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Endocrinology Vol. 144, No. 5 1718-1725
Copyright © 2003 by The Endocrine Society

The Cysteine-Rich Amino Terminus of the Thyrotropin Receptor Is the Immunodominant Linear Antibody Epitope in Mice Immunized Using Naked Deoxyribonucleic Acid or Adenovirus Vectors

Lise Schwarz-Lauer, Pavel N. Pichurin, Chun-Rong Chen, Yuji Nagayama, Charmaine Paras, John C. Morris, Basil Rapoport and Sandra M. McLachlan

Autoimmune Disease Unit (L.S.-L., P.N.P., C.-R.C., C.P., B.R., S.M.M.), Cedars-Sinai Research Institute and UCLA School of Medicine, Los Angeles, California 90048; Department of Pharmacology 1 (Y.N.), Nagasaki University School of Medicine, Nagasaki 852-8523, Japan; and Division of Endocrinology and Metabolism (J.C.M.), Department of Medicine, Mayo Clinic, Rochester, Minnesota 55902

Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
Experimental Graves’ disease is more effectively produced by immunization approaches involving in vivo TSH receptor (TSHR) expression than by conventional immunization with TSHR protein and adjuvant. Unlike conformational epitopes that are extremely difficult to define, linear epitopes can be readily assessed using synthetic peptides. TSHR linear epitopes are well characterized in conventionally immunized animals, but there is no information for animals vaccinated with TSHR DNA in plasmid or adenovirus vectors. We used synthetic peptides to characterize linear epitopes in mice immunized by in vivo expression of TSHR DNA. TSHR adenovirus-injected mice had higher antibody levels than TSHR DNA-vaccinated mice. However, the dominant peptide recognized in both groups was the TSHR cysteine-rich N terminus (residues 22–41). Sera from TSHR adenovirus-immunized (but not TSHR DNA-vaccinated) mice interacted to a lesser extent with peptides encompassing residues 352–401, which include the region deleted following TSHR cleavage as well as the ectodomain juxta-membrane region. Although antibodies characterized using synthetic peptides are probably TSH blockers or nonfunctional, stimulating antibodies may recognize linear components in a conformational epitope. The cysteine-rich TSHR N terminus is functionally important in the action of stimulating TSHR autoantibodies in humans. The immunodominance of the same region in immunized mice suggests that this region may also be immunodominant in humans.


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
IMMUNIZATION OF MICE with TSH receptor (TSHR) protein together with adjuvant induces high titers of TSHR antibodies. However, these antibodies are generally nonfunctional and do not induce Graves’-like hyperthyroidism (reviewed in Ref. 1). In contrast, disease can be induced in mice by immunizing with TSHR-expressing cells (2, 3), or by vaccination with TSHR DNA in a plasmid (4) or in an adenoviral vector (5). Comparison of the epitopes recognized by the antibodies generated in these diverse systems would be of value in understanding the pathophysiological action of TSHR autoantibodies.

Much information is available on the first-mentioned type of antibodies, namely nonstimulatory TSHR antibodies in mice immunized with TSHR protein and adjuvant. Using TSHR ectodomain synthetic peptides, several investigators (6, 7, 8, 9) identified linear epitopes at the extreme amino terminus of the protein (amino acid residues 22–41, after removal of the 21 residue signal peptide; summarized in Fig. 1Go). Immunodominance of the TSHR N-terminal region was observed with different mouse strains, different TSHR ectodomain preparations (bacterial or baculovirus) and different adjuvants. Hyperimmunized mice also recognized an overlapping peptide (residues 37–56; Refs. 7 and 8). The latter epitope became dominant when mice were immunized with TSHR fragments lacking the first 22 amino acids (10). Sera from some mice also recognized a second epitope encompassing amino acid residues 322–371, albeit to a lesser extent. Early studies referred to this peptide as the TSHR "immunogenic region" (11).



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  		Figure 1. Linear epitopes recognized by sera from TSHR immunized mice. Antibody binding was assessed using a panel of synthetic epitopes (20-mers) corresponding to the sequence of the TSHR ectodomain (ECD; Ref. 17 ). Mice of different strains were immunized with the full-length receptor (amino acids 22–794) expressed in fibroblasts or with preparations of the TSHR ectodomain (amino acids 22–415 or ectodomain fragments) prepared in bacteria or baculovirus administered with adjuvant(s). CT-B, Cholera toxin B subunit; TM, Titer Max.

