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B Rapoport

Publications and source records attributed to B Rapoport.

At least 19 recordsLinked to original sources
PubMed1995

Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins.

Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for expressing a thyrotropin (TSH) receptor capable of ligand and Graves' disease autoantibody binding comparable to the receptor produced in mammalian cells. Because of the clinical importance of high level expression of this protein, we reassessed the baculovirus system using a new transfer vector (pAcMP3) containing the late basic protein promoter, which functions earlier than the classical polyhedrin promoter. Maximal synthesis of the [35S]methionine-labeled TSH receptor extracellular domain, affinity-purified using a 6-histidine tag, occurred earlier (1 day after insect cell infection) than with a vector (pVL1393) containing the polyhedrin promoter. The pAcMP3-derived TSH receptor extracellular domain was larger (approximately 68 kDa) than the pVL1393-derived protein (approximately 63 kDa). Only the 68-kDa product was secreted, albeit in trace amounts detectable only by precursor labeling. Enzymatic deglycosylation reduced both 68- and 63-kDa cellular proteins to approximately 54 kDa, indicating that the pAcMP3 vector generated a protein with greater carbohydrate content. However, despite its greater degree of glycosylation, most of the 68-kDa protein remained within the cell, almost entirely in the particulate fraction. Remarkably, the trace amounts of 68-kDa receptor protein affinity-purified from the soluble cytosolic fraction of infected insect cells completely neutralized TSH receptor autoantibodies in patients' sera and partly inhibited TSH binding. In conclusion, a baculovirus vector with a promoter active earlier than the conventional polyhedrin promoter generates a more glycosylated and functional TSH receptor extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of functional, highly glycosylated proteins.

Animals↗
PubMed1995

Thyrotropin binding specificity for the thyrotropin receptor.

Recently, highly purified bovine thyrotropin (bTSH) of pituitary origin, as well as recombinant human (h) TSH free of lutropin (LH) contamination, has been reported to activate the LH/choriogonadotropin receptor (LH/CGR). These data challenge the concept of TSH specificity for its own receptor. We, therefore, re-evaluated these data using, as targets, the recombinant hTSH and rat LH/CGRs stably expressed in Chinese hamster ovary (CHO) cells. Partially purified bTSH (2 IU/mg protein) and, to a lesser degree, highly purified bTSH (30 IU/mg protein) increased intracellular cAMP levels in CHO-LH/CGR cells (an EC50 of 0.2 and > 20 mIU/ml, respectively). In contrast, recombinant hTSH (up to 1 IU/ml) did not. All three TSH preparations increased cAMP levels to the same extent in CHO-TSHR cells (an EC50 of 0.3 mIU/ml). Furthermore, we observed only nonspecific, low affinity TSH binding for CHO-LH/CGR cells and also for CHO cells transfected with the expression vector alone (a Kd of 100 nM), although both high and low affinity TSH binding was demonstrated in CHOT-SHR cells (a Kd of 0.3 and 100 nM, respectively). These data indicate that even highly purified bTSH of pituitary origin contains significant amounts of LH, and that TSH itself does not appear to activate the LH/CGR.

Animals↗
PubMed1995

Combinatorial libraries: new insights into human organ-specific autoantibodies.

The recent application of immunoglobulin (Ig) gene combinatorial library technology has led to a logarithmic increase in information concerning human, disease-associated, organ-specific autoantibodies of the IgG class. As reviewed here by Basil Rapoport, Stefano Portolano and Sandra McLachlan, the molecular cloning, analysis and expression of the genes for increasing numbers of these human, monoclonal autoantibodies is providing new insight into the genetic background and epitopic repertoires of such molecules.

Antibodies, Monoclonal↗
PubMed1995

Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response.

TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with VH gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production.

Animals↗
PubMed1995

Thyroid peroxidase autoantibody fingerprints. II. A longitudinal study in postpartum thyroiditis.

