Binding characteristics of antibodies to the TSH receptor.

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries.

Thyroid lymphocyte RNA from a Hashimoto patient with high serum levels of autoantibodies to thyroid peroxidase (TPO) was used to construct a phage display antibody library in the phagemid vector pComb3. The library (100,000cfu) encoded IgG1 heavy chains together with kappa light chains. Selection of the phages displaying TPO antibody on TPO-coated ELISA plates yielded a phage population enriched for surface expression of TPO antibody Fabs. 3 different Fabs specific for TPO were subsequently isolated with affinities in the region of 10(9) molar-1. 2 of the Fabs recognised the same, or closely related, epitopes on TPO whereas the third Fab recognised a different epitope. These 2 epitopes were recognised by TPO autoantibodies in the serum of the lymphocyte donor and a series of 10 patient sera. Available sequence data showed that several non-self antibodies and non-thyroid autoantibodies use the same V kappa and VH germline genes as TPO autoantibodies. There appeared to be no clear relationship between gene sequence or gene family usage by TPO autoantibodies of the same or similar epitope specificity.

Cross-reactive idiotypes on high affinity IgG class human monoclonal thyroglobulin autoantibodies.

Anti-idiotypic antibodies have been developed in rabbits against three high affinity IgG class monoclonal human autoantibodies to thyroglobulin (Tg), which resemble polyclonal Tg antibodies in patients with autoimmune thyroid disease. Antibodies to 1E10 monoclonal anti-Tg (IgG2 kappa) recognised a cross-reactive idiotype (CRI) also present on 1D3 monoclonal anti-Tg (IG1 lambda) and on VB5 monoclonal anti-Tg (IgG2 lambda). The determinant to which anti-1E10 binds appears to involve, at least in part, the binding site for Tg. In contrast, anti-idiotypic antibodies raised against VB5 failed to bind to either 1E10 or 1D3, a finding consistent with previous studies on serum polyclonal Tg antibodies, which suggested that such antibodies exhibit a mixture of private and cross-reactive idiotypes. The observed sharing of idiotypic determinants was not related to subclass, light chain type or expression of a particular VH gene family in the heavy chain. Although binding of the anti-idiotypic antibodies to Tg antibodies in a panel of patients (including the donors of the lymphocytes used to produce the monoclonal antibodies) could not be detected, the monoclonal antibodies are representative of the donor patients' serum Tg antibodies, both in terms of IgG subclass and functional affinity. Thus these idiotypes may be present in patients' sera at levels below the detection limits of the assays employed. Cross-reactive regulatory idiotypes in mice often constitute a minor component of the anti-Tg repertoire. Consequently, it is possible that low levels of a CRI, such as the 1E10 CRI, may be involved in the regulation of the autoimmune response to Tg in man.

Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies.

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.

Human monoclonal thyroglobulin autoantibodies of high affinity. II. Interaction between thyroglobulin and thyroglobulin autoantibodies of different IgG subclasses.

The interaction of human thyroglobulin (Tg) autoantibodies of different IgG subclasses with Tg was investigated using four high affinity human monoclonal thyroglobulin (Tg) autoantibodies, secreted by human-mouse hybridomas, of subclasses IgG1 (kappa and lambda) and IgG2 (kappa and lambda) and an IgG4 kappa serum monoclonal Tg antibody. With exception of a low level of interference in binding between one IgG1 lambda Tg antibody and one IgG2 kappa Tg antibody (27% decrease), binding by human monoclonal Tg antibodies of one IgG subclass was unaffected by pre-incubation of 125-I Tg (or Tg on an ELISA plate) with a human monoclonal Tg antibody of a different IgG subclass. Furthermore, preincubation of Tg-coated ELISA plates with an IgG1 human monoclonal Tg antibody had little effect on binding to Tg by IgG2, IgG3 and IgG4 Tg antibodies present in the sera of 6 Hashimoto patients. Comparable observations were made using an IgG2 monoclonal Tg antibody and serum Tg antibodies of subclasses IgG1, IgG3 and IgG4. Binding of an IgG1 kappa Tg antibody was inhibited (> 80%) by pre-incubation of Tg with an IgG1 lambda Tg antibody derived by fusion of lymphocytes from the same Hashimoto patient. In contrast, pre-incubation of Tg with an IgG2 kappa Tg antibody had little effect on subsequent binding by an IgG2 lambda Tg antibody derived from lymphocytes of a different Hashimoto patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between thyroid autoantibody spectrotype and IgG subclass.