 
Regarding the new animal models in which stimulatory TSHR antibodies arise and induce hyperthyroidism in some animals (see above), there is little information on the linear TSHR peptides recognized by antibodies from these animals. In the Shimojo model (injection of TSHR-expressing fibroblasts), approximately 25% of mice become hyperthyroid (2, 12, 13, 14). Sera from these animals recognized poorly (if at all) the immunodominant peptide (residues 22–41). Rather, they interacted with peptide 97–116, as well as with peptides spanning residues 322–386 (12). Clearly, this difference between animals immunized conventionally (purified protein together with adjuvant) and by the Shimojo approach is of potential pathophysiological importance. No information is available on TSHR peptide recognition by serum antibodies in the more recent animal models, namely mice vaccinated with TSHR DNA in plasmid or adenovirus vectors.

In the present study, to fill this gap in our knowledge, we examined TSHR peptide recognition by antibodies from mice immunized with TSHR DNA in plasmid and adenovirus vectors. Surprisingly, with both of these forms of DNA immunization, as with conventional immunization, the major epitopic focus was amino acid residues 22–41, the cysteine-rich amino terminus of the TSHR.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Mouse immunization with TSHR DNA plasmid, TSHR adenovirus, or TSHR protein
Female BALB/c or C57BL/6 mice aged 6–8 wk (The Jackson Laboratory, Bar Harbor, ME) were vaccinated with the human TSHR DNA in the vector pcDNA3 (Invitrogen, Carlsbad, CA). As described (15), mice were pretreated in the anterior tibialis muscle with cardiotoxin (100 µl of 10 µM Naja nigricollis, Calbiochem, La Jolla, CA) and injected 5–7 d later in the same muscle with 100 µg (50 µl) TSHR DNA or empty vector cDNA. The vaccination protocol was repeated twice at three weekly intervals and mice were euthanized 1–3 wk after the third vaccination. Immunization with recombinant adenovirus expressing human TSHR (AdCMVTSHR, referred to as TSHR adenovirus) was performed as previously described (6) by im injection three times at 3-wk intervals with 50 µl PBS containing 1011 particles of TSHR adenovirus or, as a control, adenovirus expressing ß-galactosidase (AdCMV-ß-gal). Blood was drawn 1 wk after the second injection and mice were euthanized 8 wk after the third injection. Total T4 levels in serum were measured by RIA (Coat-a-Count, Total T4; Diagnostic Products Corp., Los Angeles, CA). The mean ± 2 SD for control mice was 5.9 ± 2.2 µg/dl (range 3.7–8.1c µg/dl).

Some mice were immunized conventionally using adjuvant and TSHR 289 protein, a variant of the receptor expressed in eukaryotic cells that corresponds to the extracellular A subunit (14). Using TSHR-289 affinity purified from culture medium (16), the following immunizations were performed: 1) Priming with TSHR 289 (50 µg) emulsified in Complete Freund’s Adjuvant (CFA; Sigma, St. Louis, MO) and boosting 3 wk later with same dose in Incomplete Freund’s Adjuvant (IFA; Sigma); and 2) A single injection of TSHR 289 (50 µg) in IFA; 3) Two injections 2 wk apart with TSHR 289 (2 µg) in ImmunEasy Mouse Adjuvant (QIAGEN Inc., Stanford, CA), which contains short pieces of DNA with unmethylated cytosine-guanine dinucleotides. In these experiments, mice were euthanized approximately 4 wk after the final injection.

All animal studies were approved by the local Institutional Animal Care and Use Committee and were performed in accordance with the highest standards of humane care in a pathogen-free facility.

ELISA for recognition of TSHR antigen and TSHR peptides
Serum antibody binding was assessed by ELISA using TSHR 289, as previously described (15). In brief duplicate, serum aliquots (1:200 dilution) were incubated on wells coated with TSHR 289 (1 µg/ml) and antibody binding was detected with antimouse IgG coupled to horseradish peroxidase (Sigma). The signal was developed using o-phenylene diamine + H2O2 and OD read at 490 nm.

