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Jadwiga Furmaniak

Publications and source records attributed to Jadwiga Furmaniak.

18 recordsLinked to original sources
PubMed2006

Estimated risk for developing autoimmune Addison's disease in patients with adrenal cortex autoantibodies.

CONTEXT: Patients with adrenal cortex autoantibodies (ACA) without overt autoimmune Addison's disease (AAD) are at risk of adrenal failure. DESIGN: To assess the contribution of different clinical, immunological, genetic, and functional factors in the progression to AAD, we followed up 100 ACA-positive and 63 ACA-negative patients without AAD for a maximum of 21 yr (mean 6.0 yr, median 4.8). ACA were measured by immunofluorescence and 21-OH autoantibodies (Abs) by RIA. Adrenal function was assessed by measuring basal levels of cortisol, aldosterone, ACTH, renin activity, and cortisol response to ACTH. The risk of developing AAD was calculated using survival and multivariate analyses. RESULTS: AAD developed in 31 ACA-positive patients and one ACA-negative patient. The cumulative risk of disease in ACA-positive patients was 48.5% [95% confidence interval (CI) 40.8-56.1]. The cumulative risk was higher in children than adults (100 vs. 31.9%; P < 0.0001), males than females (68.6 vs. 42.7%; P = 0.006), patients with subclinical rather than normal adrenal function at entry (87.4 vs. 30.1%; P < 0.0001), patients with hypoparathyroidism and/or candidiasis than patients with other autoimmune or nonautoimmune diseases (100 vs. 29.7%; P < 0.0001), and patients with high rather than low-medium ACA titers (62.8 vs. 41.2%; P = 0.12). The presence of human leukocyte antigen (HLA)-DRB1 did not appear to contribute to the prediction of AAD. Adjusted hazard ratios by Cox model for the development of AAD were 3.37 for males (CI 1.38-8.24), 5.23 for hypoparathyroidism and/or candidiasis (CI 1.53-17.92), 3.33 for high antibody titers (CI 1.43-7.78), and 6.15 for impaired adrenal function at entry (CI 2.79-13.57). CONCLUSIONS: These results were used to construct a risk algorithm for estimating the probability of developing AAD from the combination of gender, age, adrenal function, antibody titer, and associated autoimmune disorders at entry. The values of estimated risk could be used to decide appropriate follow-up intervals and future immunointervention strategies.

Addison Disease↗
PubMed2006

Estimation of serum TSH receptor autoantibody concentration and affinity.

We have used the human monoclonal TSH receptor (TSHR) autoantibody (M22) as a labeled ligand in competition with individual patient TSHR autoantibodies (TRAb) to estimate their serum concentrations and affinities. TSHR coated tubes, (125)I-labeled M22 IgG and Fab, and patient sera IgG and Fab were used in these studies. In 15 patients with Graves' disease, TRAb concentrations ranged from 50 to 500 ng/mL of serum (5- 60 parts per million of total serum IgG) and TRAb IgG affinities from 3.0 +/- 1.0-6.7 +/- 1.54-10(10) L/mol (mean +/- SD; n=3). Fab fragment affinities were similar to those of intact IgG. Serum TRAb with blocking (TSH antagonist; 4 patients) activity had similar affinities (3.0 +/- 0.25-7.2 +/- 2.2-10(10) L/mol) to TRAb IgG from patients with Graves' disease, but blocking TRAb concentrations were higher (1.7 - 27 mg/mL of serum). The concentrations of TRAb that we observed in the sera of the 15 Graves' patient (0.33 - 3.3 nmol/L) can be compared with that of circulating TSH. In particular, a serum TSH concentration of 100mU/L (0.7 nmol/L) is in the same range as the concentrations of TRAb we observed. Such a TSH concentration (similar to that observed after injection of 0.9 mg of recombinant human TSH) would be expected to cause a similar degree of thyrotoxicosis as seen in Graves' disease. Consequently, the thyroid-stimulating potencies (i.e., activity per mol) of patient serum TRAb and human TSH appear to be of a similar magnitude in vivo as well as in vitro. Overall, our results indicate that serum TRAb affinities are high and show only limited variations between different sera whereas concentrations of the autoantibodies vary widely.