The relationship between spectrotype and IgG subclass of autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO) has been investigated using sera from Hashimoto and Graves' patients. Isoelectric focussing (IEF) was carried out in gels over the range pI 3.5-9.5 followed by transblotting to nitrocellulose and probing the filters using 125I-labelled TPO and Tg. As has been shown previously for Tg antibodies, TPO antibody focussed over different pI values in different patients but the spectrotypes for individuals were constant over 2-5 years. Further, the pI values for TPO and Tg autoantibodies appeared to be related to IgG subclass, for example IgG1 Tg antibodies tended to focus nearer the cathode (pI 8.0-9.5) than IgG4 antibodies (pI 6.5-8.5) while antibodies of subclasses IgG1 + 2 + 4 produced spectrotypes covering a broad pI range (5.7-9.5). Consequently, it seems likely that the characteristic spectrotypes described by others for Tg autoantibodies, and those we now report for TPO antibodies, reflect the IgG subclass "fingerprints" which we suggest may be a measure of the ability of an individual to respond to different epitopes on these two thyroid antigens.

Potential role of PHA in producing human monoclonal thyroid autoantibodies of different subclasses.

A human monoclonal autoantibody to thyroglobulin (Tg) of subclass IgG2 was developed by fusing a mouse myeloma with Tg antibody secreting Epstein-Barr virus (EBV)-infected B lymphocytes from a Hashimoto patient. Subsequent studies showed that EBV-infected B lymphocytes from this patient synthesized IgG2 Tg antibody while unfractionated blood lymphocytes cultured with pokeweed mitogen secreted IgG1, IgG2, and IgG4 Tg antibodies in amounts proportional to those present in the patient's serum. To investigate this discrepancy further, we cultured EBV-infected lymphocytes from blood, lymph nodes, and thyroid tissue in medium alone and with increasing concentrations of PHA. In individuals with thyroid autoantibodies predominantly of subclass IgG1, PHA enhanced the levels of total Tg antibody synthesis without affecting the IgG subclass distribution. However, in patients with serum autoantibodies of subclasses IgG1, 2, and 4, the increased levels of total Tg antibody synthesis were associated with increased amounts of thyroid autoantibodies of all of these subclasses; in some instances IgG1 and IgG4 autoantibodies were only synthesized in cultures containing PHA. These observations suggest that addition of the T-cell mitogen PHA to cultures of EBV-infected lymphocytes may ensure activation of B-cell precursors committed to synthesizing the IgG subclasses characteristic of serum antibody in the lymphocyte donor. Since Tg antibodies of subclasses IgG2 and IgG4 recognize different epitopes on Tg, the ability to produce human monoclonal antibodies of different IgG subclasses may simultaneously ensure the development of antibodies to different epitopes on the same antigen.

Human thyroglobulin autoantibodies of subclasses IgG2 and IgG4 bind to different epitopes on thyroglobulin.

IgG autoantibodies to thyroglobulin (Tg) in the serum of patients with autoimmune thyroid disease only recognize a very limited number of epitopes, probably between four and six (Nye, Pontes De Carvalho & Roitt, 1980) on the large Tg molecule (660,000 MW), but attempts to characterize the epitopes have been unsuccessful so far (Male et al., 1985). The distribution of Tg autoantibodies between the IgG subclasses also tends to be restricted and individual patients possess characteristic 'fingerprints' of high affinity IgG1 and/or IgG4 Tg antibodies with smaller amounts of IgG2 Tg antibody (McLachlan et al., 1987, 1988). We have therefore investigated the possibility that Tg autoantibodies of different IgG subclasses interact with different epitopes on Tg.

A human-mouse hybridoma which secretes monoclonal thyroglobulin autoantibody with properties similar to those of the donor patient's serum autoantibody.

Human monoclonal antibodies produced by Epstein Barr (EB) virus transformation and/or cell fusion are frequently IgM antibodies which tend to cross react with a range of antigens and often bear little relationship to the highly specific IgG antibodies associated with human autoimmune disease. By fusing EB virus transformed B lymphocytes from a Hashimoto patient with a mouse myeloma line and selecting for synthesis of IgG class thyroglobulin (Tg) antibody, we have developed a hybridoma (VB/5) secreting Tg antibody of IgG2 subclass and lambda light chain type which has the characteristics of a monoclonal antibody on isoelectric focussing. The antibody has a high affinity for human Tg and recognises Tg from other primates but not non-primate Tg. However, it does not react with human thyroid peroxidase or a panel of other autoantigens. In terms of affinity constant, functional affinity and affinity heterogeneity, the antibody closely resembles the IgG2 lambda Tg antibody present in the serum of the Hashimoto patient whose B lymphocytes were used to develop the hybridoma. In addition to providing a useful reference standard for Tg antibody IgG subclass assays, VB/5 antibody and the hybridoma line provide a valuable starting point for detailed studies of Tg autoantibodies and the genes coding for the variable regions of their heavy and light chains.

Photoaffinity labelling of the TSH receptor on FRTL5 cells.

An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.

IgG thyrotrophin receptor antibody activity in Graves' disease; a study of TSH agonist and antagonist activities by isoelectric focusing.

