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S Ohtaki

Publications and source records attributed to S Ohtaki.

158 records · Page 9Linked to original sources
PubMed1979

[Study on enzyme immunoassays for the measurement of thyroglobulin and anti-thyroglobulin autoantibody in human serum (author's transl)].

Enzyme-linked sandwich immunoassays for the measurement of thyroglobulin and anti-thyroglobulin autoantibody in human serum using silicone rod and beta-D-galactosidase were studied. These methods showed excellent results in specificity, sensitivity, precision and clinical application. 1) A method using silicone rod coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin G (Fab') conjugated with beta-D-galactosidase was developed for the measurement of circulating thyroglobulin. The sensitivity of the assay with as little as 2 microliter of serum was 10.7 amoles/tube corresponding to 3.5 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The correlation coefficient between values determined by the present assay and a double-antibody radioimmunoassay was 0.99 (n = 63, p less than 0.001). Circulating thyroglobulin was detectable in 90% of 146 normal subjects, the concentration being 13.3 +/- 10.3 ng/ml (mean +/- S.D.). Interference of anti-thyroglobulin autoantibody with the assay for thyroglobulin was smaller than that in radioimmunoassay. 2) Another method using human thyroglobulin conjugated with beta-D-galactosidase and silicone rod coated with human thyroglobulin was developed for the measurement of circulating (anti-human thyroglobulin) autoantibody. The sensitivity of the assay with as little as 5 microliter of serum was 7 fmoles/tube corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The highly significant correlation was observed between the concentrations of anti-thyroglobulin autoantibody determined by the present assay and a radioimmunoassay (r = 0.80, n = 74, p less than 0.001) and also between those by the present assay and those by tanned red cell hemagglutination (r = 0.78, n = 199, p less than 0.001). No effect of thyroglobulin on the present assay was observed unless the ratio of the amount of thyroglobulin to that of (anti-human thyroglobulin) immunoglobulin G was higher than a tenth.

Autoantibodies↗
PubMed1979

[Partial purification of hog thyroid peroxidase using detergent treatment and its spectral changes induced by hydrogen peroxide (author's transl)].

Thyroid peroxidase (TPO) was partially purified from hog thyroid microsomes after solubilization by means of deoxycholate treatment followed by ammonium sulfate farctionation and affinity chromatography with Con A Sepharose. The absorption spectra of the preparation showed the maxima at around 410 nm for oxidized form, 422 nm for dithionite-reduced form and 422 nm for CO complex of reduced form. The cyanide difference spectrum showed a peak at 431 nm and a trough at 403 nm. This preparation was contaminated with little cytochrome b5 and it was shown that the TPO preparation was able to be used for the following spectrophotometric experiments. The addition of H2O2 to the TPO preparation induced the characteristic change in the difference spectrum with a peak at 430 nm and a trough at 407 nm, which was gradually disappeared in a few minutes, but at the high concentration of H2O2 (35 muM) the trough at 411 nm was observed after decomposition of H2O2, accompanying loss of peroxidase activity. This deepening of the trough caused the heme degradation which was dependent with the concentration of H2O2 added and to less extent at the low concentration of H2O2 (3.5 MUM). Since the difference spectrum produced by the addition of small amount of H2O2 disappeared rapidly after the addition of KI or ascorbate and resembled the spectral change due to the formation of Compound II in the reaction of other perioxidases, it was concluded that the difference spectrum with a peak at 430 nm and a trough at 407 nm observed after the addition of H2O2 was ascribable to the formation of Compound II of TPO. Although Compound I was not observed under the experimental conditions used, the results were accounted for the presence of I bound to TPO or other endogeneous reducing agents. We tentatively concluded that Compound I and Compound II are formed in the reaction of TPO with H2O2 as well as in that of horseradish peroxidase.

Animals↗
PubMed1979

An enzyme immunoassay for the measurement of thyroglobulin in human serum.

An enzyme-linked sandwich immunoassay using silicone rods coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin F (Fab') conjugated with beta-D-galactosidase was developed for the measurement of thyroglobulin in human serum. The volume of serum needed for the assay was as little as 2 microliters. The sensitivity of the assay was 3.5 ng/ml, which is equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by the following observations: (1) The absence of crossreaction of thyroxine and triiodothyronine, (2) non-detectability of thyroglobulin in the sera of patients who underwent total thyroidectomy, (3) parallelism of the standard curve with dilutions of reference serum. The precision of the assay was proven by the demonstration of the sufficient recovery of human thyroglobulin added to sera (92--99%) and coefficients of variance in within and between assays were 6.2--9.3 and 2.5--5.3%, respectively. Furthermore, a highly significant correlation was observed between thyroglobulin concentrations measured by our enzyme immunoassay and those by radioimmunoassay (r = 0.99, p less than 0.001, n = 63). Human thyroglobulin in serum was detectable in 90% of 146 normal subjects, the concentration (mean +/- S.D.) being 13.3 +/- 10.3 ng/ml.

Female↗
PubMed1979

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.

Animals↗
PubMed1978

Suppressive effects on simian virus 40-induced oncogenesis of several immunosuppressive agents and hormonal modifications applied during the latent period.

