Fukuko Watanabe

Fluorescence polarization immunoassay for cortisol

A fluorescence polarization immunoassay for serum cortisol was established using cortisol 21-amine which was readily coupled with fluorescein isothiocyanate. The proposed method is sufficiently sensitive, reliable, specific and simple for routine determination of serum cortisol. The assay is rapid, without separation of antibody-bound and free ligands. The minimal amount of cortisol detected was 1.5 ng/tube and the measurable range was from 1.5 to 100 micrograms/dl. There was a good correlation between values obtained from radioimmunoassay and the proposed method.

The analysis of alkaline phosphatase isoenzyme using 4-methylumbelliferyl phosphate as substrate on a cellulose acetate membrane

A new procedure is established for the analysis of alkaline phosphatase isoenzymes. The electrophoretic separation on cellulose acetate membrane coupled with the detection of alkaline phosphatase activity with 4-methyl-umbelliferyl phosphate as a substrate is described. The proposed method would be useful for the analysis of sample of micro-scale quantities and low activities.

Enzyme immunoassay for serum 18-hydroxycorticosterone and its clinical application

An enzyme immunoassay for serum 18-hydroxycorticosterone was established using alkaline phosphatase as a label. The antiserum for 18-hydroxycorticosterone was produced by immunization of rabbits with 18-hydroxycorticosterone 3-(O-carboxymethyl)oxime conjugated to bovine serum albumin. Sephadex LH-20 column chromatography was used to separate 18-hydroxycorticosterone from other steroids in serum samples. The minimal detectable amount of 18-hydroxycorticosterone was 50 pg/tube and the measurable range was from 5 to 1000 ng/dl when a 1.0 ml serum sample was used. Intra- and inter-assay coefficients of variance were 5.0% (n = 6) and 5.8% (n = 6), respectively. Four of 5 patients with aldosterone-producing adenoma had above-normal serum 18-hydroxycorticosterone levels.

Clinical evaluation of serum guanase activity in liver diseases

We measured serum guanase (EC 3.5.4.3) activity in patients with various diseases and in healthy controls, and evaluated the clinical usefulness of this enzyme in liver diseases. The reference range, which showed no significant difference between sexes and ages over the range studied, was 0 to 1.8 U/L. The mean guanase activities for patients with various liver diseases, including acute hepatitis, chronic hepatitis, liver cirrhosis, hepatoma and metastatic carcinoma, were above the upper limit of the reference range. In acute hepatitis and metastatic carcinoma of the liver, the activities were especially high. Validity (sensitivity + specificity) of guanase, which in all tests was above 1.66, was compared to that of AST and ALT in liver diseases. With guanase, the highest validity (1.98) was found in acute hepatitis and metastatic carcinoma. Specificity of guanase was 0.98, whereas sensitivity of AST was 1.00 in all diseases. Sensitivity and specificity of ALT were 0.85 to 0.97 in all diseases. As guanase was specific, including this enzyme with other liver function tests, such as AST and ALT, may decrease false-positive results and may be effective for prediction of liver disease.

A solid phase fluoroimmunoassay of serum cortisol

Direct solid phase fluoroimmunoassay of serum cortisol was established by using fluorescein isothiocyanate labelled anti-cortisol antibody and cortisol conjugated polyacrylamide beads. Sodium salicylate was used as a blocking agent for cortisol binding proteins. The sensitivity of this assay was 0.2 ng/assay tube and the measurable range was from 2 to 100 micrograms/dl using 10 microliters of serum. Intra- and inter-assay coefficients of variation were 11.6% (mean +/- SD 19.8 +/- 2.3 micrograms/dl, n = 5): and 12.1% (mean +/- SD 22.8 +/- 2.8 micrograms/dl, n = 5), respectively. THe accuracy was estimated from recovery study and the average recovery was 100.2%. Cortisol values determined by the present method correlated well with those determined by radioimmunoassay (r = 0.98, y = 0.98 x + 0.04, n = 35). The proposed assay satisfied the standard criteria of dilution, accuracy, and precision, and is applicable to routine measurement of serum cortisol.

Decreased serum 19-nor-deoxycorticosterone (21-hydroxy-19-nor-4-pregnene-3, 20-dione) level in adrenal regeneration hypertensive rats

An enzyme immunoassay of 19-nor-deoxycorticosterone in rat serum was established. The normal value of 19-nor-DOC in rat serum obtained from 9:00 am to 10:00 am was 148 +/- 30 ng/dl (mean +/- SE,n = 10). Serum levels of this steroid decreased in rats with adrenal regeneration hypertension during the course of the experiment up to 8 weeks, while systolic blood pressure rose progressively. We concluded that this mineralocorticoid is not involved at least as a circulating hormone in the pathogenesis of adrenal regeneration hypertension in rats.

Analysis of guanase by agarose gel electrophoresis and activity staining.

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.

Fluorescence Polarization Immunoassay Theory and Application

The principles of fluorescence polarization were first developed by Perrin (1926). About 30 years later the technique was applied to biological system by Weber (1953), and its application to the antigen-antibody reaction was first described by Dandliker and Feigen (1961). Since then, the principles and practice of fluorescence polarization and fluorescence polarization immunoassay have been described in a number of review articles (Dandliker et al., 1964; Parker, 1973; Dandliker and de Saussure, 1970).

Determination of serum glucose with o-toluidine without glacial acetic acid

Abstract In the determination of serum glucose with o -toluidine, the glacial acetic acid medium generally adopted is irritant. A mixture of glycollic acid and propylene glycol is described as an alternative to glacial acetic acid. This mixture is not irritant, is colourless, and stable. Good agreement was observed in the results obtained by this method and by the ultraviolet method using a hexokinase-glucose-6-phosphate dehydrogenase mixture.