The distribution of TSH receptor antibody activity in the 7S and 19S fractions of Graves' sera has been re-evaluated. Serum fractions were obtained by gel filtration from 12 Graves' sera and assayed for TSH receptor binding activity in a radioreceptor assay. Thyroid stimulating activity was determined in a cultured porcine thyroid cell bioassay. In apparent contrast to the findings of Baker et al. (1983) TSH receptor binding activity was confined to the 7S gel filtration fraction, containing IgG, and was not detected in the 19S fraction, containing IgM. Similarly thyroid stimulating activity was detected only in the 7S fraction. 7S fractions from seven Graves' sera were fractionated by isoelectric focusing and the fractions analysed for TSH receptor binding activity and TSH agonist and antagonist activities. Five of the IgGs showed TSH agonist activity and in all five, the peak thyroid stimulating activity (measured by stimulation of cyclic AMP release from isolated porcine thyroid cells) was in fractions with a pI of between 8.0 and 9.5. In four of these five IgGs, TSH receptor binding activity showed similar isoelectric distribution to the thyroid stimulating activities. High levels of TSH receptor binding activity without associated TSH agonist or antagonist activity were however observed in some isoelectric fractions of the fifth stimulating Graves' IgG studied. All the isoelectric fractions from the fifth IgG with thyroid stimulating activities contained TSH receptor binding activity. Two of the Graves' IgGs showed TSH antagonist activity and both the TSH receptor binding and TSH antagonist activities of these IgGs showed similar isoelectric distribution with the peak activities at a pI of around 9.0. Consequently, it was not possible to separate TSH agonist or TSH antagonist activities from TSH receptor binding activity in seven Graves' sera by isoelectric focusing although in one IgG several isoelectric fractions contained isolated receptor binding activity. These findings are in keeping with the hypothesis that the biological activities of Graves' IgGs are intimately related to their ability to bind to the TSH receptor.

Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor.

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves' serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes' radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

Thyrotropin receptor antibodies.

The thyrotropin (TSH) receptor is an integral membrane protein which contains 2 subunits linked by a disulphide bridge. The A subunit (mol. wt. 50,000) is water soluble and forms the binding site for TSH, whereas the B subunit (mol. wt. 30,000) penetrates the lipid bilayer and probably forms the site for interaction with adenylate cyclase. Autoantibodies to the TSH receptor are found in the sera of patients with Graves' disease. The antibodies bind to the same region of the receptor's A subunit as TSH and usually act as TSH agonists, causing hyperthyroidism. Occasionally, TSH receptor autoantibodies are found which can act as TSH antagonists. Isoelectric focusing and binding studies indicate that these antibodies also bind to the same region of the receptor A subunit as TSH.

Solubilization and partial characterization of human and porcine thyrotrophin receptors.

Thyrotrophin (TSH) receptors have been extracted from human and porcine thyroid membranes by treatment with Triton X-100. 125I-Labelled bovine TSH was used to monitor receptor activity. Analysis by gel filtration and electrophoresis on acrylamide gels containing sodium dodecyl sulphate suggested that Triton extracts of human thyroid membranes contained TSH receptors with a molecular weight in the region of 50 000 closely associated with Triton micelles of approximate molecular weight 300 000. Isoelectric focusing studies indicated that the Triton-solubilized TSH binding activity had an isoelectric point of pH 4--4.5. The soluble TSH receptors were heat-labile, showed optimum TSH binding at pH 7.4 and reduced hormone binding at high ionic strength. The TSH binding characteristics of membrane-bound and solubilized human TSH receptors were similar and both preparations gave curved Scatchard plots. Solublized porcine TSH receptors appeared to have a similar molecular weight to the human receptors and were also closely associated with Triton micelles of approximate molecular weight 300 000. Scatchard analysis of TSH binding to membrane-bound or solubilized porcine TSH receptors gave approximately linear plots with association constants of 2.8 +/- 0.95 (S.E.M.) X 10(9) and 1.7 +/- 0.27 x 10(9) l/mol respectively. Comparison of the binding capacities of the solubilized and membrane-bound porcine receptors indicated that the 0.5% Triton extracts contained 40% of the original TSH binding activity and that this was present at a concentration of 25 ng/ml.

The secretion of thyrotrophin with impaired biological activity in patients with hypothalamic-pituitary disease.

We describe here two patients with hypothyroidism due to pituitary-hypothalamic disease in whom basal thyrotrophin (TSH) levels measured by radioimmunoassay (RIA) were elevated yet when measured by a cytochemical bioassay (CBA) were found to be normal. This finding and the absence of the normal rise of thyroid hormones in response to thyrotrophin-releasing hormone (TRH) mediated release of TSH confirms for the first time the secretion of TSH with impaired biological activity. Primary thyroid disease as a cause for the elevated immunoreactive TSH was excluded by the absence of circulating thyroid antibodies and by a normal thyroidal radioiodine uptake response to exogenous TSH.