Antibodies, Monoclonal↗
PubMed2006

Effects of TSH receptor mutations on binding and biological activity of monoclonal antibodies and TSH.

The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.

Animals↗
PubMed2005

A sensitive non-isotopic assay for acetylcholine receptor autoantibodies.

BACKGROUND: Detection and measurement of serum acetylcholine receptor autoantibodies (AChRAb) are useful in the diagnosis and management of myasthenia gravis (MG). An immunoprecipitation assay (IPA) based on AChR labelled with 125I-alpha bungarotoxin is widely used for measurement of AChRAb, but a non-isotopic assay of sensitivity and specificity comparable to IPA is not available as yet. METHODS: A new AChRAb ELISA, which is based on the ability of AChRAb to compete with 3 different AChR monoclonal antibodies (MAbs 1-3) for binding sites on affinity purified fetal and adult-type AChR preparations, is described. The sensitivity and specificity of the ELISA were assessed by comparing assay results with a conventional IPA. RESULTS: There was good agreement between the IPA and the ELISA for measurement of AChRAb (r = 0.85; n = 83; p <0.001). 76/83 MG sera were positive in the ELISA, whilst 72/83 were positive by IPA. Eight sera were positive in the ELISA (inhibition range 18%-46%) but negative by IPA (0.33-0.47 nmol/L toxin bound) and 4 sera were negative in the ELISA (inhibition range -1% to15%) whilst positive by IPA (0.56-2.9 nmol/L toxin bound). Overall 80/83 (96%) of the MG sera were AChRAb positive in one or both assays. In addition, all 191 control serum samples which were negative for AChRAb by IPA were below or equal to 16% of inhibition in our ELISA. The AChRAb ELISA also showed good inter-assay and intra-assay precision. CONCLUSION: The AChRAb ELISA we have described has sensitivity and specificity at least as high as our current radioactive IPA. It has good precision and good handling characteristics making it suitable for routine use.

Adult↗
PubMed2005

Sensitive non-isotopic assays for autoantibodies to IA-2 and to a combination of both IA-2 and GAD65.

BACKGROUND: A sensitive ELISA for measurement of IA-2 autoantibodies has been developed and assessed. Also, a combination ELISA for detection of both GAD65 autoantibodies and IA-2 autoantibodies is described. METHODS: The IA-2 autoantibody assay is based on the ability of IA-2 autoantibodies to form a bridge between IA-2 intracellular fragment coated onto ELISA plate wells and liquid-phase IA-2 labelled with biotin. The combination ELISA uses plates coated with both IA-2 and GAD65 and a mixture of IA-2-biotin and GAD65-biotin. Assay sensitivity was assessed using the WHO reference (NIBSC 97/550) for islet cell antibodies. IA-2 autoantibody measurements by ELISA were compared with measurements in immunoprecipitation assays (IPAs) based on 125I or 35S labelled IA-2. Combination ELISA results were compared with results obtained for individual autoantibodies. RESULTS: As little as 15 units/mL of NIBSC 97/550 was detectable in the IA-2 autoantibody ELISA compared to 125 units/mL by 125I-IA-2 IPA. 110/216(51%) sera from patients with type 1 DM were positive in the IA-2 autoantibody ELISA while 97/216 (45%) and 91/216 (42%) were positive in the 125I-IA2 and 35S-IA-2 IPAs, respectively. The IA-2 autoantibody ELISA showed 100% specificity for type 1 DM. The combination ELISA was able to detect GAD65 and/or IA-2 autoantibodies in 183/216 (85%) diabetic sera and 183/216 (85%) were also found positive for autoantibodies to IA-2 and/or to GAD65 in the assays for individual antibodies. Autoantibody measurements in the individual autoantibody assays and in the combination ELISA showed good agreement by Pearson correlation (r=0.92, n=216, p<0.001) and by Bland and Altman analysis. CONCLUSIONS: Sensitive and specific ELISAs for measurement of autoantibodies to IA-2 and to a combination of IA-2 and GAD65 have been developed. These assays are suitable for screening large numbers of samples in diabetes prediction and prevention trials.

