Thyroid Peroxidase Autoantibodies Research Articles

High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line.

Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotinlabelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r = 0.96, P < 0.001, n = 50 and r = 0.99, P < 0.001, n = 80 respectively). In addition, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor delta-aminolevulinic acid was included in the culture medium. Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO-TPO autoantibody complexes.

Majority of thyroid peroxidase autoantibodies in patients with autoimmune thyroid disease are directed to a single TPO domain.

Murine monoclonal antibodies (mAb) produced against native human thyroid peroxidase (TPO) are powerful tools for analyzing the autoantibody (Aab) epitopes on TPO. Binding sites of thirteen mAbs cover all or most antigenic regions on TPO. We determined the competition between Aabs from 75 AITD patients and 13 mAbs in binding to TPO. Autoantibodies recognize predominantly the TPO area close or identical to mAb#9 epitope. All sera tested inhibited this mAb binding by 92.9 +/- 14.8 (mean +/- SD), range from 69-100%. AITD patients' sera with low Aabs titer up to 1/2,000 inhibited mAb#9 binding to TP0 by 85 +/- 11.5% (mean +/- SD) and did not influence remaining mAbs binding to TPO. With elevated Aab levels the inhibition of other mAbs binding was higher, but never exceeded 35%. The amount of Aabs yielding 50% inhibition of mAbs binding was lowest for mAb#9. In order to obtain this degree of inhibition for other mAbs 5 to 25 times more Aabs were needed. Our results demonstrate that the majority of autoantibodies in sera of patients with AITD recognize a single immunodominant region on the TPO mapped by mAb#9. They account for about 80-90% of serum TPO autoantibodies. The autoimmune response to other regions on TPO molecule is directed to several other epitopes, but represents quantitatively a minority of autoantibodies. This response intensifies with increasing Aabs level in the serum.

Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type

IgG class thyroid peroxidase (TPO) autoantibodies with kappa light (L) chains predominate in serum and the genes for a large repertoire of such autoantibodies have been characterized. The present study was performed to clone and characterize TPO autoantibodies with lambda L chains which comprise ∼20% of serum TPO autoantibodies. From a combinatorial IgG H/lambda L chain cDNA library in the phage display vector pComb3, 24 TPO-binding clones with lambda L chains were isolated, comprising three different heavy (H) and light (L) chain combinations. These combinations utilized two genes from the Vlambda II and IIIb families (closest germline genes DPL11 and hsigg11150) and three genes from the VH1, VH3 and VH4 families ( VH26, 4.34 and hv1L1). The deduced amino acid sequences of these H chains were quite different from those of kappa F(ab) isolated using the same H chain library. We expressed the proteins for these three lambda F(ab), as well as for a lambda F(ab) (Humlv318 L chain/DP10-like H chain) previously isolated from another patient. The affnities for TPO of the lambda F(ab) ( K d 8 × 10 −10 M to 10 −7 M) were lower than those of the kappa F(ab) ( K d ∼ 10 −10 M). For two lambda F(ab), both H and L chain genes were close to germline configuration, but there was no straightforward relationship between the extent of somatic mutation from germline configuration and affinity for TPO. All four lambda F(ab) bound less well to denatured TPO as to native TPO. The three F(ab) for which sufficient protein could be expressed for competition studies all recognized domain B within the immunodominat region on TPO previously identified using F(ab) with kappa L chains. Aside from these TPO-specific F(ab), only a few other human IgG class, organ-specific autoantibodies with lambda L chains have been characterized at the molecular level. Our study significantly augments the small database on this category of autoantibodies in general.

Role of complement in the pathogenesis of postpartum thyroiditis: relationship between complement activation and disease presentation and progression

The aim of this study was to assess whether the presentation and progression of autoimmune postpartum thyroiditis (PPT) was related to the degree of thyroid peroxidase autoantibody (TPO-ab)-mediated activation of the complement cascade. One hundred and forty-eight thyroid autoantibody-positive women have been followed during their postpartum year. Seventy-five women remained euthyroid during this time whilst the remaining 73 showed one or more episodes of thyroid dysfunction. Fourteen women showed hyperthyroid PPT, 23 showed a biphasic PPT and the remaining 36 showed hypothyroid PPT. Hyperthyroid PPT was always transient but 29 of the 59 women with hypothyroidism remained hypothyroid or still required thyroxine replacement therapy at the conclusion of the study. Thyroid autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA), free triiodothyronine and free thyroxine by the Amerlex M methods and thyrotrophin by the Amerlite TSH (monoclonal) assay. Complement component C3b, immobilized as a result of classical complement pathway activation in the presence of TPO/TPO-ab complexes in vitro, was measured by ELISA. Bioactive TPO-ab were calculated as the product of the C3 index and TPO-ab level. Basal levels of complement C3 activation were seen in the euthyroid TPO-ab-positive women (C3 index 0.06 at delivery rising to 0.36 at 12 months postpartum; bioactive TPO-ab activity 0.4 kIU/l at delivery rising to 10.4 kIU/l at 12 months' postpartum (N = 75). These parameters were elevated progressively as the severity of the clinical syndrome increased. In 14 hyperthyroid PPT women the C3 index was 0.47 at 8 months' postpartum (bioactive TPO-ab activity = 20 kIU/I; p vs euthyroid group, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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

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

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.