It is not known whether epitopes recognized by autoantibodies in an individual remain constant or change over time, especially during perturbations of the humoral immune response. To address this question, we studied the epitopic profile ("fingerprint") of autoantibodies to thyroid peroxidase (TPO) in the sera of 19 women during the postpartum period. Fingerprints were determined in competition studies using 4 recombinant F(ab). At delivery and at 3 time intervals over the subsequent 9-12 months, the pool of F(ab) inhibited autoantibody binding to TPO by 80-100%, consistent with the definition by these F(ab) of a TPO immunodominant region (A1, A2, B1, and B2 domains). Despite a wide spectrum among individuals, the TPO epitopic fingerprints for all 19 women were relatively unchanged throughout the postpartum period. Fingerprint constancy occurred regardless of fluctuations in serum TPO autoantibody levels. When assessed numerically as a ratio of inhibition by the A domain F(ab) to inhibition by the B domain F(ab), the A/B domain ratios in individual women ranged from 0.2 (predominantly B domain) to more than 3.0 (predominantly A domain). However, for each individual woman, the A/B epitopic ratio was conserved throughout the study interval. Our TPO autoantibody epitopic fingerprint data have potential implications for understanding the humoral autoimmune response in man. First, the present study indicates a remarkable lack of spreading of B cell epitopes during a state of perturbation of the immune system over a period of 1 yr. Second, and perhaps more important, despite marked variations in TPO epitopic profiles among different individuals, their constancy over time suggests that TPO autoantibody fingerprints may be inherited.

Autoantibodies↗
PubMed1995

Thyroid peroxidase autoantibody fingerprints in hypothyroid and euthyroid individuals. I. Cross-sectional study in elderly women.

Human monoclonal immunoglobulin G-class autoantibodies to thyroid peroxidase (TPO), expressed as recombinant F(ab), are powerful tools for analyzing the individual components of polyclonal serum TPO autoantibodies. Four TPO-specific F(ab) interact with epitopes in two closely related domains (A and B) in the immunodominant region on TPO. In the present study, these TPO F(ab) were used to compete for serum autoantibody binding to [125I]TPO to determine the "epitopic fingerprints" in two groups of carefully controlled individuals. All individuals (14 hypothyroid and 32 euthyroid) were elderly women (60-71 yr old) with similar genetic and environmental backgrounds as well as comparable levels of serum TPO autoantibodies. Using the pool of four F(ab), serum TPO autoantibody binding was inhibited to the same extent (approximately 90%) in hypothyroid and euthyroid individuals, demonstrating that the majority of TPO autoantibodies in both groups recognize the TPO immunodominant domain. When tested individually, the F(ab) produced a spectrum of inhibition patterns, ranging from sera preferentially inhibited by domain A F(ab) to sera preferential inhibited by domain B F(ab). The ratio of inhibition by domain A F(ab) to inhibition by domain B F(ab) was similar in hypothyroid (0.11-1.39) and euthyroid (0.21-1.79) women. In conclusion, no difference in TPO autoantibody epitopes was observed in this cross-sectional study of hypothyroid and euthyroid individuals. Longitudinal studies are required to address the question of whether TPO autoantibody epitopic fingerprints are stable over time.

Adult↗
PubMed1995

Genetic alterations in thyroid hyperfunctioning adenomas.

Thirty-seven thyroid autonomously hyperfunctioning adenomas were screened for mutations in the TSH receptor (TSHR), G alpha s (gsp), and ras genes. Polymerase chain reaction-amplified fragments of the TSHR C-terminal part (exon 10), the G alpha s (exons 8 and 9), and the three ras genes were obtained from the genomic DNA extracted from 37 tumors and their adjacent normal tissues and were studied by direct nucleotide sequencing and hybridization with synthetic probes. A point mutation in the third intracellular loop (codon 623) of the TSHR was found in 3 of 37 adenomas studied. This mutation codes for a change (Ala to Ser) in the TSHR structure and is somatic and heterozygotic. Constitutive activation of the TSHR was demonstrated by an increase in basal cAMP levels after transfection of Chinese hamster ovary cells with a mutated Ser623-TSHR complementary DNA. Nine gsp[00ae]MDRV[00af]- and one ras-activating mutations were also detected. No simultaneous alteration of the studied genes was present. Thus, in hyperfunctioning thyroid adenomas, our data suggest that a mutational activation of the TSHR and gsp genes may play a tumorigenic role through constitutive activation of the cAMP pathway.

