An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

In vitro responses of luteinizing rat granulosa cells to human thyroid-stimulating hormone

The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.     

Evaluation of an immunoenzymatic method for determining TSH and establishment of reference values]

In the present work we reported the results of the valuation of IMx Ultrasensitive hTSH assay which is a Microparticle Enzyme ImmunoAssay (MEIA) for quantitative determination of human stimulating hormone (hTSH) in the human serum or plasma. We have determined the method's precision, within run and between run, sensitivity and recovery. This method has been compared with another one (Immuno RadioMetric Assay). Also reference values have been calculated in the "normal" male and female population and shortly commented.     

Dissociation of thyrotropin and prolactin responsiveness to thyrotropin releasing hormone stimulation in L-dopa treated parkinsonian patients

The hPRL, hTSH and T3 response to thyrotropin releasing hormone (TRH) stimulation (200 microgram i.v.) were studied in 8 parkinsonian patients under chronic L-dopa-carbidopa therapy. In 6 out of the 8 patients studied, treatment was stopped for a period of 2 weeks and the TRH stimulation test was repeated under similar experimental conditions. In the L-dopa-carbidopa treated patients basal hTSH levels and the hTSH response to TRH were significantly suppressed. By contrast, in the 6 patients 2 weeks after cessation of treatment, although basal hTSH levels were still suppressed, a normal hTSH response to TRH was observed. Neither the basal T3 and T4 concentrations, nor the T3 response to TRH were affected by the L-dopa-carbidopa treatment. In addition, basal hPRL levels as well as the hPRL: response to TRH were within the normal range in the two groups of patients studied. Our study provides further support for a dopaminergic inhibitory action on the hypothalamo-hypophyseal-thyroidal axis (HHTA). The inhibition of basal hTSH secretion and th hTSH response to TRH by L-dopa, suggest that the blocking action of dopamine is exerted at the hypothalamic as well as at the pituitary level. In our hands, chronic administration of L-dopa did not affect either tonic hPRL secretion of the hPRL response to TRH. The dissociation or response to TRH under the same inhibitory action of dopaminergic stimulation can be interpreted as demonstrating a greater sensitivity of the pituitary thyrotrophs, than the prolactin secreting cells, to the blocking effect of dopamine.     

Fractionation of polyclonal antibody by isoelectric focusing--differences in cross-reactivity and affinity of rabbit clonotype anti-human thyrotropin antibody

Immunoglobulin G (IgG) fractions prepared from three different batches of rabbit antihuman thyrotropin (hTSH) antisera were fractionated by agarose isoelectric focusing (IEF) in the pH ranges 3 to 10 and 5 to 8. Staining of protein in agarose gel after IEF showed that polyclonal IgG separated into more than 20 protein bands with isoelectric points (pIs) ranging from 6 to 9. The clonotype antibodies to hTSH were recovered from the fractions and subjected to radioimmunoassay for determination of the binding-affinity for hTSH and the cross-reactivity with human chorionic gonadotropin (hCG). The affinity constants of the antibodies recovered ranged from 6.4 X 10(9) M-1 to 3.1 X 10(10) M-1, and the cross-reactivities of the clonotype antibodies differed greatly. A good correlation was observed between the pIs of antibody molecules and their cross-reactivities: antibodies with higher pIs bound hCG more strongly than those with lower pIs. The correlation coefficients between the pIs and cross-reactivities were 0.83, 0.84, and 0.87 in three batches of antibody.     

The role of the carboxyl-terminal 6 amino acid extension of human TSH beta subunit

The nucleotide sequence of human thyroid stimulating hormone (hTSH) gene can encode a protein of 138 amino acids. However, the mature polypeptide is lacking 6 amino acids of the carboxyl-terminus (C-terminus), suggesting posttranslational cleavage of these residues. To analyze a possible function of these 6 amino acids, we expressed two hTSH beta cDNAs with or without the 6 codons for C-terminal extension, together with alpha subunit cDNA in CHO cells, and determined the amino acid sequence of C-terminus of hTSH beta. hTSH beta propeptides without C-terminal extension were glycosylated, associated with alpha subunit and secreted, as normal propeptides were, and its heterodimer with alpha subunit showed normal TSH bioactivity in FRTL-5 bioassay. These data indicate that the 6 amino acid C-terminal extension is not necessary for the hTSH maturation in the process of the biosynthesis and for its bioactivity.     

