The extract from isolated rabbit aortae were added to a Krebs solution for 1 h to secrete enzymes. Then, the arginine amidase activity identified in the obtained sample solution was separated using affinity adsorption on lysine-, anti-thrombin lll- and aprotinin conjugated columns. Enzymes adsorbed on lysine and aprotinin affinity columns were found in the extract of isolated rabbit aortae. On the other hand, few enzymes showing affinity with the anti-thrombin lll column were detected. Two forms of arginine amidases with different affinities were isolated and these showed different substrate specificities. The arginine amidase obtained from the lysine affinity column was supposed to be plasmin because it was completely inhibited by antiplasminogen.

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An antibody-lectin sandwich enzyme immunoassay for analysis of altered sugar chains in serum transferrin of patients with hepatoma was developed. Anti-transferrin lgGcoated polystyrene beads incubated with neuraminidase-treated serum samples were reacted with peroxidase (POD)-conjugated lectins such as ricinus communis agglutinin (RCA), lens culinaris agglutinin (LCA), phytohemagglutinin-E₄ (EPHA), and phytohemagglutin-L₄ (LPHA). The amounts of lectins bound to the sugar chains in transferrin were determined by measurement of the POD activities. The LCA/RCA, EPHA/RCA, and LPHA/RCA POD activity ratios were increased significantly in sera from patients with hepatoma in comparison with those in healthy individuals. These results indicated that fucosylation at the reduced end of the N-acetylglucosamine residue, bisecting N-acetylglucosamine at the trimannosyl core, and highly-branched complex-type sugar chains were increased in serum transferrin of patients with hepatoma.

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We developed an anti-GOR assay using the Luminomaster TM system, a fully automated, random access chemiluminescent enzyme immunoassay system. GOR antibody, which is often detected in the serum of patients infected with Hepatitis C virus (HCV), is closely associated with HCV infection.
This new assay provides detection of GOR antibody in 45 min, about half the time of existing EIA methods. The standard curve is 0.1-300 GOR unit/ml (1/10th-300 times cut-off value), givinga detection range about 30 times greater than that of existing assays. This greater detection range allows quantitative determination of low-titer samples without the need for additional detectionprocedures. This assay can also be used to evaluate the clinical efficacy of interferon (IFN) therapy in patients with HCV.

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Dry chemistry technology is well established in the clinical chemistry laboratory because of its simplicity and rapidity. Without preparation of reagents, the concentrations of blood components can easily be determined.
CRP is measured for diagnosis of early inflammation and tissue injury. We have developed a multilayer dry film for quantification of serum CRP which is measured on a Fuji DRI-CHEM Analyzer. The principle involved is that of homogeneous enzyme immunoassay. The hydrolyzing activity for insoluble starch of amylase conjugated to an antibody is modulated by binding of macromolecular antigen (CRP) to the conjugate.
The film has three major layers: a reaction layer for immunological and enzymatic reactions, a barrier layer, and a color formation layer.
Precision and comparison studies gave good results. The lower detection limit was approximately 0.5mg/100ml.

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The Central Committee of Preventive Medicine Project has been undertaking a nationwide health check-up study on cardiovascular diseases in children, in which the serum total cholesterol (TC) and other related analytes were measured. In these nationwide examinations, it is a prerequisite to ensure the accuracy of the measured values from different district branch laboratories and maintain the interchangeability of data from these laboratories to determine any differences in TC values in various districts. To improve the interchangeability of the measured values among the laboratories and correct interlaboratory differences, we analyzed the control survey results which had been carried out yearly from 1988 to 1992. Compared with the first survey, subsequent survey results showed gradual improvements each time in both inter- and intralaboratory variation, eventually reaching a CV of 2.2-2.6%. Frozen pooled sera and lyophilized sera were used for these surveys, but lyophilized samples were found unsuitable. It was important to use the survey samples the accuracy of which were assured by the use of certified reference materials or secondary reference materials as calibrators. These reference materials could contribute significantly to obtain a correction factor and to correct the measured values in each laboratory. So, these control survey samples with defined target values are valuable for improvement of the accuracy of measurement of TC concentration.

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The zinc sulfate turbidity test (ZTT) is a convenient assay procedure to follow qualitative and quantitative changes in serum proteins, and it has been used clinically as an important di-agnostic test. The standard procedure of ZTT uses a Kunkel reagent which is a solution of zinc sulfate in 2.6mM barbiturate buffer. However, it is difficult for this buffer to maintain its pH in solution which is important for the test. The pH in the solution decreases due to absorption of carbon dioxide from the air when the vial is opened and due to instability of barbiturate even when the vial is capped. As a result, the sensitivity increases with use leading to variation between assays and between laboratories. For these reasons, we developed a new Kunkel reagent using a Bis-Tris buffer which has a low dissociation constant and is chemically stable. The results using the new Kunkel reagent correlated with those using the standard Kunkel reagent, and the former showed good stability even when the vial was opened to the air. This new reagent may contribute to renewed interest in the clinical significance of ZTT.

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The levels of urinary total sulfated bile acids (U-TSBA) in 12 social alcohol drinkers and 20 heavy drinkers with liver diseases were investigated. In comparison with the reference value, no significant elevation of U-TSBA was found in either group regardless of the severity of alcoholic liver injury. Two of the three heavy drinkers with abnormal values of UTSBA were positive for hepatitis C virus antibody and showed corresponding histological changes compatible with viral hepatitis. The results show that alcoholic liver injury per se may have minimal effects, if any, on urinary sulfated bile acid excretion, while other hepatobiliary diseases including viral hepatitis result in significant increases.

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A reference method for the determination of creatinine in serum is described. The method is based on the use of high performance liquid chromatography (HPLC), employing phosphate buffer (pH 5.75) as a mobile phase at a flow rate of 0.7 ml/min. A serum sample is deproteinized with trichloroacetic acid solution. Creatinine is separated from coexisting substances in serum by cation-exchange chromatography on Shimadzu Shim-pak ISC 05/S0504 column. The absorbance of creatinine in the eluate is monitored at 234 nm. Sera and reference standards (NIST, SRM914) are assaied in every 7 minutes. The relationship between the concentration of creatinine reference standard and peak area is linear up to 1327.4 μmol/l. The coefficient of variation (CV) is 1.0 to 1.8% at 56.6 to 511.5 μEmol/l of creatinine. Between-day precision studies for the levels of 91.1 and 515.9 μmol/l provided CV of 2.7 and 0.9%, respectivity. The recovery rate of creatinine added to serum was observed in the range 99.6 to 102.0%. None of 24 kinds of endogenous and exogenous substances exerted interferences. It was demonstrated that the alkaline picrate procedure exhibits a statistically significant bias in interlaboratory and survey studies employing this procedure as a reference.

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Guideline I. JSCC concensus method. This method was established in order to avoid discrepancies between JSCC recommended methods and routine methods in terms of assay temperature. The assay conditions for the JSCC consensus method are those recommended by JSCC, except that incubation is carried out at 37°C. Guideline II. Reagents prepared according to the recommendation of JSCC. These are reagent kits for automated analyzers which are prepared in such a way that the final concentrations of each reagent in the reaction mixture remain the same as those used by the JSCC recommended method, and preservatives or stabilizing agentsadded to the reagents have no influence on enzyme assays. Guideline III. Quality assurance survey. We are hoping that more institutions will report their results according to this method and that the accuracy of enzyme assays will be controlled by the JSCC consensus method.

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