Antibody Specificity↗
PubMed2005

Autoimmune oophoritis as a mechanism of follicular dysfunction in women with 46,XX spontaneous premature ovarian failure.

OBJECTIVE: To assess the association between serum adrenal cortex autoantibodies and histologically confirmed autoimmune lymphocytic oophoritis. DESIGN: Controlled, prospective. SETTING: Tertiary research center. PATIENT(S): Two hundred sixty-six women with 46,XX spontaneous premature ovarian failure. INTERVENTION(S): Ovarian biopsy in 10 women. MAIN OUTCOME MEASURE(S): Serum adrenal cortex autoantibodies assessed by indirect immunofluorescence and autoimmune oophoritis assessed by immunohistochemical lymphocyte markers. RESULT(S): We obtained a histologic diagnosis of autoimmune oophoritis in four women who tested positive for adrenal cortex autoantibodies and excluded this diagnosis in ovarian biopsies from six women who tested negative for adrenal cortex autoantibodies (4/4 vs. 0/6). Women with histologically confirmed autoimmune oophoritis had a greater total ovarian volume as assessed by transvaginal sonography (11.4 +/- 5.6 mL vs. 1.5 +/- 0.4 mL) (mean +/- SEM). They were also more likely to have subclinical adrenal insufficiency and clinical signs of androgen deficiency (3/4 vs. 0/6). Overall, 10/266 women tested positive for adrenal cortex autoantibodies (3.8%, 95% confidence interval: 1.8%-6.5%). CONCLUSION(S): In women who present with 46,XX spontaneous premature ovarian failure as their primary concern there is a clear association between serum adrenal cortex autoantibodies and the presence of histologically confirmed autoimmune oophoritis.

Adolescent↗
PubMed2005

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling.

OBJECTIVE: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. DESIGN: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH-21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. METHODS: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. RESULTS: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% +/- 24% (n = 6), 86.7% +/- 7.6% (n = 6) and 86.5% +/- 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 +/- 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. CONCLUSIONS: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

Animals↗
PubMed2004

Muscle-specific receptor tyrosine kinase autoantibodies--a new immunoprecipitation assay.

BACKGROUND: It has been reported that measurement of autoantibodies to a muscle-specific receptor tyrosine kinase (MuSK) may be useful in the assessment and management of patients with acetylcholine receptor antibody (AChRAb)-negative myasthenia gravis (MG). METHODS: A new and convenient assay for MuSKAb measurement based on 125I-labeled purified recombinant MuSK is described. In the assay, serum samples (5 microl) were incubated with 50 microl of 125I-MuSK and any immune complexes formed precipitated with antihuman IgG (50 microl). RESULTS: With this assay, MuSKAbs were detected in 8/33 (24%) sera from AChRAb-negative MG patients studied. In contrast, no MuSKAb was detected in 53 AChRAb-positive MG patient sera; furthermore, 0/18 Lambert-Eaton myasthenic syndrome sera, 0/5 non-MG neuromuscular disease sera, 0/95 control autoimmune disease sera, and 0/50 healthy blood donor sera contained detectable MuSKAb. In this assay, inter-assay coefficients of variation (n = 5) were 6.8%, 5.9%, and 3.6% for samples with MuSKAbs at 0.73, 0.32, and 0.11 nmol/l, respectively. Similar results were obtained with 125I-labeled rat and human MuSK. CONCLUSION: The 125I-MuSK immunoprecipitation assay we have described provides a simple, specific, and precise procedure for detecting MuSKAbs.

Animals↗
PubMed2004

Epitopes recognised by tissue transglutaminase antibodies in coeliac disease.

