Glycoprotein hormone isomorphism and assay discrepancy: the paradigm of luteinizing hormone (LH).
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Carbohydrate Sequence↗
Biomedical subjects
S Costagliola
Publications and source records attributed to S Costagliola.
Recombinant thyrotropin receptor and the induction of autoimmune thyroid disease in BALB/c mice: a new animal model.
In a preliminary study, we observed the production of TSH binding-inhibiting (TBII) and thyroid-blocking (TBAb) antibodies accompanied by lymphocytic infiltration of the thyroid in a pool of male BALB/c mice immunized with the extracellular domain (ECD) of the human TSH receptor (TSHR) expressed as a maltose-binding protein (MBP) fusion in bacteria. In the present study we evaluated the humoral response to the same antigenic preparation in a new series of individual male and female BALB/c mice immunized ip on day 0 with 100 micrograms MBP-ECD and days 25, 39, and 53 with 50 micrograms MBP-ECD in an adjuvant composed of aluminum oxide, magnesium hydroxide, and Bordetella pertussis vaccine. Mice immunized with MBP served as control. Individual sera and immunoglobulins were tested for TBII, TBAb, and thyroid-stimulating antibodies (TSAb) on days 0, 32, 46, and 60, and total circulating T4 levels were measured by RIA. Animals were killed on day 120, their thyroids were examined histologically, the infiltrates were characterized using monoclonal antibodies specific for T-cells (total, activated, helper, and suppressor), B-cells, and macrophages. Sera and immunoglobulins G of the MBP-treated control group were all negative for TSAb, TBAb, and TBII activity. The receptor-immunized mice, despite having high titers of antibodies to the immunogen in an enzyme-linked immunosorbent assay, displayed a heterogeneous response in terms of biological activity, with 3 of 7 female and 4 of 8 male mice having TBAb/TBII activities that persisted and whose activity increased throughout the experiment. No significant TSAb antibody activity was observed. Total T4 levels were also heterogeneous even before immunization, but 9 of 15 MBP-ECD-treated mice had levels below the normal range after immunization, and 7 of these also had TBII/TBAb activities. At the end of the experiment, only 4 of the MBP-ECD-treated female mice survived, but all of them had a severe lymphocytic infiltration of their thyroid, composed mostly of activated T-cells, although B-cells and macrophages were also present. A similar infiltrate was seen in 4 of 8 male MBP-ECD-treated mice. No infiltrate was observed in male or female MBP-treated mice. The model described demonstrates the feasibility of using the TSHR as an immunogen to overcome tolerance and mimics some characteristics of human autoimmune disease of the thyroid.
Animals↗
Overexpression of the extracellular domain of the thyrotrophin receptor in bacteria; production of thyrotrophin-binding inhibiting immunoglobulins.
The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP-Binding Cassette Transporters↗
Molecular genetics of the thyrotropin receptor.
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Autoimmunity↗
Recombinant TSH-receptor for determination of TSH-receptor-antibodies.
We have expressed the complete coding sequence of the human TSH-R in eucaryotic cells. Limiting dilution enabled us to select two clones, JP09 and JP26, which have formed the basis of binding and bioassays respectively, for TSH-R antibodies. Results obtained in the binding assay correlated well with those obtained in the TRAK (Henning Berlin) assay, while the bioassay was at least as sensitive as measurements made with FRTL5 or human thyroid cells. These cell lines provide a reliable source of human TSH-R which will be useful in routine diagnosis.
Animals↗
Binding assay for thyrotropin receptor autoantibodies using the recombinant receptor protein.
We have characterized a transfected Chinese hamster ovary cell line, JP09, which expresses high levels of the human TSH receptor (TSH-R). Based on a theoretical biological activity for TSH of 40 IU/mg, JP09 has approximately 90,000 receptors per cell, having a dissociation constant of 1.64 x 10(3) mU/L or 1.47 x 10(-9) mol/L. We have used JP09 to prepare solubilized TSH-Rs which have formed the basis of a binding assay for thyroid-binding inhibiting immunoglobulins in unfractionated sera. We have compared the JP09 assay with the TRAK assay (which is based on solubilized porcine TSH-R) and found a highly positive correlation between the two assays, r = 0.83 P < 0.0001, in 55 sera from patients with autoimmune thyroid disease. JP09 can be adapted to growth in suspension culture, permitting large scale production. The tracer in the assay is bovine [125I]TSH; surprisingly, despite the use of a hTSH-R, hTSH had no effect on the binding of the tracer up to 10(3) mU/L and only a minor effect at 10(4) mU/L.
Animals↗
Monoclonal antiidiotypic antibodies interact with the 93 kilodalton thyrotropin receptor and exhibit heterogeneous biological activities.