Circulating concentrations of calcitonin gene-related peptide (CGRP) in normal man determined with a new, highly sensitive radioimmunoassay

A highly sensitive and specific radioimmunoassay for human calcitonin gene-related peptide (CGRP) has been developed and used for determination of serum CGRP levels in 232 normal individuals, 123 females and 109 males, range of age 18 to 79 years. Mean serum concentration was 36.3 pmol/l +/- 6.2 (SD), and serum levels were unrelated to age and sex. The specificity of the assay was confirmed by gel chromatography, followed by HPLC analysis of extracts from normal serum using synthetic alpha- and beta-CGRP as standards.     

Radioimmunoassay for estimation of thyroglobulin in human serum.

A specific double antibody radioimmunoassay has been develop for the measurement of thyroglobulin in human serum. Human thyroglobulin was purified by combined DEAE-cellulose and affinity chromatography using Sepharose 4B-bound Concanavalin A. Sensitivity of test serum was 10 ng/ml. Thyroglobulin was not detectable in half of normal subjects, and half showed values between 10 and 180 ng/ml. In the patients with simple goiter and secondary hypothyroidism, serum thyroglobulin was usually in the normal range. In Hashimoto's thyroiditis, many sera having precipitating antibodies or high hemagglutination antibodies for thyroglobulin showed a high thyroglobulin concentration in serum probably due to a false positive reaction. In hyperthyroidism, an increased thyroglobulin level was observed in 64% of patients. However, there was no correlation between serum thyroglobulin and thyroxine levels in untreated hyperthyroidism. Serum thyroglobulin was increased significantly in some cases for several weeks after isotope therapy for the hyperthyroidism.     

Monoclonal antibody approach to the relationship between immunological structure and biological activity of thyrotropin

In order to locate the domains involved in the biological activity of TSH and to get some insight in the relationship between immunological and biological properties of TSH, 24 monoclonal antibodies (mAb) to 11 different antigenic regions of hTSH were tested for both binding to hTSH and inhibition of hTSH stimulation of adenylate cyclase in human thyroid membranes. These mAb were also investigated for binding to bovine TSH (bTSH), and interference with bTSH binding to the receptor and stimulation of adenylate cyclase. Radioiodinated human TSH (hTSH) was incubated with increasing concentrations of mAb. Maximum hTSH binding by the various mAb ranged from 15-75% and was not related to the apparent affinity of the mAb for hTSH. Maximum inhibition by the mAb of hTSH stimulation of adenylate cyclase ranged from 3-92%. As compared to the antigenic map of hTSH, it was observed that mAb reacting with the same antigenic regions might display varying inhibition of hTSH. Nevertheless, it was clearly shown that the most potent inhibitors of hTSH stimulatory activity interacted with epitopes located on the alpha- and beta-subunits or expressed only by holo hTSH. Only 11 of the 24 mAb cross-reacted significantly with bTSH. Seven exhibited the same inhibition of hTSH and bTSH stimulatory activity; the four remaining mAb rather than to inhibit adenylate cyclase stimulation as observed with hTSH, did not interfere or even increased adenylate cyclase stimulation by bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of the human thyrotropin beta-subunit gene and transient expression of biologically active human thyrotropin after gene transfection

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.     