The interaction between IgA tissue transglutaminase (tTG) antibodies (Abs) and 35S-labelled tTG produced in a transcription/translation (TnT) system with various amino acid (aa) deletions has been studied. These experiments showed that the tTG N-terminal aa 1-89 were important for tTG Ab binding in all 15 coeliac disease sera studied and the central residues (aa 401-491) were important for binding of tTG Abs in all but one sera. The contribution of C-terminal residues to tTG Ab binding varied in different coeliac sera but overall was less than the contributions of the N terminal and central regions. Mouse monoclonal antibodies (MAbs) to tTG were produced and the tTG aa sequences recognised by the MAbs determined using modified 35S-labelled tTG proteins. Analysis of the inhibiting effects of patient sera tTG Ab on binding of tTG MAbs to tTG confirmed the importance of the N-terminal and central regions of tTG in forming serum tTG Ab binding sites. Recombinant human tTG was expressed in yeast and purified to better than 95% homogeneity using MAb affinity chromatography as a final purification step. This material was highly suitable for use in an ELISA for tTGAb.

Adolescent↗
PubMed2004

A new assay for thyrotropin receptor autoantibodies.

A new procedure for measuring patient serum thyrotropin receptor (TSHR) autoantibodies is described in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 (labeled with biotin) to TSHR-coated enzyme-linked immunosorbent assay (ELISA) plate wells. In the assay, M22-biotin binding is detected by addition of streptavidin peroxidase. The M22 based assay was more sensitive than a similar ELISA based on inhibition of TSH-biotin binding to TSHR coated wells with 1 U/L of NIBSC 90/672 giving approximately 35% inhibition in the M22-based system compared to approximately 15% inhibition in the TSH-based ELISA. This had an important impact on the precision of the 2 assays with the M22-based ELISA showing an interassay coefficient of variation (CV) of 10% at 1 U/L whereas the TSH-based ELISA had an interassay CV of 20% at 1 U/L. Analysis of sera from 307 control subjects without a diagnosis of Graves' disease indicated that only 2 (0.65%) gave inhibition of M22 binding values of greater than 10% (11% and 12% inhibition). In the case of sera from 108 patients with Graves' disease (treated and untreated) 103 (95%) gave inhibition of M22 binding values of 14% or greater. Receiver operating characteristic (ROC) plot analysis showed that 100% specificity for TSHR autoantibody detection in Graves' disease was obtained at 95% sensitivity for the M22-based ELISA and 89% sensitivity for the TSH-biotin-based ELISA. Inhibition of M22 binding to the TSHR was closely correlated to inhibition of TSH binding in the 108 Graves' sera (r = 0.99). However, inhibition of M22 binding was almost always greater resulting in improved sensitivity and precision.

Adult↗
PubMed2004

Analysis of the thyrotropin receptor-thyrotropin interaction by comparative modeling.

We have used the most advanced programs currently available to construct the first three-domain structure of the human thyrotropin receptor (TSHR) using comparative modeling. The model consists of a leucine-rich domain (LRD; amino acids 36-281; porcine ribonuclease inhibitor used as a template for modeling), a cleavage domain (CD; amino acids 282-409; tissue inhibitor of matrix metalloproteinases 2 as template) and transmembrane domain (TMD amino acids 410-699; bovine rhodopsin as template). Models of human, porcine, and bovine TSH were also constructed (human chorionic gonadotropin [hCG] and human follicle stimulating hormone [hFSH] as templates). The LRD has a characteristic horseshoe shape with 10 tandem homologous repeats. The CD consists of beta-barrel and alpha helix structures (OB-like fold) with two disulfide bridges and the structure around these disulfide bridges remains stable after cleavage. The TMD presents the typical seven membrane-spanning helices. The TSH, LRD, CD, and TMD models were brought together in an extensive series of docking experiments. Known features of the TSH-TSHR interaction were used for selection of appropriate complexes that were then validated using a different set of experimental data. A similar approach was used to build a model of a complex between the TSHR and a monoclonal TSHR antibody with weak thyroid stimulating activity. Human thyrotropin (hTSH) alpha chains were found to make contact with many amino acids on the LRD surface and CD surface whereas no interaction between the beta chains and the CD were found. The higher affinity of bovine thyrotropin (bTSH) and porcine thyrotropin (pTSH) (relative to hTSH) for the TSHR is explained well by the models in terms of charge-charge interactions between their alpha chains and the receptor. Experimental observations showing increased sensitivity of the TSHR to hCG after mutation of TSHR Lys209 to Glu are explained well by our model. Furthermore, several mutations in the TMD that are associated with increased TSHR basal activity are predicted from our model to be caused by the formation of new interactions that stabilize the activated form of the TMD.

