[Evaluation of one of the third generation reagents for anti-HCV antibody measurement in comparison with three second generation methods].

It is important to measure anti-HCV antibody(HCV.Ab) in blood to diagnose and treat viral hepatitis C patients. We evaluated one of the third generation reagents, Entymun-Test anti-HCV(Boehringer mannheim), which was developed to minimize false-positive reactions, and compared these results to findings obtained with three second generation tests for HCV.Ab. Coefficients of variation in the Entymum-Test assay were good(within-run, 1.1 approximately 1.8%; between-run, 2.5 approximately 7.5%). Four interfering substances(bilirubin C, bilirubin F, lipid and hemoglobin) hardly affected the HCV.Ab measurement. The HCV.Ab indices of healthy donors(n = 61) were distributed below the cut-off index(C.O.I). Sensitivity of the third generation test was the same as that of second generation tests, calculated from the results of dilution tests. Index of the dilution test in the third generation test formed a plateau until four-fold dilution, but there was no prozone phenomenon. Among the third generation test and three second generation tests, the former had the lowest positive percentage in serum HCV.Ab. Moreover, findings of HCV-RNA agreed more with the results of the third generation test among the four tests. As a result, the third generation test appeared to minimize false-positive results and improve specificity.

[Evaluation of type IV collagen in patients with various thyroid disease].

Serum level of type IV collagen was measured in 104 patients with various thyroid disease, and the relationship between its level and thyroid hormone level was examined. The type IV collagen was measured by the method of one step sandwich enzyme immunoassay (EIA) using two distinct monoclonal antibodies recognized triple-helical (TH) domain and 7-S domain, respectively. The serum level of type IV collagen was significantly high in the hyperthyroid patients compared with that in normal controls, and a significant positive correlation was found between its value and thyroid hormone levels (T3, T4, FT3, FT4). The elevated level of type IV collagen in hyperthyroid patients was decreased to normal level, when they became to euthyroid after antithyroid drug therapy for hyperthyroidism. The serum level of type IV collagen was in normal range in hypothyroid patients, but the value was increased to high normal range after T4-replacement therapy for hypothyroidism. This evidence indicates that the serum level of type IV collagen is closely related to thyroid hormone level in patient with various thyroid disease. Type IV collagen concentration might be one of the useful variables for evaluating the thyroid function, although its mechanism is not elucidated.

[Estimation of anti-thyroid peroxidase autoantibody (TPOAb) and anti-thyroglobulin autoantibody (TgAb) in patients with various thyroid disease--comparison between histopathological findings and serological results in patients with Hashimoto's thyroiditis].

We compared and evaluated titers of AMC (anti-microsome antibody), and ATG (anti-thyroglobulin antibody) by passive gelatin-agglutination and by radioimmunoassay in 170 sera from 129 patients with various thyroid diseases and 41 normal subjects. The results of conventional ATG and TgAb by RIA correlated (r = 0.731) and those of conventional AMC and TPOAb correlated well (r = 0.907), with discrepancies mostly limited to sera with low antibody titers. Five patients with Hashimoto's thyroiditis showed positive results in AMC, whilst negative in TPOAb. These sera had positive ATG autoantibody and preincubation with thyroglobulin inhibited the agglutination reaction of AMC tests, suggesting ATG producing false positive results in AMC assay. The prevalences of positive TgAb rates were higher than (p < 0.001) ATG in patients with Hashimoto's disease (96.6% vs 50.0%) and Graves' disease (76.9% vs 46.1%). However, the prevalences of positive TPOAb were not different from AMC (Hashimoto's disease 75.9% vs 81.0%: Graves' disease 80.8% vs 76.9%). In patients with Hashimoto's thyroiditis, our study demonstrates the results of TgAb by RIA reflects the pathological findings and the diagnostic sensitivity increases by using in combination with TPOAb.

[Clinical evaluation of serum 1,5AG levels in patients with liver cirrhosis].