Adenoma↗
PubMed1995

Human monoclonal autoantibodies against the immunodominant region on thyroid peroxidase: lack of cross-reactivity with related peroxidases or thyroglobulin and inability to inhibit thyroid peroxidase enzymatic activity.

Thyroid peroxidase (TPO) autoantibodies are heterogeneous and have been classified in terms of whether they cross-react with myeloperoxidase (MPO), lactoperoxidase (LPO), or thyroglobulin (Tg) as well as by whether they inhibit TPO enzymatic activity. Four human monoclonal TPO autoantibodies, generated using combinatorial immunoglobulin gene libraries and expressed as F(ab), have been used to investigate these properties of TPO autoantibodies. The binding of F(ab) WR1.7, TR1.8, TR1.9, and SP1.4 to 125I-labeled recombinant TPO was inhibited 50% by approximately 10(-10) mol/L unlabeled TPO, reflecting the high affinities of these F(ab) for TPO. In contrast, F(ab) binding to TPO was unaffected by human MPO (both native and reduced), bovine LPO, or human Tg at concentrations up to 10(-8) mol/L. Further, TPO enzymatic activity, measured by guiacol oxidation, was unaffected by preincubation with the four F(ab) individually or as a pool (each at 10(-8) mol/L). In conclusion, four human TPO monoclonal autoantibodies do not cross-react with related peroxidases or Tg, nor do they inhibit TPO enzymatic activity. These monoclonal immunoglobulin G class autoantibodies define the immunodominant region on TPO and represent about 85% of TPO autoantibodies in an individual patient's serum. Consequently, our data suggest that TPO autoantibodies that cross-react with MPO, LPO, or Tg, or inhibit TPO enzymatic activity are likely to bind outside the immunodominant region.

Antibodies, Monoclonal↗
PubMed1994

Cleavage of the thyrotropin receptor does not occur at a classical subtilisin-related proprotein convertase endoproteolytic site.

The human thyrotropin receptor (TSHR) undergoes proteolytic cleavage closely upstream to amino acid 317. Between residues 261 and 313 are three clusters of positively charged amino acids, arginines (Arg) and lysines (Lys), which are potential subtilisin-related proprotein convertase sites. We used oligonucleotide-directed mutagenesis to perform conservative amino acid substitutions within these regions (Arg or Lys to glutamine, Gln). Chinese hamster ovary cells stably transfected with mutant receptor cDNA TSHR-CS1 (Gln261) and TSHR-CS3 (Gln312, Gln313) bound radiolabeled TSH with an affinity similar to the wild-type TSHR. Mutant cDNA TSHR-CS2 (Gln290, Gln291) and TSHR-CS4 (Gln261, Gln290, Gln291, Gln312, Gln313) did not express a protein on the cell surface capable of specific TSH binding. After covalent cross-linkage of radiolabeled TSH to TSHR-CS1 and TSHR-CS3, the mutant receptors dissociated into two subunits under reducing conditions. The most prominent cluster of basic amino acids in the TSHR extracellular region (residues 287-293) was studied in a second series of mutations designed to eliminate the classical proprotein convertase sites in this region and yet be compatible with TSHR function. All three mutant receptors, TSHR-CS5 (Gln290), TSHR-CS6 (Gln291), and TSHR-CS7 (Gln291, Gln293) bound TSH with an affinity similar to that of wild type, and none of these amino acid substitutions prevented proteolytic cleavage of the extracellular domains of the TSHR. Thus, cleavage of the TSHR extracellular domain does not involve a classical subtilisin-related proprotein convertase cleavage site, raising the possibility that TSHR cleavage occurs after processing and trafficking of the protein to the plasma membrane.

