Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for alpha-fetoprotein from hepatocellular carcinoma.

Alpha-fetoprotein in sera from patients with hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak), which passed through the column without adsorption, was found in both healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma. The second and third peaks were reactive with L. culinaris agglutinin and found only in patients with hepatocellular carcinoma. For α-fetoprotein in the second and third peaks, a novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) was developed. Alpha-fetoprotein in test serum was reacted with dinitrophenyl affinity-purified anti-α-fetoprotein IgG, and the complex formed was trapped onto affinity-purified (antidinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. The polystyrene balls were washed to eliminate substance(s) other than a-fetoprotein in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine. The eluted complex containing α-fetoprotein in the second and third peaks was trapped onto L. culinaris agglutinin-coated polystyrene balls and reacted with affinity-purified anti-α-fetoprotein Fab'-β-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene balls was assayed by fluorimetry. The maximal volume of serum that could be used without interference was 20μl, which was 100-fold larger than that in the previous enzyme immunoassay. As a result, the minimal detectable serum concentrations of α-fetoprotein in the second and third peaks were 0.1 mg/L and 3.5 μg/L, respectively, which were 35- and 29-fold lower than those obtained by the previous enzyme immunoassay. It was possible to confirm the presence of α-fetoprotein in the second and third peaks by a significant difference between bound β-D-galactosidase activities in the absence and presence of α-methyl-D-mannoside or D-glucose. This assay may be more useful for diagnosis of hepatocellular carcinoma than was the previous enzyme immunoassay. However, the maximal serum concentration of a-fetoprotein in the first peak that did not affect the detection limits of α-fetoprotein in the second and third peaks was 0.35 mg/L, which was also 29-fold lower than that obtained by the previous enzyme immunoassay. The detection limits of a-fetoprotein in the second and third peaks remain to be lowered without lowering the maximal serum concentration of α-fetoprotein in the first peak that does not affect the detection limit of serum α-fetoprotein in the second and third peaks.