Development of des-gamma-carboxy prothrombin (DCP) measuring reagent using the LiBASys clinical analyzer.

BACKGROUND: Des-gamma-carboxy prothrombin (DCP), Lens culinaris agglutinin-reactive alpha-fetoprotein ratio (AFP-L3) and total alpha-fetoprotein (AFP) are tumor markers useful for diagnosing and determining the prognosis of hepatocellular carcinoma (HCC). There is a real need for measurement of these three markers on a one-assay platform. METHODS: A method of DCP measurement in human serum was developed using liquid binding assay (LBA), which enables rapid antigen-antibody reaction and bound/free separation on the LiBASys clinical analyzer. RESULTS: The dilution curve for DCP was linear up to 500 ng/mL. The limit of detection of DCP concentration was 0.5 ng/mL. Intra- and inter-assay coefficients of variation of DCP were 0.7%-2.4% and 2.2%-6.5%, respectively. This method was free from interference by hemoglobin, bilirubin, ditaurobilirubin, intrafat, ascorbate, galactose, glucose and rheumatoid factor. The analytical recoveries of DCP added to serum were 91.7%-108.2%. DCP concentration measured with the LBA method was linear and was significantly correlated with that measured with the ELISA method. CONCLUSIONS: The LiBASys clinical analyzer made possible measurement of the complementary tumor markers, HCC, total AFP, AFP-L3 and DCP.

Determination of procalcitonin concentration using the SphereLight 180 clinical auto-analyzer.

BACKGROUND: Procalcitonin (PCT) is a biomarker for the diagnosis of sepsis and bacterial infection diseases. METHODS: A new fully automated SphereLight PCT (SL-PCT) assay system for PCT concentration in human serum or plasma by using SphereLight 180 (SL180, Olympus Corp.) analyzer was developed. The SL-PCT assay is based on chemiluminescent enzyme immunoassay. RESULTS: A linear dose response relationship was observed up to 200 ng/ml PCT concentration. The detection limit of PCT concentration was 0.06 ng/ml. Endogenous substances, anticoagulants, sodium fluoride and drugs did not interfere with assay results. There was a good correlation between the present method and the manual method in serum and plasma samples. CONCLUSIONS: These results indicate that the SL-PCT assay showed good performance in terms of the linearity, detection limit and precision. Use of this PCT measurement may improve the detection of sepsis and infectious disease.

Discrimination of thyroglobulin from thyroid carcinoma tissue and that from benign thyroid tissues with use of competitive assay between lectin and anti-thyroglobulin antibody.

Thyroglobulin is produced only by thyroid follicular cells, and has a molecular weight of 660,000 and carbohydrate content of approximately 10%. The composition of carbohydrate chains on thyroglobulin from thyroid carcinoma has been reported to differ from that in normal thyroid tissue. In this study, heterogeneities of carbohydrate chains on thyroglobulin obtained from thyroid tissues were investigated by competitive reaction between lectin and anti-thyroglobulin monoclonal antibody. Concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin-120 and Datura stramonium agglutinin were compared. The ratio of Lens culinaris agglutinin-reactive thyroglobulin to thyroglobulin was significantly lower in thyroid carcinoma than in normal thyroid tissue, Graves' disease and benign thyroid tumor. However, no differences between malignant and benign tissues were observed with the other lectins tested. Differences in carbohydrate chain on thyroglobulin were observed between malignant and benign thyroid tissues.

The clinical utility of Lens culinaris agglutinin-reactive thyroglobulin ratio in serum for distinguishing benign from malignant conditions of the thyroid.

BACKGROUND: Traditionally, the follow-up of differentiated thyroid carcinoma consists of periodic withdrawal from L-T4-suppressive therapy to allow performance of a highly sensitive serum Tg measurement to detect recurrences. We investigated Lens culinaris agglutinin-reactive thyroglobulin ratios in serum to evaluate in usefulness for detection of thyroid carcinoma. METHODS: The study was conducted on 93 serum sample from 23 healthy volunteers, 32 patients with benign thyroid tumor, 28 patients with thyroid carcinoma without metastasis, and 10 patients with thyroid carcinoma with lymph node metastasis. RESULTS: The Lens culinaris Agglutinin reactive thyroglobulin ratio in patients with thyroid carcinoma was significantly lower than in patients with benign thyroid tumor with serum thyroglobulin concentration >200 ng/ml. Among cases of thyroid carcinoma with lymph node metastasis, Lens culinaris Agglutinin reactive thyroglobulin ratios were significantly lower than in patient with thyroid carcinoma without metastasis and those with benign tumor regardless of serum thyroglobulin concentration. CONCLUSION: Measurement of Lens culinaris Agglutinin reactive thyroglobulin ratio in serum may be useful for distinguishing between thyroid carcinoma and benign thyroid tumor.

Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats.

Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

Discovery of the neutralizing epitope common to influenza B virus victoria group isolates in Japan.

Monoclonal antibody 9B2 possesses hemagglutination inhibition activity against all the 2002/2003 influenza B virus Victoria group isolates in Kobe, Japan, as well as representative strains isolated between 1987 and 1997. The 9B2 epitope localizes three-dimensionally in the vicinity of antigenic site A of the hemagglutinin molecule, and amino acid substitutions in this region affected the binding of 9B2.

Increased soluble FcgammaRIIIa(Mphi) in plasma from patients with coronary artery diseases.

Atherosclerosis is the underlying disease process in patients affected with coronary artery diseases (CAD). Macrophages play a major role in the development of vascular lesions in atherogenesis. The cells express Fcgamma receptor type IIIa (FcgammaRIIIa: CD16) identical to that in natural killer cells (NK cells), but with a cell type-specific glycosylation. In contrast, neutrophils express FcgammaRIIIb. These FcgammaRIIIs are released from the cell surface on activation, and these soluble forms (sFcgammaRIII) are present in the plasma. We measured sFcgammaRIIIa(Mphi) in the plasma with a newly developed anti-FcgammaRIII mAb, MKGR14, which recognizes FcgammaRIIIa(Mphi) specifically. The level of sFcgammaRIIIa(Mphi), as well as the level of sFcgammaRIIIa (sFcgammaRIIIa(Mphi) plus sFcgammaRIIIa(NK)) or the level of total sFcgammaRIII (sFcgammaRIIIa plus sFcgammaRIIIb), were significantly increased in patients with CAD, but not in patients with vasospastic angina (VSA) or intact coronary arteries, compared with age-matched healthy donors. The sFcgammaRIIIa(Mphi) level was related to the number of significantly affected coronary arteries, and positively correlated with LDL-cholesterol to HDL-cholesterol ratios, but negatively with HDL-cholesterol. No correlation among the levels of three sFcgammaRIIIs was observed in CAD patients, as well as in healthy donors. The macrophages are activated during the process of atherosclerosis, and sFcgammaRIIIa(Mphi) may serve as a novel marker for atherosclerosis.

Quantitative microarray-based DNA-DNA hybridization assay for measuring genetic distances among bacterial species and its application to the identification of family Enterobacteriaceae.

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.