Amino Acid Sequence↗
PubMed1994

Immunoglobulin A in Graves' orbital tissue: deoxyribonucleic acid amplification by polymerase chain reaction.

A role for IgA autoantibodies in Graves' ophthalmopathy is suggested by the presence of immunoglobulins of this class in Graves' orbital tissue, as detected by immunohistochemistry. We, therefore, investigated the possibility of using the polymerase chain reaction (PCR) to amplify IgA immunoglobulin genes from plasma cells infiltrating Graves' eye tissue. Template cDNA was reverse-transcribed from orbital muscle (M) mRNA of one patient (#7) and from orbital connective tissue/fat (F) mRNA of two patients (#1 and #7), both undergoing surgery for exophthalmos because of severe infiltrative ophthalmopathy. Preliminary studies to establish the PCR procedure were performed for kappa light chain DNA amplification. With the very small amount of orbital tissue template available, the sensitive "hot start" modification of the PCR was necessary to amplify significant amounts of kappa light chain DNA. Using this procedure, IgA heavy chain DNA was amplified from both connective tissue/fat (F7) and muscle (M7) cDNA of patient #7. The DNA yield was less for IgA than for IgG using the same template. There was no significant IgA (or IgG) DNA product using the connective tissue/fat cDNA of patient #1. While not implying that IgA-infiltrating plasma cells are specific for Graves' orbital tissue, our studies nevertheless demonstrate the feasibility of amplifying the genes coding for IgA antibodies from Graves' orbital tissue plasma cells. Expression of these immunoglobulin genes in future studies will make it possible to determine the antigen specificity of the antibodies expressed by Graves' orbital tissue plasma cells.

Aged↗
PubMed1994

Profile of lambda light chain variable region genes in Graves' orbital tissue.

Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2/3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in both framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.

Adipose Tissue↗
PubMed1994

Isolation and characterization of a monoclonal human thyroid peroxidase autoantibody of lambda light chain type.

Thyroid peroxidase (TPO) autoantibodies, a hallmark of human autoimmune thyroid disease, may have kappa or lambda light chains. Monoclonal human TPO autoantibodies with kappa light chains have previously been developed by cloning and expressing "combinatorial" libraries of immunoglobulin genes in bacteria. In the present study, an IgG1/lambda combinatorial library was generated from thyroid cDNA of a Graves' patient whose serum contained lambda TPO antibodies. Screening the bacteriophage library with 125I-TPO yielded one clone, TR1.41. The oligonucleotide sequence of TR1.41 was determined and the nature of its interaction with TPO was investigated. The affinity of TR1.41 for TPO is high (Kd approximately 10(-9) M), comparable to that of monoclonal kappa TPO autoantibodies derived from the same patient. The genes encoding the heavy and light chains of TR1.41 differ in a number of respects from the closest available germline genes. Such differences are consistent with somatic mutation in a high-affinity antibody. An important characteristic of TR1.41 is its interaction with the immunodominant domain on TPO recognized by approximately 80% of serum TPO autoantibodies. The frequency of TPO-specific F(ab) generated from the thyroid gland of patient TR was much lower for F(ab) with lambda light chains (1:150,000) than for F(ab) with kappa light chains (1:13,000). Despite this low frequency, the high affinity of TR1.41 and its recognition of the immunodominant region on TPO indicate that lambda autoantibodies of this type may represent an important constituent of the TPO autoantibody response in man. In conclusion, this is the first report on the molecular cloning and characterization of a thyroid autoantibody of lambda L chain type by the combinatorial library approach.

Amino Acid Sequence↗
PubMed1994

Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition.