Ten antiidiotypic monoclonal antibodies (AI mAb) reacting with the TSH receptor were produced by immunization of mice with a mouse mAb directed to a TSH epitope involved in the binding of the hormone to the receptor. The AI mAb were tested for their effects on the TSH receptor-adenylate cyclase system. Four AI mAb behave as agonists of TSH as they compete with TSH for binding to the receptor and stimulate the adenylate cyclase activity. Five AI mAb inhibited both TSH binding to the receptor and adenylate cyclase activity in the presence and absence of TSH; they were thus considered as antagonists of the hormone. One mAb inhibited only slightly the binding of TSH to the receptor and did not interfere significantly with the adenylate cyclase activity. The 10 AI mAb bound with various apparent affinities to solubilized thyroid membrane proteins but not to kidney, spleen, and liver membranes. Membrane desialylation did not significantly alter the binding of the AI mAb whereas deglycosylation modified more or less strongly the binding of mAb to thyroid membranes. It was also shown that the binding to solubilized thyroid membranes of the 10 mAb was inhibited by anti-TSH receptor autoantibodies present in patients with autoimmune disease. The mAb were further analyzed by Western blot analysis. One mAb did not react with the antigen, which suggested that it was directed to a conformational epitope. The other 9 mAb reacted with a protein of 70 kilodaltons and 5 revealed another band of 93 kilodaltons in accordance with the TSH receptor size deduced from the recently molecular cloning.
Adenylyl Cyclases↗
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)
Adenylyl Cyclases↗
Clinical usefulness and limitations of serum thyrotropin measurement by 'ultrasensitive' methods. Comparisons of five kits.
Five different ultrasensitive thyrotropin (TSH) assay kits (Boots-Celltech, Immunotech, ORIS-CIS, Travenol and Boehringer) have been used for TSH measurements in various conditions. All the kits were based on an immunometric method but differed with regard to components and procedure. The sensitivity appeared essentially the same for the five kits (0.10 microU/ml) as well as the intraassay precision (coefficient of variation less than 12%). In contrast, the interassay coefficients of variation in the low TSH range varied from 12.8 to 21.3%. Discrepancies from kit to kit were observed and accounted for by differences in the components and procedure of the kits. Basal serum TSH was determined in normal subjects (n = 261) and in patients with thyroid dysfunction (n = 392). No overlap was shown between normals and patients with overt hypothyroidism. In contrast, an overlap existed between normals and hyperthyroids for all the kits but one. Measurements in patients with nontoxic goiter showed that TSH may be undetectable in clinically euthyroid patients, whatever the kit used. After TRH stimulation, 95% of the 375 patients tested associated either an absence of response to TRH with undetectable basal TSH values, or a blunted response with low basal TSH levels or normal response with normal basal TSH concentrations. However, 9 patients with suppressed TSH showed a response to TRH and 7 patients with normal basal TSH levels presented an exaggerated response to TRH. Taken together, these results demonstrate that even though ultrasensitive measurements of TSH do not meet the expectation of completely discriminating euthyroid from hyperthyroid patients, ultrasensitive TSH assay kits represent a powerful tool in the diagnosis of thyroid dysfunction, which would eliminate, in most instances, the need for TRH test and diminish thyroid hormone assay requests.
Adolescent↗
Monoclonal antibody mapping of the antigenic surface of human thyrotropin and its subunits.
Although the amino acid sequence of the alpha- and beta-subunits of glycoprotein hormones in various species has been deciphered, data on their tertiary structure are not abundant. This impedes correlation between structure and function. The availability of monoclonal antibodies to human TSH (hTSH) offers the opportunity to enumerate the antigenic determinants present on the surface of hTSH and its subunits and to examine their spatial relationships. Twenty-eight monoclonal antibodies to hTSH were obtained from several fusions, and screens carried out separately in the laboratories involved in this study. Affinities for hTSH ranged from 10(8)-10(11) M-1. Cross-reactivity with bovine TSH (bTSH), human gonadotropins (hLH, hFSH, and hCG), and the alpha- and beta-subunits of hTSH distinguished 10 groups of monoclonal antibodies (mAb) according to their main cross-reactions: 1) hTSH alpha, hLH, hFSH, and hCG; 2) hTSH alpha, bTSH, hLH, hFSH, and hCG; 3) hFSH; 4) bTSH and hFSH; 5) bTSH, hLH, and hFSH; 6) bTSH, hLH, hFSH, and hCG; 7) hTSH beta; 8) hTSH beta and bTSH; 9) hTSH beta and hFSH; and 10) hTSH beta, hLH, hFSH, and hCG. mAb were incorporated into 2-site binding assays to probe hTSH by a 28 X 28 matrix, the free alpha-subunit by a 4 X 4 matrix, and the free beta-subunit by a 18 X 18 matrix. Regarding intact hTSH, 12 different clusters of mAb were distinguished and interpreted as reflecting 12 distinct antigenic regions on the surface of the hTSH molecule. Two of them were localized on the alpha-subunit, and 6 on the beta-subunit; 4 were only expressed by the holo-hormone and, thus were designated conformational antigenic regions (alpha beta). Surface mapping of the free alpha- and beta-subunits was virtually identical to that observed with the holo-hormone. Modification of the operative conditions of mAb reacting only with holo-hTSH shows that they recognize the alpha-subunit, but not the beta-subunit of hTSH. These results indicate that 1) hTSH beta presents epitopes that are evolutionary conserved; 2) hTSH alpha presents several epitopes that are species specific and 2 that are not hormone specific; 3) dissociation of hTSH does not modify the antigenic surface expressed by both subunits when they are associated; and 4) some of the conformational determinants expressed only by holo-hTSH are more likely derived from the alpha-subunit than from the beta-subunit.
Antibodies, Monoclonal↗