Exclusion of two major areas on thyroid peroxidase from the immunodominant region containing the conformational epitopes recognized by human autoantibodies.

We have used a chimeric molecule between thyroid peroxidase (TPO) and myeloperoxidase (MPO) as well as new information on the three-dimensional structure of MPO to refine further our understanding of the location of the TPO-immunodominant region recognized by TPO autoantibodies in patients' sera. In TPO-MPO chimera A, the amino-terminal 146 amino acids of MPO substitute for the amino-terminal 121 amino acids of TPO. We performed fluorescence-activated cell sorter analysis of Chinese hamster ovary cells expressing TPO-MPO-A on their surface using four monoclonal human autoantibody F(ab) (WR1.7, TR1.8, TR1.9, and SP1.4) that define the immunodominant region. All four F(ab) recognized the TPO-MPO-A chimeric molecule to the same extent. In a second approach to refine the location on the TPO-immunodominant region, we compared the ability of the TPO autoantibody F(ab) to inhibit the binding of serum autoantibodies to the monomeric and dimeric forms of human TPO. The F(ab) inhibited equally (approximately 80%) the binding to the TPO monomer and dimer by autoantibodies in the sera of six individual patients. The present observations exclude two major regions of TPO from the autoantibody-immunodominant region, namely the amino-terminal 121 amino acids of the TPO extracellular domain and the contact region between the two TPO monomers. These findings together with previous data on the Mab47/C21 region of TPO and the recently elucidated 3-dimensional structure of highly homologous MPO, narrow, by a process of exclusion, the site on TPO comprising the immunodominant region. The data provide further support for the thesis, still controversial, that the majority of TPO autoantibodies recognize the native molecule.

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.

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.

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.

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.

Isolation and characterization of a monoclonal human thyroidperoxidase 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 ( K d ∼ 10 t-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 ∼ 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 (1:150000) than for F(ab) with kappa light chains (1:13000). 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.

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).

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

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

Human thyroid peroxidase-myeloperoxidase chimeric molecules: tools for the study of antigen recognition by thyroid peroxidase autoantibodies.

We constructed seven chimeric molecules in which sequential segments in the cDNA for thyroid peroxidase (TPO) were replaced with the homologous regions of myeloperoxidase (MPO) cDNA. The sizes of the translated cDNA segments A through G ranged from 23-175 amino acid residues in length. The TPO-MPO cDNA chimeras, inserted into an eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Protein expression was examined by immunoblotting under reduced/denaturing conditions with a murine monoclonal antibody to denatured wild-type TPO. Expression (at a low level) was confirmed for TPO-MPO chimeras A, B, F, and G. The amino acid substitutions in TPO-MPO-C eliminate the monoclonal antibody epitope, and this chimera, therefore, provides a negative control. TPO-MPO-D and TPO-MPO-E did not generate detectable levels of protein. To study TPO autoantibody interaction with native protein, we performed fluorescence-activated cell sorter analysis using intact Chinese hamster ovary cells stably transfected with the wild-type and TPO-MPO chimeric cDNAs. Of the chimeras, only cells transfected with TPO-MPO-A (N-terminal 146 amino acids of MPO substituted for the N-terminal 121 amino acids of TPO) were recognized by TPO autoantibodies, although to a lesser degree than cells expressing wild-type TPO. In conclusion, the present data indicate that TPO autoantibodies can interact with TPO molecules in which the amino-terminus is replaced with the homologous MPO prosequence region, not normally present in mature MPO. Our study provides a foundation for designing future TPO mutants that may be of value for characterizing disease-associated B-cell epitopes in autoimmune thyroid disease.