Animals↗
PubMed2004

Autoantibodies to human tryptophan hydroxylase and aromatic L-amino acid decarboxylase.

OBJECTIVE: To assess the prevalence of autoantibodies (Abs) to tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC) in patients with different autoimmune diseases and to analyse their respective epitopes. DESIGN: TPH and AADC Abs were measured in an immunoprecipitation assay using (35)S-labelled full-length and fragments of TPH and AADC. METHODS: Patients with different autoimmune adrenal diseases (n=84), non-adrenal autoimmune diseases (n=37), idiopathic vitiligo (n=8) and 56 healthy blood donors were studied. RESULTS: Fourteen of twenty-three (61%) of patients with autoimmune polyglandular syndrome (APS) type I and 1/34 (3%) of patients with isolated Addison's disease (AD) were positive for TPH Abs. None of the patients with APS type II (n=27), coeliac disease (n=10), autoimmune thyroid disease (AITD) (n=11), type 1 diabetes mellitus (DM) (n=16) or idiopathic vitiligo (n=8) was positive for TPH Abs. AADC Abs were detected in 12/23 (52%) patients with APS type I, in 1/29 (3%) patients with APS type II and 1/34 (3%) patients with isolated AD. None of the patients with coeliac disease, type 1 DM, AITD or idiopathic vitiligo was positive for AADC Abs. TPH Abs were found to interact with the C-terminal amino acids (aa) 308-423, central aa 164-205 and N-terminal aa 1-105 of the TPH molecule. AADC Ab binding epitopes were within the C-terminal aa 382-483, the central aa 243-381 and the N-terminal aa 1-167. CONCLUSIONS: Our study suggests that TPH Abs and AADC Abs react with several different epitopes and that different epitopes are recognized by different sera. The prevalence of TPH Abs and AADC Abs in patients with APS type I in our study is in agreement with previous reports. TPH Abs and AADC Abs were found very rarely in patients with other forms of autoimmune adrenal disease and were not detected in patients with non-adrenal autoimmune diseases.

Adolescent↗
PubMed2003

A sensitive non-isotopic assay for GAD65 autoantibodies.

BACKGROUND: A new ELISA for 65 kDa isoform of glutamic acid decarboxylase autoantibodies (GAD(65) Abs), which depends on GAD(65) Ab acting divalently and forming a bridge between immobilized GAD(65) and liquid-phase GAD(65)-biotin, is described. METHODS: Sera (25 microl) were incubated in GAD(65)-coated ELISA plate wells followed by washing and incubation with GAD(65)-biotin. After a further wash step, GAD(65)-biotin bound was quantitated by addition of streptavidin peroxidase followed by tetramethylbenzidine. Assay calibration was with WHO reference preparation 97/550. RESULTS: Using a cut-off for positivity of 5 units/ml, sera from 0.7% (2/300) healthy blood donors (HBDs), 100% (39/39) selected type 1 diabetes mellitus (DM) patients, 1.6% (1/62) type 2 diabetes mellitus patients and 3% (4/119) autoimmune disease controls were GAD(65) Ab positive in the ELISA. Levels of positivity in an immunoprecipitation assay (IPA) based on 125I-labelled GAD(65) (cut-off 25 units/ml) were 1%, 82%, 0% and 3%, respectively. ELISA inter-assay coefficients of variation (n=12) were 7.3%, 3.8%, 7.2% and 6.3% at 5.4, 40.8, 137 and 382 units/ml, respectively. CONCLUSIONS: The ELISA we have described has sensitivity and specificity at least as high as current radioactive assays. It has good precision and handling making it suitable for routine use.

Autoantibodies↗
PubMed2002

Thyroid-stimulating monoclonal antibodies.

