For sensitive measurement of the anti-thyroglobulin antibody in serum, a two-step sandwich chemiluminescence enzyme immunoassay (CLEIA) was developed. This method uses a microplate as a solid phase, peroxidase as a labeling enzyme, and luminol and an enhancer as chemiluminescence reagents. The CLEIA allowed absolute levels of the antibody to be measured over a wide range (from 100 to 100,000 ng/ml). The results of this assay correlated with those from a dual antibody radioimmunoassay (RIA) 1), revealed a correlation coefficient of 0.64 and regression equation of Y=21.6X-160 (N=20). There was poor agreement between the results from the two assays at high concentrations. The upper limit of the normal range for blood anti-thyroglobulin antibody was 195 ng/ml. Antibody levels significantly lower than this concentration were found to cause underestimation of serum thyroglobulin levels. Serum anti-thyroglobulin antibody levels were determined for 30 patients who had been checked periodically for serum thyroglobulin after surgical resection of thyroid cancer. Levels higher than 200 ng/ml were found for 6 of these patients. In these cases, the serum thyroglobulin levels seemed to be underestimated because of the presence of high levels of anti-thyroglobulin antibody. These findings indicate the necessity of measurement of the levels of both anti-thyroglobulin antibody and thyroglobulin for early detection of recurrence after surgical resection of thyroid cancer.

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A chemiluminescent enzyme immunoassay (CLEIA) was developed for measurina thyroglobulin in serum. This assay is rapid, easy to perform, and highly sensitive. A microplate was used as the solid phase, peroxidase as the labeling enzyme, and luminol and enhancer as the chemiluminescence reagents. The minimum concentration of thyroglobulin detectable by this CLEIA was 0.05 ng/ml. The results obtained with the CLEIA were in good agreement with those obtained by the standard RIA, the correlation coefficients being r=0.98, Y=0.97X-5.1. where Y is the CLEIA value and X the RIA value. The CLEIA was used to measure thyroglobulin serum levels as a means to monitor thyroid cancer. In most patients, the high preoperative levels dropped to nearly 0 ng/ml after total thyroidectomy, and then increased again upon recurrence. In conclusion, measurement of the thyroglobulin serum level changes by this highly sensitive assay is effective for early discovery of thyroid cancer recurrence postoperatively.

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The effects of treatment with valproic acid (VPA) and other anticonvulsants such as phenobarbital, phenytoin, and carbamazepine on serum carnitine levels were studied. Considering the influence of gender and age on the reference intervals of serum carnitine levels, the levels in patients receiving anticonvulsant therapy (>10 years old) were compared with those in gender-matched controls. VPA therapy alone reduced the serum free carnitine (FC) levels in both male and female groups. Moreover, VPA polytherapy caused marked decrease of serum FC levels and increased the incidence of hypocarnitinemia. The non-VPA anticonvulsant therapies also reduced serum FC levels, however, the incidence of hypocarnitinemia was unchanged in the non-VPA polytherapy groups.

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We described the diazo reaction of bilirubins IIIα, IXα and X IIIα, using various diazonium salts. These bilirubin isomers reacted with diazonium salts to form an azo dye with a slightly different absorption peak. There was little difference in the rate constant between these three isomers, while a significant difference in the color intensity of the azo dyes was observed. It has been reported that serum bilirubin in human adults is bilirubin IXα, but in patients' sera after storage bilirubins IIIα and X IIIα were also detected by means of thin layer chromatography. The relative proportions of formation of these three isomers were correlated with serum total bilirubin concentration.

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We investigated the determination of bone-type alkaline phosphatase (ALP) with densitometric scan using polyacrylamide disc gel electrophoresis (PAGE). The CV obtained in precision study was less 2% for within-run assay, and the linearity extended down to 20 mU/ml. The measured values with the PAGE method were correlated well with cellulose acetate membrane (CA) electrophoresis method, but it was appeared that the PAGE method with densitometric scan was more correctly detected bone-type ALP than CA method because of good separating liver-and bone-type ALPs. Moreover, the normal reference values of bonetype ALP (mean±2SD), obtained 68 healthy adults was 53±36.6 mU/ml. This result was similar to that of WGA affinity electrophoresis as described earlier. We conclude that the precision determination of bone-type ALP is useful metabolic bone marker for diagnosis of bone disease.

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We have demonstrated the separation of human serum protein with capillary electrophoresis (CE) system. A untreated fused silica capillary, 20μm (I. D.) ×20cm, was assembled in the CE cartridge format. On-line detection of CE system was at 200nm. The capillary temperature was maintained at 23°Cduring electrophoresis. Human serum samples were diluted 11-fold in 20mM PBS (pH7.0). Diluted samples of about 0.2nl were introduced by pressure injection for 10 s. The analysis buffer was used 150mM borate buffer (pH 10.0) and electrophoresis was performed at 10 KV for 6.5 min.
Human serum sample was separated five fractions by CE and was identical with those of cellulose acetate membrane electrophoresis. We present our method for separation of serum proteins into 10 peaks. These protein peaks were detected immunoglobulins, complement C3, transferrin, α₂-macroglobulin, haptoglobin, α₁-antitrypsin, albumin, preablumin, respectively, according to fast migration time with CE. Moreover, capillary electrophoretic patterns in various patient sera were in complete agreement with the results shown by cellulose acetate membrane electrophoresis.
From these results, it is suggested that CE system is useful techonology for clinical diagnosis and suitable tool for routine work in the future.

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[in Japanese]

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