Human thyroid peroxidase-myeloperoxidase chimeric molecules: tools for the study of antigen recognition by thyroid peroxidase autoantibodies

We constructed seven chimeric molecules in which sequential segments in the cDNA for thyroid peroxidase (TPO) were replaced with the homologous regions of myeloperoxidase (MPO) cDNA. The sizes of the translated cDNA segments A through G ranged from 23-175 amino acid residues in length. The TPO-MPO cDNA chimeras, inserted into an eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Protein expression was examined by immunoblotting under reduced/denaturing conditions with a murine monoclonal antibody to denatured wild-type TPO. Expression (at a low level) was confirmed for TPO-MPO chimeras A, B, F, and G. The amino acid substitutions in TPO-MPO-C eliminate the monoclonal antibody epitope, and this chimera, therefore, provides a negative control. TPO-MPO-D and TPO-MPO-E did not generate detectable levels of protein. To study TPO autoantibody interaction with native protein, we performed fluorescence-activated cell sorter analysis using intact Chinese hamster ovary cells stably transfected with the wild-type and TPO-MPO chimeric cDNAs. Of the chimeras, only cells transfected with TPO-MPO-A (N-terminal 146 amino acids of MPO substituted for the N-terminal 121 amino acids of TPO) were recognized by TPO autoantibodies, although to a lesser degree than cells expressing wild-type TPO. In conclusion, the present data indicate that TPO autoantibodies can interact with TPO molecules in which the amino-terminus is replaced with the homologous MPO prosequence region, not normally present in mature MPO. Our study provides a foundation for designing future TPO mutants that may be of value for characterizing disease-associated B-cell epitopes in autoimmune thyroid disease.

The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope.

We performed studies to determine whether the binding sites on thyroid peroxidase (TPO) of immunoglobulin antigen binding fragments (Fabs) representing more than 80% of the human autoantibody repertoire overlap with the binding site of monoclonal antibody (Mab) 47, the only Mab whose partial epitope has been defined at the amino acid level (residues 713-721). We also investigated whether these Fabs preferentially recognize native or denatured TPO. None of the Fabs, when bound to radiolabeled TPO, interfered with the ability of Mab 47 to bind to this material. In enzyme-linked immunosorbent assay experiments, the binding of TPO autoantibody Fabs SP1.5, WR1.7, TR1.8, and TR1.9 was greatly diminished by denaturation of TPO. In contrast, binding of Mab 47 was higher to denatured TPO than to intact TPO. Our studies indicate that the Mab 47/C21 epitope lies outside the immunodominant region on TPO. Further, the data confirm that the majority of epitopes for TPO autoantibodies are highly conformational (dependent on the three-dimensional structure of the native protein). Native TPO will be needed to complete the mapping of the epitopes for TPO autoantibodies as well as to determine the amino acids at the autoantibody-antigen-binding sites.

Autoantibodies in the sera of patients with autoimmune thyroid disease recognize a secreted form of human thyroid peroxidase generated in a baculovirus system

We used a baculovirus vector to express the cDNA for a truncated (amino acid residues 1–848), secreted form of thyroid peroxidase (TPO) in Sf9 insect cells. Immunoreactive TPO was detected in pooled conditioned media from 10 clones using polyclonal TPO autoantibodies in the sera of patients with autoimmune thyroid disease. As a further test of TPO immunogenicity, the pooled media completely inhibited autoantibody binding to antigen. We used an ELISA to compare autoantibody reactivity to insect cell-derived TPO and TPO antigen produced by Chinese hamster ovary (CHO) cells, the standard form of antigen in present use. In a study of 22 TPO antibody-negative sera and 24 sera with different TPO autoantibody potencies, there was a highly significant correlation ( r = 0.977; p< 0.001) in OD values obtained with TPO from the two different sources. The highest producing baculovirus clone generated 8.5 μg TPO/ml of conditioned medium, nearly 10-fold higher than previously achieved with stably transfected Chinese hamster ovary cells. Baculovirus-derived, soluble TPO therefore is an excellent source of recombinant TPO in further studies to examine the precise B cell epitopes for human autoantibodies.

Fetal growth and autoimmune thyroid disease

To determine whether fetal and infant growth could influence susceptibility to autoimmune disease in adults, the occurrence of thyroid autoantibodies and autoimmune thyroiditis was studied in 305 women, aged 60-71, born in Hertfordshire and for whom details of birthweight, infant growth, and feeding were routinely recorded. Thyroglobulin autoantibody was detected in 37% of the women, thyroid peroxidase autoantibody in 41%, and autoimmune thyroiditis, defined as biochemical or clinical hypothyroidism in association with thyroid autoantibodies, in 5.6%. The proportion of women with thyroglobulin and thyroid peroxidase autoantibodies fell with increasing birthweight but was not related to weight at 1 year of age or the method of infant feeding. The prevalence of both autoantibodies rose with increasing adult body mass index but fell as the waist to hip ratio increased. These results demonstrate the importance of early environment in determining the susceptibility to autoimmune thyroid disease. The contrasting effects of adult body mass index and waist to hip ratio on antibody prevalence could be explained by their associations with different hormonal environments.