Specific enzyme immunoassay for alpha-fetoprotein from hepatocellular carcinoma

alpha-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak) was found in all the serum samples, but the other two peaks (the second and third peaks) were found only in patients with hepatocellular carcinoma. For alpha-fetoprotein in the second and third peaks, a specific enzyme immunoassay was developed. Lens culinaris agglutinin-coated polystyrene balls were incubated with alpha-fetoprotein and, after washing, with affinity-purified anti-alpha-fetoprotein Fab'-beta-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 0.2 microliters, and the minimal detectable serum concentrations of alpha-fetoprotein in the second and third peaks were 3.5 mg/liter and 0.1 mg/liter, respectively. The maximal serum concentration of alpha-fetoprotein in the first peak that did not affect the detection limits of alpha-fetoprotein in the second and third peaks was 10 mg/liter. It was possible to confirm the presence of alpha-fetoprotein in the second and third peaks by a significant difference between bound beta-D-galactosidase activities in the absence and presence of alpha-methyl-D-mannoside or D-glucose. This assay may be useful for diagnosis of hepatocellular carcinoma, although further improvements remain to be made.