Masayoshi Aosasa

Application of tyramide signal amplification for detection of N-glycolylneuraminic acid in human hepatocellular carcinoma.

AbstractBackground. N-Acetylneuraminic acid and N-glycolylneuraminic acid (NeuGc) are the most common sialic acids in mammals, and NeuGc has attracted attention as a tumor-associated antigen. Methods. In frozen liver sections from patients with hepatocellular carcinoma, glycolipid-type NeuGc was detected on the surface of liver cancer cells in 9 of 17 samples (52.9%) by immunostaining, using two chicken monoclonal antibodies against NeuGc and the tyramide signal amplification method. When conventional immunostaining without amplification was used, all 17 specimens tested were negative. Results. Increased serum levels of anti-NeuGc IgG and/or IgM were observed in 13 of the17 patients with hepatocellular carcinoma (76.5%). The presence of these antibodies was mostly attributed to the expression of NeuGc on hepatocellular carcinoma cells. Of the subjects with small HCCs (diameter 3 cm or less), 6 of 10 were positive for serum anti-NeuGc antibodies; however, 1 of these was negative for both serum Α-fetoprotein (AFP) and for prothrombin–induced vitamin K antagonist II (PIVKA-II). There was no correlation between serum AFP- or PIVKA-II, levels and the presence of NeuGc or anti-NeuGc IgG and/or IgM. Conclusion. The tyramide signal amplification method is useful for the immunohistochemical detection of low-level NeuGc expression by hepatocellular carcinoma cells. We therefore consider that measurement of serum levels of anti-NeuGc antibodies is clinically meaningful and that anti-NeuGc antibody may be a useful screening test, in combination with AFP and PIVKA-II, for the early diagnosis of hepatocellular carcinoma.

Generation of intracellular single-chain antibodies directed against polypeptide GalNAc-transferase using a yeast two-hybrid system

Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.