Twenty-six peptides corresponding to amino acids in the TSHR extracellular domain were synthesized using an automated 431A peptide synthesizer, HPLC purified and their structures confirmed by mass spectrometry (17). Each peptide was 20 amino acids long (20-mers) and overlapped the subsequent peptide by five residues. As in previous studies, peptides are designated as nos. 1, 2, 3, etc., corresponding to residues 22–41, 37–56, 42–71, etc. We also included three peptides (EC1, EC2, EC3) corresponding to the extracellular loops of the receptor. ELISA wells were coated with the peptides (10 µg/ml) by overnight incubation in 0.035 M NaHCO3, 0.015 M Na2CO3 (pH 9.3). Duplicate aliquots of mouse sera (diluted 1:200) were incubated on the peptides and reactivity developed with antimouse IgG and substrate as above. Peptide assays included TSHR 289-coated wells (as a positive control for potentially negative peptides) and normal mouse serum and mouse monoclonal antibodies (McAb A10, 2C11 and 3BD10; Refs. 18, 19, 20, 21) as negative and positive controls, respectively, for the peptides.


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
TSHR antibody responses
TSHR antibody responses in mice immunized according to different protocols were determined by ELISA using TSHR 289 protein. Strong TSHR antibody responses were detected in BALB/c mice after two injections, and particularly after three injections, of TSHR adenovirus (Fig. 2Go). In contrast, we have previously reported that the majority of BALB/c mice housed in the same pathogen-free facility develop low or undetectable levels of TSHR antibodies after vaccination with plasmid TSHR DNA (15). Serum from one of two rare BALB/c mice with moderately high titers of TSHR antibodies (22) was available for analysis in the present study. Unexpectedly, we found that vaccination of a different murine strain (C57BL/6) with the identical TSHR-DNA-containing plasmid induced moderately high TSHR antibody levels in four of five mice (Fig. 2Go). Very low levels of binding were observed for sera from unimmunized mice or animals vaccinated with control-DNA plasmid or control adenovirus expressing ß-galactosidase.



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  		Figure 2. Antibody responses to the TSHR in mice vaccinated with TSHR DNA in a plasmid or adenovirus vector. Responses were measured after two or three injections (2x or 3x, respectively). Controls (Con) received the plasmid alone (for DNA injections) or adenovirus expressing ß-galactosidase (for adenovirus injections). Also included are data from mice immunized conventionally with TSHR 289 and adjuvant (CFA and/or IFA). IgG class serum binding (dilution 1:200) was assessed using ELISA plates coated with TSHR 289, a recombinant protein produced in mammalian cells corresponding approximately to the shed A subunit of the receptor (21 ). The number of mice studied is in parentheses, and data for groups of three or more mice are presented as mean + SEM. Binding for one of the two BALB/c mice with relatively high levels of TSHR antibodies (22 ) is shown as the mean of duplicate serum aliquots.

 
We also studied mice conventionally immunized with TSHR 289 protein and adjuvant. Comparable high levels of antibody were induced by immunizing BALB/c mice with two consecutive injections of TSHR 289 in IFA or with a single dose of TSHR 289 in IFA (data not shown). We therefore pooled the data for TSHR antibodies in these two groups of mice (Fig. 2Go).

TSHR peptide recognition in TSHR DNA plasmid-vaccinated mice
Peptide no. 1 was the major peptide recognized by antibodies from mice vaccinated with TSHR DNA plasmid (OD values > 1.00; Fig. 3Go, top two panels). This was the case for the four C57BL/6 mice vaccinated with the identical plasmid as well as for the rare BALB/c mouse previously found to have moderately high levels of TSHR antibody (22). Much lower signals (OD values ~0.3) were obtained for several other peptides (for example peptide nos. 17 and 18 in the BALB/c mouse). Incidentally, none of the TSHR DNA plasmid vaccinated had elevated serum T4 levels.



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  		Figure 3. Recognition of TSHR peptides by mice vaccinated with TSHR DNA in a plasmid. IgG class antibody binding was measured using ELISA wells coated with 29 peptides, 26 peptides (20-mers) covering the TSHR ectodomain (peptides 1–26), and 3 peptides corresponding to the extracellular loops (EC1, EC2, EC3). Data are reported as mean + SEM for sera (1:200 dilution) from mice vaccinated three times with TSHR DNA (C57BL/6 mice, n = 4) or with control-DNA (n = 4, pooled data from two BALB/c and two C57BL/6 mice). Peptide recognition is also shown (mean of duplicate aliquots) for serum available from one of two TSHR DNA-vaccinated BALB/c mice with high TSHR antibody binding (22 ). Peptides recognized by TSHR immunized mice are indicated by #1, etc. An arrow indicates the peptide (no. 6) that gave a low but elevated nonspecific signal by sera from all control and immunized mice.

