Nobuto Koyama

A one step sandwich enzyme immunoassay for γ-carboxylated osteocalcin using monoclonal antibodies

A highly sensitive, simple and reliable one-step sandwich enzyme immunoassay (EIA) for the gamma-carboxylated form of osteocalcin (Gla-OC) has been developed using a monoclonal antibody. The minimum amount of Gla-OC detected by this EIA was approximately 0.2 ng/ml when a 10 microliter aliquot of the sample was used. The serum Gla-OC level in 30 healthy subjects was 3.6 +/- 2.19 ng/ml (mean +/- SD). A significant increase was seen in patients with chronic renal failure (20.3 +/- 4.60 ng/ml), atherosclerosis (8.3 +/- 4.94 ng/ml) and osteoporosis (10.1 +/- 4.60 ng/ml). The correlation between the values obtained by the sandwich EIA and competitive RIA methods was given by the linear regression equation, y = 2.896 + 0.759 chi, for which the correlation coefficient (r) was 0.815 (n = 58). This newly developed Gla-OC specific EIA may be useful for the diagnosis of metabolic bone disease and ectopic calcification.

Identification of an inducible factor secreted by pancreatic cancer cell lines that stimulates the production of fucosylated haptoglobin in hepatoma cells.

Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer.

Reevaluation of a lectin antibody ELISA kit for measuring fucosylated haptoglobin in various conditions.

BACKGROUND Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker in a variety of pathological conditions. We previously found that the level of Fuc-Hpt is increased in the sera of patients with pancreatic cancer, and established a lectin antibody ELISA using Aleuria aurantia lectin, which specifically binds to fucosylated residues on oligosaccharides. METHODS To apply this assay system to the clinical detection of several diseases, several assay conditions such as serum dilutions and inhibitory factors were investigated. The Fuc-Hpt kit was available for 25-625 fold serum dilution. RESULTS While the values of Fuc-Hpt assay using sera and plasma were different, they showed positive correlation. The addition of bilirubin and formagine did not influence on Fuc-Hpt assay, but hemoglobin inhibited this assay in a dose-dependent manner. CONCLUSIONS We reevaluated this lectin antibody ELISA kit for measuring fucosylated haptoglobin in various conditions in this study.

High levels of E4-PHA-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells

Oligosaccharides serve as markers of the cell surface and have been used as certain kinds of tumor markers. In the present study, we established a simple method for isolating hepatic progenitor cells using a lectin, which recognizes a characteristic oligosaccharide structure. Rat liver epithelial (RLE) cells, which have been established as a hepatic stem-like cell, were used to identify characteristic oligosaccharide structures on hepatic stem cells. As a result from lectin micro array, several types of lectin including E4-PHA were identified to bind RLE cells specifically. Furthermore, lectin blot and lectin flow cytometry analyses showed that binding to E4-PHA lectin was significantly increased in RLE cells, compared to hepatocytes, and hepatoma cells. The induction of differentiation into a hepatocyte lineage of RLE cells by treatment with Oncostatin M and dexamethasone resulted in a decrease in E4-PHA binding. Using an E4-PHA column, we succeeded in isolating hepatic stem cells from LEC (Long-Evans with cinnamon coat color) rat livers with fluminant hepatitis. The characteristics of the established cells were similar to RLE cells and had a potential of proliferating in rat liver. These results suggest that oligosaccharides can serve as a novel marker for the isolation of the hepatic progenitor cells.