We measured serum 1,5 anhydroglucitol (1,5AG) levels by HPLC in 32 patients with liver cirrhosis, 32 with diabetes mellitus and 61 normal subjects. Serum 1,5AG was significantly lower in patients with diabetes mellitus and liver cirrhosis compared with that in normal subjects. Serum levels of type IV collagen were higher in patients with liver cirrhosis than in those without liver cirrhosis. A negative correlation was observed between serum 1,5AG and type IV collagen in patients with liver cirrhosis (r = -0.37, p < 0.05), but not in patients with diabetes mellitus. These data suggest that serum 1,5AG levels reflect the degree of liver cirrhosis.

[Recent developments of measurements for pituitary-thyroid hormones: evaluation of five immunometric assay methods (RI and non RI methods].

We evaluated five commercially available immunoassay kits for pituitary-thyroid function tests. The possible interference in analysis by immunoglobulin G (IgG) has also been examined. The BeriLux TSH assay kit (Hoechst co. FRG) was compared with two other non-RI methods (AIA-1200 and IMx) and two other immunoradiometric assays (RIA-gnost TSH IRMA and EIKEN IRMA kits) in 32 normal subjects and 92 patients with Graves' disease. The new ICMA BeriLux kit had a marked improved analytical and clinical sensitivity. The minimal detectable level of TSH in the assay was 0.006 mU/l. The precision was 2.8% and 6.1% at 0.093 +/- 0.003 mU/l and 0.028 +/- 0.002 mU/l, respectively, whereas the levels for the other methods were above 17.2% and 59.4% respectively. The TSH pattern was always less than 0.006 mU/l with the BeriLux kit, before and after TRH administration whereas the other methods showed random fluctuations which indicated their low accuracy at this concentration. This new ICMA BeriLux kit appears far more reliable than the ordinary immunometric assay kits.

[A predictive and prognostic study on two cases of transient hyperphosphatasemia of infancy].

We presented two cases with transient hyperphosphatasemia (TH) of infancy, whose serum alkaline phosphatase (EC 3.1.3.1, ALP) activity showed markedly increased and the atypical isoenzyme fractions were seen by electrophoresis. These isoenzymes migrated into normal bone ALP region (alpha 2-beta globulin fraction) and fast liver ALP region (fast alpha 2 globulin fraction). From various investigation such as, heat stability, inhibition test by amino acid, neuraminidase treatment and Triton X-100 treatment, former fraction seemed to derive from bone ALP and later fraction from liver ALP. From our study, increment of the activity of alpha 2-beta gl fraction was in advance one month before maximum ALP activity stage, and fast alpha 2 gl fraction followed increasing 3 weeks after that. On the other hand, decreasing of fast alpha 2 gl fraction showed a shorter delay than alpha 2-beta gl fraction. These results suggest that a differential exchange of sugar chain or an impaired clearance of the enzyme from circulation was possibly occurred. It seemed to be important to increase a study of such a predictive and prognostic change of ALP activity and isoenzyme fraction in TH cases.

Evaluation of a new chemiluminescence technique for human thyrotropin (BeriLux hTSH): diagnostic value of five immunometric assay methods.