The long latent period required for tumor induction with Simian virus 40 (SV40) in the subcutis of hamsters can be used to investigate whether host factor(s) participate in oncogenesis. Several treatments were applied during this period, and the results were compared with those of the same treatment applied in another series of experiments, homologous SV40 tumor grafting in hamsters. The results obtained were as follows: (a) the latent period for SV40 tumor induction in the female was shorter than that in the male; tumor development was delayed significantly by oophorectomy but was little affected by orchiectomy. Tumor development was markedly delayed in animals of both sexes by estrone given at birth but was accelerated by testosterone given in the adult male; (b) by each of these hormonal modifications, growth of transplanted SV40 tumors was influenced in a different way from that of tumor induction; (c) immunosuppressive treatments, such as thymectomy, administration of antilymphocyte sera, cyclophosphamide, or cortisone acetate delayed and decreased tumor development when applied in the latent period, and the degree or pattern of this effect varied from one procedure to another, depending on the sex and age of the animals; (d) in contrast, tumor growth was markedly accelerated in thymectomized or cortisone-treated hosts irrespective of sex. The different and almost reverse effect of the same procedure in the two phases of tumorigenesis may indicate two discriminating mechanisms operating in the host during these phases. This different effect may be due to virtual absence of any "mature" neoplastic cell in the latent period, except for a few weeks before the appearance of a palpable tumor. These results suggest that the long period of latency may be spent to complete SV40-induced neoplastic conversion of cells, receiving some help by host factors.

Age Factors↗
PubMedDate not supplied

The same chemicals induce different neurotoxicity when administered in high doses for short term or low doses for long term to rats and dogs.

Dose- and term-dependent differences in the location and nature of brain lesions induced in rats and dogs by 2,5-hexanedione (2,5-HD), misonidazole, clioquinol, and acrylamide are reported. Subchronic neuropathies ("distal axonopathy") were induced by low-dose administration of these neurotoxicants and at high doses, lesions caused by acute or subacute neurotoxicity were found in the central nervous system (CNS). In rats, 2,5-HD induced extracellular edema, nerve cell degeneration, and axonal degeneration in the cerebellar and vestibular nuclei. Similar lesions were observed in misonidazole-treated dogs and clioquinol induced nerve cell degeneration in the hippocampus and malacia in the piriform lobes of these animals. In rats, acrylamide induced degeneration of Purkinje cells. Although the mechanism(s) underlying the differential neurotoxicity of high and low doses of these neurotoxicants remains unclear, we suggest certain biochemical mechanisms, cytotoxic edema and excitotoxicity, as factors in the production of such lesions after high-dose treatment.

Acrylamide↗
PubMedDate not supplied

Modulation of T cell function by alpha-fetoprotein: An in vivo study on porcine thyroid peroxidase-induced experimental autoimmune thyroiditis in transgenic mice producing human alpha-fetoprotein.

Experimental autoimmune thyroiditis was induced in transgenic mice (TG-3) producing human alpha-fetoprotein (AFP) and in control mice by immunization of porcine thyroid peroxidase (TPO). Development of thyroiditis accompanied with mononuclear cell infiltration was observed in 6 out of 8 treated control mice. In contrast, the development was significantly suppressed in TG-3 mice and mild histologic change was observed in only 1 out of 8 TG-3 mice (p < 0. 05). Serum anti-TPO antibody titers gradually increased in TG-3 mice as well as in control mice and there were no significant differences. In vitro phytohemagglutinin response of splenocytes from TG-3 mice was lower than that from control mice. Significantly reduced numbers of CD4+ and CD8+ splenocytes, total thymocytes, and CD4+ thymocytes were found in the nontreated TG-3 mice as compared with those in control mice. These results suggest that ubiquitously produced AFP modulates in vivo T cell development and/or T cell-dependent immune responses.

Animals↗
PubMedDate not supplied

Application of the avidin-biotin-complex method for the light microscopic analysis of lymphocyte subsets with monoclonal antibodies on air-dried smears.

A study was undertaken of the application of the avidin-biotin-peroxidase complex (ABC) method to the monoclonal antibody MAbs staining of mononuclear cells in hematologic and cytodiagnostic materials. Satisfactory cell morphology and immunoreactivity of surface antigens were observed when the slides were fixed in 80% acetone in phosphate-buffered saline or in 60% acetone in 0.03 M citric acid buffer solution (pH 5.4). Unstained air-dried preparations could be preserved for two weeks at room temperature in a desiccator and for one year at -70 degrees C after fixation. An excellent immunoreaction, even with a weak surface antigen, was observed by inhibition of endogenous peroxidase after the secondary antibody reaction; reactions of weak antigens tended to be obscured when the inhibition was performed before the first antibody reaction. Use of the Giemsa stain as a counterstain made it possible to readily observe the cell morphology; therefore, white blood cell analysis could be performed simultaneously when peripheral blood smears were studied. The positive rate of immunoreaction by an immunofluorescent method was well correlated with that obtained by the ABC method. The ABC method proved to be an excellent immunocytochemical technique for detecting cell surface antigens with high sensitivity and specificity; furthermore, it is useful for cell morphology studies and yields permanent preparations.

Antibodies, Monoclonal↗
PubMedDate not supplied

Immunohistochemical demonstration of thyroid peroxidase (TPO) in human thyroid tissues from various thyroid diseases.

Human thyroid peroxidase (hTPO) was detected immunohistochemically in 61 normal, benign and malignant thyroid tissues with a monoclonal antibody (38E) against hTPO using paraffin-embedded sections. Thyroid peroxidase was expressed intensely not only in tissues from Graves disease and hyperfunctioning adenomas, but also in those from carcinomas, adenomas and Hashimoto's thyroiditis. The staining pattern of TPO in the follicular cells was a diffuse and fine granular one in the normal thyroids, Graves' thyroids and adenomas, but abnormal coarse granular deposits of TPO were characteristically identified in most of the papillary carcinomas and Hashimoto's thyroiditis. These coarse granular deposits of TPO suggested that qualitative or structural changes in the TPO molecule are present in carcinomas and Hashimoto's thyroiditis. Consequently, these qualitative changes in TPO may be responsible for the lack of biochemically measured TPO activity in these diseases. This finding may also be useful in distinguishing between benign and malignant thyroid tumors.

Humans↗