Most thyroid peroxidase (TPO) autoantibodies in man recognize closely associated epitopes in two domains (A and B) on TPO. These epitopes were defined by recombinant monoclonal human autoantibodies expressed as antigen-binding fragments [F(ab)]. Only five heavy (H) and light (L) chain gene combinations encoded 34 F(ab), all of which have high affinity (Kd, approximately 10(-10) M) for TPO. We, therefore, investigated the roles of H and L chain genes in TPO domain recognition in two ways. First, we created hybrid F(ab) by forced recombination of H and L chain genes from 4 F(ab) recognizing the A or B domains. These hybrid F(ab) proteins, expressed in bacteria, bound extremely poorly (or not at all) to TPO, even at concentrations more than 100-fold higher than those required for detection of TPO binding by the original F(ab). Nucleotide sequencing of the cDNA as well as gel electrophoresis of the expressed proteins confirmed that poor hybrid F(ab) binding to TPO was not the result of cloning artifacts. Therefore, contrary to prevailing views on combinatorial libraries, we found no tolerance for H and L chain cross-combinations in high affinity TPO binding. These observations strengthen the likelihood that the H and L chain combinations from combinatorial libraries reflect those of TPO autoantibodies in vivo. In a second approach to examine the roles of H and L chains in TPO binding, we focused on three original F(ab) with similar L chains (encoded by KL012-like germline genes) and similar H chains (encoded by V1-3B-like germline genes), but different diversity (D) regions. All F(ab) bound predominantly to TPO domain A, as observed previously for a F(ab) with a KL012 L chain and a different H chain. Conversely, a F(ab) with a V1-3B-like H chain but a different L chain (A') bound to TPO domain B. These data indicate that the L chain plays a major role in defining TPO epitope recognition.

Amino Acid Sequence↗
PubMed1994

Recombinant thyroid peroxidase-specific autoantibodies. I. How diverse is the pool of heavy and light chains in immunoglobulin gene libraries constructed from thyroid tissue-infiltrating plasma cells.

Thyroid peroxidase (TPO) autoantibodies are a distinguishing feature of autoimmune thyroid disease. We have previously constructed immunoglobulin G heavy (H) and light (L) chain cDNA libraries from intrathyroidal B-cells. TPO-selected autoantibodies expressed by combined H and L chain libraries (combinatorial libraries) recognized a limited number of epitopes on TPO and used only a few of the many H and L chain variable region genes present in the genome (germline genes). One possible explanation for this restriction is a lack of diversity in the parental H and L chain gene libraries used to construct the combinatorial library. To address this issue, we determined the nucleotide sequences of randomly selected H and kappa L chain variable region genes from a pair of H and L chain libraries. The 12 H chain gene sequences analyzed were highly diverse, and none resembled the genes of TPO-selected autoantibodies. The sequences of 14 randomly selected kappa L chain genes were less diverse; 12 of 14 were closely related to the same germline gene (KL012) used by TPO-specific autoantibodies. However, we observed previously that only about 1 in 500 of the L chains in this library can pair with an H chain and bind TPO. We now find that, with 1 exception, the randomly selected KL012-like genes in the L chain library differ significantly from the antigen-specific KL012-like genes, particularly in the antigen-binding regions. In summary, the present data indicate that 1) the restricted number of H chain genes used by TPO-specific autoantibodies cannot be ascribed to limited H chain gene diversity in the parent library; and 2) L chains from combinatorial libraries (even when related to the same germline gene) cannot simply be regarded as plastic, or promiscuous, partners for high affinity antigen binding by a particular H chain.

Amino Acid Sequence↗
PubMed1994

Studies on homologous desensitization of the thyrotropin receptor in 293 human embryonal kidney cells.