A new commercially available human thyrotropin immunochemiluminometric assay (ICMA) kit was evaluated. The BeriLux assay (Hoechst Co., Germany) was compared with two other non-radioisotopic methods (AIA-1200 and IMx) and two other immunoradiometric assays (RIA-gnost TSH IRMA and EIKEN IRMA kits) in 32 normal subjects and 104 patients with Graves' disease, divided into seven groups: 1) untreated hyperthyroidism; 2) hyperthyroidism during treatment; 3) euthyroid with negative thyroliberin test (subclinical hyperthyroidism); 4) euthyroid with low thyroliberin test; 5) euthyroid with normal thyroliberin test; 6) euthyroid with high thyrotropin level (subclinical hypothyroidism); and 7) primary hypothyroidism. Patients in groups 2-6 were undergoing treatment with mercazole and propylthiouracil. The new immunoluminometric assay (ILMA) BeriLux kit was shown to have a remarkably improved analytical and clinical sensitivity. The minimal detectable level of thyrotropin in the assay was 0.006 mU/l. The precision was 2.8% and 6.1% at 0.093 +/- 0.003 mU/l and 0.028 +/- 0.002 mU/l, respectively, whereas the precision of the other methods was above 17.2% and 59.4% respectively. Seven patients from the untreated hyperthyroid group were given 500 micrograms thyroliberin i.v. (the thyroliberin test). The thyrotropin pattern before and after thyroliberin administration was always less than 0.006 mU/l with the BeriLux kit, whereas the other methods showed random fluctuations indicating their low accuracy at this concentration. Using the BeriLux kit, 7 of the 16 overt hyperthyroid patients undergoing treatment showed a measurable thyrotropin level below 0.01 mU/l but a negative thyroliberin test.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.

Clinical evaluation of accuracy in determining serum free thyroxine and free triiodothyronine in patients with non-thyroidal illness: immunoglobulin effect on T3/TBG ratio and T4/TBG ratio.

We examined the effect of endogenous immunoglobulins (G, A and M) and albumin on the measurement of thyroid hormones by different methods, including a new non-isotopic immunoassay of free thyroxine (FT4) and free triiodothyronine (FT3), in a large number of patients with non-thyroidal illness (NTI). Variations in serum protein concentrations can affect the results of radioimmunoassay of human thyroid hormones and thyroxine binding globulin (TBG). Our data revealed that in patients with non-thyroidal illness, when fluctuations in serum gamma-globulin occurred the T3/TBG and T4/TBG ratios altered. Consequently, when patients are suffering from non-thyroidal illness with changing gamma-globulin levels, clinical scientists should take care when they use T3/TBG and T4/TBG ratios as a substitute for FT3 or FT4 estimation. We found FT4 and FT3 (determined with Amerlex-M kits) T3 and the T3/TBG ratio were altered inversely due to the difference in the serum gamma-globulin levels. A recently developed enhanced luminescence enzyme immunoassay for FT3 and FT4 (Amerlite FT3 and FT4 kits) provides more reliable and accurate results, because of its resistance to interference, especially from albumin and gamma-globulin.

Interference of immunoglobulins in the radioimmunoassay of human beta-endorphin.

The interference of immunoglobulins in the radioimmunoassay (RIA) of human beta-endorphin was investigated. Human IgM showed no cross-reactivity. Human IgA showed a weak cross-reaction, but the dilution curve of IgA did not show parallelism with the standard curve of beta-endorphin, thus indicating its antigenic difference. The dilution curves of human IgG showed 0.18% displacement with respect to the human beta-endorphin standard curve, with good parallelism. Moreover, five patients with multiple myeloma of the IgG type showed falsely elevated beta-endorphin levels. We investigated the possibility that certain IgGs may be responsible for the displacement of [125I]beta-endorphin in the beta-endorphin kit. The apparent beta-endorphin level of plasma from multiple myeloma patients was markedly decreased after affinity chromatography of the serum on protein A-Sepharose. In another 3 patients with multiple myeloma, we examined IgG interference by measuring the beta-endorphin levels in their lyophilized IgG diluted with saline. The results demonstrated high values of 20.2, 25.5 and 21.2 pmol/l respectively, also showing good parallelism. These immunological parallels to human beta-endorphin verify that a part of the amino acid sequence of human IgG is similar to that of human beta-endorphin. Consequently, in the measurement of beta-endorphin with polyclonal antibody, the results may sometimes be spuriously high due to cross-reaction with IgG, e.g., in patients with IgG myeloma. To avoid IgG interference, a specific monoclonal antibody to synthetic beta-endorphin should be used rather than polyclonal antibodies.