Hideo Katoh

Comparison of carbohydrate structures of serum α-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis

Serum alpha-fetoprotein (AFP) is a glycoprotein of which the sugar chain is considered to show structural changes with malignancies. Microheterogeneity of the serum AFP carbohydrate structure was studied in samples from 35 patients with benign and malignant diseases. Sera were digested directly, extensively, and sequentially with sialidase. beta-galactosidase and beta-N-acetylhexosaminidase. Before and after digestion, sera were examined by means of lectin affinity electrophoresis using eight lectins. Relationships between AFP carbohydrate structures and liver diseases were elucidated by the lectin-reactive profiles and the effect of glycosidase digestion. More than 94% of the AFP carbohydrate structures found in patients with benign and malignant liver diseases were biantennary complex-type oligosaccharides. Changes in the AFP carbohydrate structures at the early stage of hepatocellular carcinoma revealed the addition of alpha 1-->6 fucose to the reducing terminal N-acetylglucosamine and monosialylated AFPs. In both advanced hepatocellular carcinoma and AFP producing extrahepatic malignancies, AFP carbohydrate structures were characterized as the further addition of beta 1-->4 N-acetylglucosamine and heterogeneity in the galactose and N-acetylglucosamine residues. Sequential glycosidase digestion and lectin affinity electrophoresis is useful for analysing the carbohydrate structures of serum glycoprotein.

Determination of α-fetoprotein concentration based on liquid-phase binding assay using anion exchange chromatography and sulfated peptide introduced antibody

A new immunoassay for alpha-fetoprotein based upon liquid-phase binding reactions is described. In this procedure, a sample that contains alpha-fetoprotein is mixed with a solution of two anti-alpha-fetoprotein monoclonal antibodies complexed with peroxidase and sulfated peptide, respectively. After incubation, immune complex is separated from other components by anion exchange column chromatography. Immune complex is quantified using fluorometric detection by peroxidase enzymatic activity. Peroxidase activity correlated with a alpha-fetoprotein with a 1:1 relationship. The stoichiometric immunoreaction allowed a large analytical range (0.4-7500 ng/ml) with a linear dose-response relationship, high sensitivity and good precision. Endogenous substances did not interfere with assay performance. Assay results showed good correlation with other established methods. These results indicate that the method is useful for clinical alpha-fetoprotein determinations.