Thyrotropin (TSH) receptor monoclonal antibodies (TSHR mAbs) were obtained from cDNA-immunized NMRI mice. Three mAb immunoglobulin Gs (IgGs) (TSmAbs 1-3) that had distinct V(H )and V(L) region sequences stimulated cyclic adenosine monophosphate (cAMP) production in isolated porcine thyroid cells greater than 10x basal and as little as 20 ng/mL (0.13 nmol/L) of TSmAb 1 IgG caused a 2x basal stimulation. TSmAb 1 and 2 Fab fragments were also effective stimulators and thyroid-stimulating activities of the IgGs and Fabs were confirmed using TSHR transfected Chinese hamster ovary (CHO) cells. The TSmAbs also inhibited (125)I-labeled TSH binding to TSHR-coated tubes by 50% or more at concentrations of 1 microg/mL or less and gave 15%-20% inhibition at 20-50 ng/mL. (125)I-labeled TSmAbs bound to TSHR-coated tubes with high affinity (approximately 10(10) L/mol) and this binding was inhibited by TSHR autoantibodies with both TSH agonist and antagonist activities. Inhibition of labeled TSmAb binding by Graves' sera correlated well with inhibition of TSH binding (r = 0.96; n = 18; p < 0.001 for TSmAb 2). The TSmAbs have considerable potential as (1) new probes for TSHR structure-function studies, (2) reagents for new assays for TSHR autoantibodies, and (3) alternatives to recombinant TSH in various in vivo applications.

Animals↗
PubMed2002

Characterization of the thyrotropin binding pocket.

A panel of monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) was prepared using three different immunization strategies. The mAbs obtained (n = 138) reacted with linear epitopes covering most of the TSHR extracellular domain and with conformational epitopes. mAbs that bound to five different regions of the TSHR (amino acids [aa] 32-41, aa 36-42, aa 246-260, aa 277-296, and aa 381-385) were able to inhibit (125)I-labeled thyrotropin (TSH) binding to solubilized TSHR preparations. Fab and immunoglobulin G (IgG) preparations were similarly effective inhibitors for mAbs reactive with aa 246-260, aa 277-291 and aa 381-385 suggesting that these three regions of the TSHR are involved in TSH binding. In contrast mAbs reactive with aa 32-41 and aa 36-42 were not effective at inhibiting TSH binding when Fab preparations were used, suggesting that these N terminal regions of the TSHR were less critical for TSH binding. Our studies suggest that three distinct and discontinuous regions of the TSHR (aa 246-260 and 277-296 on the TSHR A subunit) and aa 381-385 (on the TSHR B subunit) fold together to form a complex TSH binding pocket. Alignment of the aa sequences of these three regions in TSHRs from different species indicates that they are highly conserved.

Animals↗
PubMedDate not supplied

A mouse monoclonal antibody to the TSHR.

INTRODUCTION: Mouse monoclonal antibodies (mAbs) with the ability to inhibit thyrotropin (TSH) binding to the TSH receptor (TSHR) are useful tools to study TSH-TSHR interaction. The 3C3 mAb we produced was found to inhibit binding of TSH to human (h)TSHR but not to porcine (p)TSHR. MATERIAL/METHODS: Purified 3C3 immunoglobulin G (IgG) and its antibody-binding fragment were prepared using standard methods and their ability to inhibit TSH binding to hTSHR or pTSHR was analyzed using a coated tube assay. The TSHR epitope reactive with 3C3 IgG was determined using Western blotting, ELISA based on peptides corresponding to the TSHR sequence, and the SPOT synthesis technique. RNA was isolated from 3C3 hybridoma cells and the mAb variable (V) region genes were sequenced and analyzed. RESULTS: 3C3 mAb had a 1 x 108 l/mol binding affinity to the hTSHR as assessed by Scatchard analysis. 3C3 reacted with the hTSHR region between amino acids (aa) 212-230, and two aa differences were found between the corresponding regions in the hTSHR and pTSHR. The light chain (LC) genes of 3C3 were derived from the Vk21 germ-line (97.6% homology) and Jk2 genes. The heavy chain (HC) genes were from the V130 germ-line (94.6% homology) combined with a D gene (not identified) and JH3 gene. The replacement/ silent mutation ratios of 6.0 and 6.5 for the LC and the HC V regions, respectively, indicated that 3C3 underwent antigen-driven maturation. CONCLUSIONS: Mouse mAbs of this type should be useful in studying the interactions between the TSHR, TSH, and mAbs in more detail.

Amino Acid Sequence↗