 
IgG class antibodies in sera from control DNA-vaccinated mice interacted very poorly with most TSHR peptides (OD values < 0.1; sera 1:200; Fig. 1Go, bottom panel). However, binding to peptide no. 6 (Fig. 3Go, arrow) was significantly higher than to adjacent peptides (P < 0.01) in all DNA-vaccinated animals regardless of the presence of TSHR DNA.

Positive controls in these assays included several mouse McAb with defined epitopes. As expected, McAb A10 and 3BD10 (18, 21) bound strongly to peptide no. 1 (residues 22–41; OD values > 3.0) and McAb 2C11 (19, 20) bound to peptide no. 23 (residues 352–371; OD value > 1.5). Binding to all other peptides was negative (OD values < 0.3).

TSHR peptide recognition in TSHR adenovirus immunized mice
As in TSHR DNA-vaccinated mice, the major peptide recognized by BALB/c mice vaccinated two or three times with TSHR adenovirus was the amino terminal peptide no. 1 (Fig. 4Go, top two panels). Three of these mice developed hyperthyroidism: T4 levels 9.0, 10.8, and 11.3 µg/dl (mean + 2 SD for control mice 8.1 µg/dl). Of importance, all mice recognized the same, dominant peptide (no. 1). These sera also bound, albeit to a lesser degree, to C-terminal regions of the TSHR ectodomain (peptide nos. 23–25). The magnitude of the error bars reflects variable recognition by individual TSHR adenovirus-vaccinated mice of peptides within this region. Sera from control-adenovirus injected animals (Fig. 4Go, second panel from the bottom) bound at very low levels to all peptides other than no. 6 (arrow) and peptide nos. 25 and 26 and EC1 (one of three mice each binding to one of the three peptides). It should be noted that the maximum binding to peptide no. 25 by control mouse sera (OD 0.60) was much lower than the maximum value observed found for the same peptide in TSHR adenovirus-vaccinated mice (OD > 1.50).



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  		Figure 4. Recognition of TSHR peptides by mice after two or three injections of TSHR adenovirus or control adenovirus. IgG class antibody binding was measured using ELISA wells coated with 29 peptides, 26 peptides (20-mers) covering the TSHR ectodomain (nos. 1–26), and 3 peptides corresponding to the extracellular loops (EC1, EC2, EC3). Data are reported as mean + SEM for sera (1:200 dilution) from BALB/c mice (n = 4) after two or three injections of TSHR adenovirus (2x or 3x, respectively) and after three injections of control adenovirus (n = 3 mice). Also included are the pooled data for BALB/c mice (n = 6) immunized conventionally with TSHR 289 and adjuvant (CFA and or IFA). Peptides recognized by TSHR immunized mice are indicated by no. 1, etc. An arrow indicates the peptide (no. 6) giving nonspecific binding by sera from all control and immunized mice.

 
TSHR peptides recognized by conventionally immunized mice
We compared epitopes of TSHR adenovirus-vaccinated mice with those of mice conventionally immunized with TSHR protein because of their comparable TSHR antibody levels (Fig. 2Go). Despite using a truncated TSHR, animals injected with TSHR 289 and Freund’s adjuvant interacted with a more diverse set of peptides than mice of the same strain vaccinated with TSHR adenovirus (Fig. 4Go, lower panel vs. top two panels). Although peptide no. 1 was the dominant linear epitope, antibodies in TSHR 289 immunized mice also bound to overlapping peptide no. 2. Other peptides recognized included peptide no. 7 and the cluster of peptide nos. 13 and 15 and especially peptide no. 14. Similar observations were made for sera from two mice immunized with TSHR 289 and cytosine-guanine dinucleotides as adjuvant (data not shown). As expected, no binding was detected to peptides downstream of residue 289.


