcolumn (0.1 ml) containing Sepharose 4B with immobilized goat antibodies to rabbit IgG. Each serum sample or standard alpha-fetoprotein was incubated with the enzyme-labeled antibodies and the F(ab')2-labeled antibodies, then the reaction mixture was passed through the column. alpha-Fetoprotein, which linked with both the enzyme-labeled antibody and the F(ab')2-labeled antibody, bound to the antibody to rabbit IgG immobilized on Sepharose 4B in the column. The column was washed to remove the unbound label, and the buffer in the column was replaced by a solution of omicron-nitrophenyl-beta-D-galactoside. After 60 min at 30 degrees C, the enzyme reaction was stopped by washing the column with sodium carbonate solution. From the absorbance of the eluate at 420 nm, alpha-fetoprotein in the sample could be determined. Values obtained by this method and those obtained by a radioimmunoassay correlated well (slope = 0.989, y-intercept = -0.327 micrograms/l, r = 0.995).