A newly identified TSHβ splice variant is involved in the pathology of Hashimoto’s thyroiditis

Thyrotropin (TSH) is a protein that plays a key role in the control of thyroid function. TSH consists of a common α-subunit and a unique β-subunit; the latter is responsible for hormone specificity. A novel splice variant of human TSHβ was identified in 2009. To date, only the tissue distribution of the human TSHβ splice variant mRNA has been studied. Therefore, we aimed to characterize the protein translated from this splice variant. Salting-out, dialysis and concentration of serum proteins were followed by immunoprecipitation to identify the hTSHβ splice variant in serum. Stable CHO cell lines expressing the hTSHβ splice variant and V5-hTSHα were generated. After co-culture, co-immunoprecipitation was used to determine if the hTSHβ splice variant can dimerise with TSHα. We showed for the first time that the hTSHβ splice variant exists in human serum and dimerises with TSHα. To explore the relationship between the TSHβ splice variant and the pathogenesis of autoimmune thyroiditis, we assessed variations in the mRNA expression of the TSHβ splice variant in the peripheral blood leukocytes (PBLs) of Hashimoto’s thyroiditis (HT) patients using quantitative RT-PCR. We found that the mRNA expression levels of the TSHβ splice variant were higher in the PBLs of HT patients who were not undergoing prednisone therapy (n?=?10, P?<?0.0001) and in the PBLs of HT patients with a longer duration of illness (>18?months) who were undergoing prednisone therapy (n?=?5, P?=?0.023) than in those of the control group. This pattern was reversed in the PBLs of HT patients with a shorter duration of illness (<9?months) who were undergoing prednisone therapy (n?=?8, P?<?0.0001). Dexamethasone inhibition of the TSHβ splice variant mRNA expression occurred in a dose- and time-dependent manner. These results demonstrated that the TSHβ splice variant may participate in the pathogenesis of HT.     

III. Serum and sputum immunoglobulin E levels in respiratory diseases in adults

Using a solid-phase radioimmunoassay, serum IgE level was determined in 46 normal subjects, 53 patients with bronchial asthma, 44 patients with chronic bronchitis and / or emphysema, and 19 patients with restrictive lung disease. Sputum IgE was measured simultaneously in 51 of the subjects. The range of serum IgE concentration in the normal subjects was wide. It varied between 15 and 750 ng/ml with a mean of 135 ng. Asthmatic patients had significantly higher levels of serum IgE with a mean of 579 ng/ml, but only 30% fell outside the normal 95% confidence limits. Patients with chronic bronchitis, emphysema and restrictive lung diseases had normal IgE levels. There was a significant correlation between serum and sputum IgE levels.     

Thyroglobulin levels in serum and saliva of patients with differentiated thyroid carcinoma

Serum thyroglobulin levels in 39 patients with differentiated thyroid carcinoma and in 10 healthy volunteers were studied by radioimmunoassay. Sera from two of these patients were analysed preoperatively. Both of these sera showed thyroglobulin levels higher than that obtained from normal individuals. Serum thyroglobulin levels of 10 normal subjects varied between 1·0 to 20·0 ng/ml, Thirteen patients who were in remission showed serum thyroglobulin levels between 0·1 to 18.5 ng/ml which is within the normal range. Patients with bone metastasis had elevated serum thyroglobulin levels while those with lung metastasis had normal serum thyroglobulin levels. Salivary secretion from normal subjects showed thyroglobulin levels between 0·8 to 7·0 ng/ml., while that from thyroid cancer patients ranged between 0.4 to 27.5 ngjml, It appears that salivary thyroglobulin is atpari passu with serum thyroglobulin levels.     

Production of human thyroid-stimulating hormone in Chinese hamster ovary cells

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.     

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.     

Radioimmunoassay of Triiodothyronine in Unextracted Human Serum

Serum triiodothyronine (T-3) concentrations have been estimated by radioimmunoassay using unextracted serum. The serum T-3 concentrations have been shown to be similar in two separate European populations (0·76-1·67 ng/ml). Raised T-3 values have been observed in all subjects with hyperthyroidism. Low values are seen in hypothyroidism although there is some overlap with the normal range. There is a good correlation between serum T-3 and serum thyroxine (T-4) concentrations, and estimation of T-3 seems likely to prove a practical and reliable test of thyroid function.     