   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
In the early years after cloning of the TSHR, animals were immunized with various forms of recombinant or synthetic TSHR protein in attempts to develop animal models of Graves’ disease. There was no difficulty in generating antibodies to the TSHR but, in general, these antibodies did not stimulate the thyroid and produce hyperthyroidism (reviewed in Ref. 1). Using arrays of TSHR synthetic peptides, the immunodominant epitopic region of these antibodies was found to be at the extreme N terminus of the TSHR (amino acid residues 22–41), with a secondary epitopic region at the C terminus of the TSHR ectodomain (Fig. 1Go; and Refs. 6, 7, 8, 9, 10, 11). Development of the Shimojo model of Graves’ hyperthyroidism in mice by a novel form of immunization (intact syngeneic cells expressing the TSH holoreceptor) was a major advance in studying the pathogenesis of Graves’ disease (2). Apparently consistent with the functionality of the TSHR antibodies in this model, peptide scanning revealed a different immunodominant region (amino acid residues 97–116; Ref. 12).

More recently, the focus of investigation has turned to antibodies induced in mice by in vivo expression of TSHR DNA in a plasmid (4) or adenovirus (5) vectors. In the present study, we used TSHR ectodomain synthetic peptides to determine the linear components of antibody epitopes recognized by mice immunized using these procedures. Surprisingly, and in contrast to the data reported with the Shimojo model (12), the extreme amino terminus of the TSHR contained the major linear epitope of antibodies in both the TSHR DNA plasmid- and adenovirus-immunized models (summarized in Fig. 5Go and Table 1Go). This was the case even with very high titer TSHR antibody sera from adenovirus immunized animals. For comparison, we performed TSHR peptide scanning of antibodies from mice conventionally immunized with the TSHR A subunit (TSHR 289; Ref. 23) together with different adjuvants. In these animals, TSHR antibodies had more diverse epitopes. Nevertheless, a major component of the humoral immune response was also to the TSHR N terminus. Taken together, our data are in good agreement with other studies indicating the immunodominance of residues 22–41 in conventionally immunized mice (6, 7, 8, 9), in mouse monoclonal antibodies derived independently in different laboratories (18, 21, 24), as well as in rabbits (25, 26).



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  		Figure 5. Schematic representation of the TSHR to illustrate the linear antibody epitopes recognized by mice immunized with TSHR DNA in a plasmid or in an adenoviral vector or using TSHR 289 protein and adjuvant. Linear epitopes recognized (boxed) are shown in terms of amino acid residues in their approximate locations of the receptor. The C peptide region deleted from most cell-surface receptors is indicated with a dashed line. TSHR structure is modified from Ref. 48 with permission.

 

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  		Table 1. TSHR peptides recognized by TSHR antibodies induced in mice by in vivo TSHR DNA expression using plasmid or adenoviral vectors

 
Besides the TSHR amino terminus, sera from TSHR adenovirus immunized mice also recognized (to a lesser extent) peptides toward the C terminus of the ectodomain. In this context, it is of interest that immunodominant epitopes of antibodies to other antigens may be located at the amino terminus (27, 28) and the carboxy terminus (29). Returning to peptides recognized by TSHR antibodies, one C-terminal peptide (residues 352–371) lies within a portion of the receptor, which is excised in the process of receptor cleavage into A and B subunits (30, 31). Other peptides recognized were in the juxta-membrane region (residues 367–386 and 382–401). These data are in agreement with previous findings for conventionally immunized mice (ectodomain protein plus adjuvant; Refs. 6, 7, 8, 9), as well as in the Shimojo approach (12). Of note, peptide 352–367 lies within a region highly immunogenic on conventional immunization of rabbits (25, 26) and is also reported to be recognized by TSHR autoantibodies in the majority of Graves’ sera (32, 33). It should be appreciated that the amino acids represented by this peptide are absent in the fully cleaved, two subunit form of the TSHR on the cell surface.

What is the reason for the high immunogenicity of peptide no. 1 encompassing amino acid residues 22–41? A potential explanation is the cluster of four cysteines (Table 1Go). Disulfide bonds are major contributors to correct protein folding and three-dimensional conformation. Morever, peptides incorporating loop-structures are more immunogenic than short flexible peptides (34) and cyclization by disulfide bonds provides stability to peptide conformation (35). Returning to the TSHR, the four cysteines within the N-terminal region TSHR are known to be crucial for trafficking of the TSHR to the cell surface, as well as for providing the optimal conformation for recognition and function of thyroid stimulating autoantibodies in Graves’ disease (36). Of the 27 peptides studied, only this amino terminal peptide contains four cysteine residues.