It is well known that the TSH receptor (TSHR) undergoes homologous desensitization. That is, prolonged stimulation of thyroid cells with TSH attenuates the cAMP response to subsequent TSH stimulation. However, the existence of homologous desensitization of the recombinant TSHR expressed in nonthyroidal eukaryotic cells is controversial. In the present studies, therefore, we first investigated whether or not the TSHR was desensitized by TSH in 293 human embryonal kidney cells, a cell line in which the LH/CG receptor (LH/CGR) is reported to undergo homologous desensitization. The wild type (wt) TSHR and the wt-LH/CGR stably expressed in 293 cells bound to their respective hormones with high affinity and produced a dose-dependent intracellular cAMP response to hormone stimulation. Pretreatment of cells expressing the TSHR or the LH/CGR with their respective hormones attenuated the cAMP response to subsequent hormone stimulation without down-regulation of the receptors, demonstrating that the TSHR, as well as the LH/CGR, undergoes homologous desensitization in 293 cells. With this cell type expressing mutant TSHRs, we then studied some aspects of the molecular mechanism of TSHR desensitization and compared our data to those obtained with the beta-adrenergic receptor (beta-AR), which is widely regarded as the prototype for receptor desensitization. We cotransfected the wt-TSHR and a chimeric receptor consisting of the LH/CGR extracellular ligand binding domain with the TSHR transmembrane/cytoplasmic signal transducing region. These two receptors have distinct hormone specificities but share common signal regulatory mechanisms. We observed that, like the beta-AR, only hormone-occupied receptor is likely to be involved in homologous desensitization. On the other hand, studies with a truncated TSHR indicated that, in contrast to the beta-AR, the serine/threonine-rich region in the carboxyl two thirds of the cytoplasmic tail of the TSHR is not involved in homologous desensitization.

Animals↗
PubMed1994

Molecular cloning and characterization of genes for antibodies generated by orbital tissue-infiltrating B-cells in Graves' ophthalmopathy.

Graves' ophthalmopathy is a distressing autoimmune disease of unknown etiology. Analysis of the genes for antibodies secreted by orbital tissue-infiltrating plasma cells might provide insight into the pathogenesis of this disease. We, therefore, constructed an immunoglobulin heavy (H) chain and an immunoglobulin kappa light (L) chain cDNA library from the orbital tissue of a patient with active Graves' ophthalmopathy. Analysis of 15 H (IgG1) and 15 L (kappa) chains revealed a restricted spectrum of variable region genes. Fourteen of 15 variable kappa genes were about 94% homologous to the closest known germline gene, KL012. Thirteen of 15 H chain genes were 91% and 90% homologous to the closest germline genes, DP10 and hv1263, respectively. Remarkably, these germline genes also code for other autoantibodies to striated muscle (KL012) and thyroid peroxidase (KL012 and hv1263). These studies raise the possibility that particular germline genes may be associated with autoimmunity in humans. Further, the present study opens the way to identifying ocular autoantigens that may be the target of an humoral immune response.

Amino Acid Sequence↗
PubMed1994

Recombinant thyroid peroxidase autoantibodies can be used for epitopic "fingerprinting" of thyroid peroxidase autoantibodies in the sera of individual patients.

Four human monoclonal antibodies (SP1.4, WR1.7, TR1.8, and TR1.9) map the immunodominant region on thyroid peroxidase (TPO) recognized by autoantibodies in patients' sera. We used a pool of these monoclonal antibodies, expressed in bacteria as antigen-binding fragments [F(ab)], to compete for TPO autoantibody binding to radiolabeled TPO. The F(ab) inhibited TPO binding by 32 patients' sera by 82 +/- 14% (mean +/- SD), with a range from 51-100%. When each F(ab) was tested individually for its ability to compete for autoantibody binding to TPO, F(ab) TR1.8 was the most potent among the 32 sera. However, there was a wide spectrum of TPO binding inhibition when each serum was considered individually, thereby allowing an epitopic "fingerprint" to be drawn for the TPO autoantibodies in a patient's serum. There was a close association between the proportions of TPO autoantibodies to the TR1.8 and TR1.9 epitopes as well as between those to the SP1.4 and WR1.7 epitopes. These associations correspond to the previously described A and B epitopic domains in the TPO immunodominant region. No TPO epitope was observed to be associated with clinically apparent ophthalmopathy of Graves' disease, nor was there an association between TPO epitopes and patient age or sex. In summary, the present study on a large sample of sera with TPO autoantibodies indicates that by using TPO-specific F(ab) selected to cover all regions of the TPO immunodominant region, it is possible to obtain a TPO epitopic fingerprint for each serum. These data open the way to future studies directed at testing the hypothesis of disease-associated TPO epitope(s).

Autoantibodies↗