Influence of a Reduced CO<Subscript>2</Subscript> Environment on the Secretion Yield,Potency and N-Glycan Structures of Recombinant Thyrotropin from CHO Cells

A consistent increase of approximately 60% in the secretion yield of CHO-derived hTSH was observed by changing cell culture CO2 conditions from 5% CO2 to an air environment. The overall quality of the products obtained under both conditions was evaluated in comparison with a well-known biopharmaceutical (Thyrogen). The N-glycans identified were of the complex type, presenting di-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing approximately 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more di-antennary structures obtained in the absence of CO2 and 7-9% more tri-antennary structures in its presence. No remarkable difference in charge isomers was observed between the three preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39-7.35 pI range. A considerably different distribution, with more forms in the acidic region, was observed, however, for two native pituitary preparations. All recombinant preparations showed a higher in vivo bioactivity when compared to native hTSH. Different production processes apparently do not greatly affect N-glycan structures, charge isomer distribution or bioactivity of CHO-derived hTSH.     

Tyrosinase of hamster melanoma: its purification and estimation by radioimmunoassay

1. Tyrosinase was purified from melanosomal fraction of hamster melanoma. 2. A radioimmunoassay was developed to quantitate the tyrosinase protein in hamster serum and hamster melanoma tissue using polyclonal anti-tyrosinase antibodies and 125I-labeled enzyme. 3. The serum tyrosinase levels were found to be about 0.24 micrograms and 1.14-4.48 micrograms/ml in normal hamsters and melanoma-bearing hamsters, respectively. 4. Tyrosinase protein in serum correlated significantly with the enzyme activity in hamsters with melanoma (r = 0.733). 5. In the cytosol fraction of hamster melanoma, a level of 2.2 micrograms of tyrosinase/mg protein was determined. 6. The usefulness and possible applications of the tyrosinase radioimmunoassay are discussed.     

Bioluminescent immunoassay of thyrotropin and thyroxine using obelin as a label

Solid-phase bioluminescent immunoassay of thyroid hormones, human thyrotropin (hTSH) and two forms of thyroxine (T4), whose determinations are vitally important for diagnostics of thyroid diseases and the efficiency of treatment, is described. The recombinant obelin, a Ca(2+)-regulated photoprotein originally derived from the luminous marine hydroid Obelia longissima, is employed as a bioluminescent label. To produce obelin conjugates with anti-hTSH, anti-T4 immunoglobulins (IgG), and T4, additional SH groups are introduced into the obelin molecule using Traut's reagent (2-iminothiolane) and then obelin possessing extra SH groups is conjugated with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate-activated IgGs or T4. The total yield of obelin conjugates determined by luminescent activity is 60-65% after all chemical and purification procedures. The obtained conjugates are stable to lyophilization and in solution for at least 9 months at 4 degrees C, with loss of activity not exceeding 10%. The application of obelin conjugates for determination of the hTSH, total T4, and free T4 in standard, control, and patient sera displays high sensitivity and reproducibility of results. The results of bioluminescent immunoassays are closely comparable to those obtained by the radioimmunoassay method (R=0.95-0.99).     

Radioimmunoassay of ursodeoxycholic acid in serum

A sensitivie and specific radioimmunoassay for the measurement of serum ursodeoxycholic acid has been developed. Ursodeoxycholic acid bound to bovine serum albumin was used as an antigen, and antiserum to this antigen was raised in the rabbit. [11, 12-3h2]Ursodeoxycholic acid was used as the radioactive tracer, and the radioimmunoassay was carried out by the method of Simmonds et al. (1973. Gastroenterology. 65: 705-711). The percentage of bound radioactivity decreased linearly with a logarithmic increase in unlabeled ursodeoxycholic acid from 10 to 200 pmol. The antiserum showed extremely high specificity for ursodeoxycholic acid (free and conjugated), and the values determined by radioimmunoassay indicated a close correlation with those found by gas-liquid chromatography. In normal Japanese subjects, a small amount of ursodeoxycholic acid in serum was detected, and the level was detected, and the level was 0.15 +/- 0.11 nmol/ml. This convenient radioimmunoassay will provide useful information about the metabolism of ursodeoxycholic acid in man.