Antibody epitopes are often conformational and usually involve discontinuous segments of the polypeptide chain (37). Conformational TSHR autoantibody epitopes in humans have been studied using chimeric TSHR LH receptor molecules (38, 39, 40). By this approach, the epitopes of stimulating antibodies in the Shimojo (41) and TSHR adenovirus (5) models were localized to the amino-terminal half of the TSHR ectodomain, as in humans. In the past, it has been difficult to use synthetic peptides to identify linear components of B cell epitopes recognized by TSHR autoantibodies in Graves’ patients. As previously reviewed, these studies yielded a number of positive peptides (1). By using synthetic peptides in the present study, we do not diverge from the concept that TSHR autoantibody epitopes are largely conformational. However, it should be appreciated that a linear peptide can be part of a conformational epitope. TSHR autoantibodies in humans are present at very low levels (23, 42, 43), and we cannot obtain specific signals using TSHR antigen-coated ELISA wells with Graves’ sera (McLachlan, S. M., and B. Rapoport, unpublished data). In contrast, specific signals on ELISA are readily detectable with sera from TSHR adenovirus, as well as with some TSHR DNA plasmid-vaccinated mice (Fig. 2Go). Clearly, the reason for this difference is the very high titer of TSHR antibodies in the mice studied. Under these circumstances, it is possible to perform peptide scanning studies.

Very recently, after the completion of our study, several murine McAb to the TSHR with stimulatory activity have been reported: four McAb were isolated by two different groups from mice vaccinated with TSHR DNA (44, 45) and one McAb was obtained from a hamster immunized with TSHR adenovirus (46). The epitope of the hamster McAb was conformational and could not be defined using the same panel of synthetic TSHR peptides employed in the present study (46). However, one of the murine McAb with agonist properties recognized a linear epitope (45). Consequently, we cannot exclude the possibility that a component of the polyclonal serum antibodies that we characterized using synthetic peptides may be stimulators, although the majority are likely to be blocking or nonfunctional antibodies.

An interesting question that arises from our study is why animals immunized by numerous approaches all develop TSHR antibodies to the extreme N terminus of the ectodomain, yet only some of these animals become hyperthyroid? One possible explanation is a subtle difference in TSHR conformation, either within this region or at another unidentified portion of a discontinuous epitope. Another contributing factor may be recognition by different antibodies of different faces of the same amino acid segment. All faces may be accessible with the free peptide, whereas accessibility may be limited by steric hindrance of this segment when part of the native holoreceptor expressed on the cell surface (47). Overall, the variable success of different immunization approaches in mimicking Graves’-like hyperthyroidism may depend on subtle variations in antibody binding sites at the extreme N terminus of the TSHR when expressed on the cell surface.

In conclusion, the cysteine-rich N-terminal amino acid region of the TSHR is the immunodominant linear antibody epitope in mice immunized with TSHR expressed in vivo by DNA-plasmid or adenovirus vectors, as previously reported in mice conventionally immunized with TSHR protein and adjuvant (6, 7, 8, 9) but not in mice injected with TSHR-expressing fibroblasts (12). Stimulating autoantibodies in humans are acutely sensitive to minor changes in the conformation of the TSHR N-terminal region as shown using TSH-LH chimeric receptors (32, 40), variable forms of TSHR A subunit protein (16), as well as by cysteine mutagenenesis (36). Taken together, the animal data and observations on human autoantibodies, suggest that this cysteine-rich N-terminal region of the TSHR may be immunodominant in humans.


   Footnotes
 
This work was supported by NIH Grants DK-54684 (S.M.M.) and DK-19289 (B.R.). The generous contribution of Dr. Boris Catz (Los Angeles, CA) is gratefully acknowledged.

Abbreviations: CFA, Complete Freund’s adjuvant; IFA, Incomplete Freund’s adjuvant; McAb, monoclonal antibodies; TSHR, TSH receptor.

Received November 21, 2002.

Accepted for publication January 23, 2003.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 References
 

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