sepharose and Graves-Disease

We have prepared a biotinylated thyrotropin receptor (TSHR-BIO), and characterized its activity in cells and when bound to solid phase (streptavidin agarose). TSHR-BIO consists of the N-terminal 725 amino acids of the human thyrotropin (TSH) receptor linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotinylation of the protein. TSHR-BIO was expressed using a vaccinia virus expression system. HeLa cells infected with recombinant virus produced large amounts of TSH receptor of approximately 120,000 molecules per cell. Vaccinia virus produced TSHR-BIO was fully functional interacting with TSH (Kd of 2.3+/-0.1 x 10(-10) M) and coupling to cyclic adenosine monophosphate (cAMP) second messenger system. The expressed protein was biotinylated with high efficiency; more than 90% of TSHR-BIO was bound to streptavidin. We have shown the application of streptavidin agarose immobilized TSHR-BIO for the detection of thyroid-binding inhibiting immunoglobulines in unfractionated sera. There was a good positive correlation between the results obtained in this assay and the commercially available TRAK assay performed with solubilized porcine TSH receptor (r = 0.71; p < 0.001, in 45 sera of patients with Graves' disease and 17 normal sera).

Topics: Autoantibodies; Bacterial Proteins; Biotin; Cyclic AMP; Genetic Vectors; Graves Disease; HeLa Cells; Humans; Receptors, Thyrotropin; Recombinant Fusion Proteins; Recombination, Genetic; Second Messenger Systems; Sepharose; Thyrotropin; Vaccinia virus

By coupling 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)dione (MECH) to divinyl sulfone activated agarose, a novel thiophilic matrix was obtained which allows the binding of immunoglobulins from different sources. In contrast to other thiophilic gels, antibodies are bound at low ionic strength and can easily be desorbed in intact form by elution with dilute alkali. The potential of using the MECH-gel was demonstrated by the purification of antibodies from human and animal (goat, rabbit, mouse) sera. The functional integrity of the purified antibodies was established with cytoplasmic islet cell antibodies from the sera of patients with type I diabetes and autoantibodies against thyroid peroxidase from patients with Graves' and Hashimoto's disease.

Topics: Animals; Autoantibodies; Chromatography, Affinity; Diabetes Mellitus, Type 1; Fluorescent Antibody Technique, Indirect; Graves Disease; Humans; Immunoglobulins; Iodide Peroxidase; Islets of Langerhans; Osmolar Concentration; Protein Binding; Quinazolines; Radioimmunoassay; Sepharose; Sulfones; Thyroiditis, Autoimmune

A thyrotropin (TSH) binding inhibiting protein (TBIP) that inhibits TSH binding to the TSH receptor, as determined by the TSH receptor assay, was purified from human and porcine thyroid. The soluble fraction (100,000 x g supernatant of Graves' thyroid homogenate) was precipitated with ammonium sulfate between 1.75 to 2.5 mol/L. TBIP was eluted by 0.5 mol/L sodium chloride (NaCl) containing 20 mmol/L Tris buffer, pH 7.5 from a Q-sepharose column. The unbound fraction from concanavalin A (Con A) and blue-sepharose was gel-filtered using sephadex G-100, and finally purified by Resource Q column chromatography. Purified TBIP was confirmed as a single protein band of 17 kDa. The TBI activity in the purified TBIP was significantly decreased by either etnylene glycol tetraacetate (EGTA) (1 mmol/L) or antibody to calmodulin (CaM) in the TSH receptor assay. The TBIP was confirmed immunologically as CaM by the Ouchterlony method using antibody for CaM. These findings demonstrated that the TBIP purified from human and porcine thyroids was, in fact, CaM. We examined the effects of TBIP purified from human thyroid on bovine TSH (bTSH) or thyroid stimulating antibody (TSAb)-stimulated cyclic adenosine monophosphate (cAMP) production in porcine thyroid cells (PTC). TBIP itself did not increase basal levels of cAMP production, but inhibited bTSH (100 mU/L)-stimulated cAMP production. However, TBIP did not inhibit cAMP production stimulated by TSAb-IgG and various thyroid stimulators (GTPgammaS, forskolin and pituitary adenylate cyclase-activating polypeptide [PACAP, 27 and 38 amino acids]). Authentic CaM purified from bovine brain behaved in a manner similar to that of TBIP. These data showed that CaM differentially affects thyroid stimulation by TSH and TSAb in intact thyroid cell experiments.

Topics: Animals; Antibodies; Calmodulin; Cattle; Chromatography; Chromatography, Gel; Concanavalin A; Cyclic AMP; Dextrans; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Graves Disease; Humans; Immunodiffusion; Iodine Radioisotopes; Protein Binding; Receptors, Thyrotropin; Sepharose; Sodium Dodecyl Sulfate; Swine; Thyroid Gland; Thyrotropin

Graves' disease is attributed to the presence of autoantibodies with agonist activity which interact with the TSH receptor causing thyroid hyperstimulation and hyperthyroidism. The degree of TSH-binding inhibition (TSH-BI) caused by a Graves' serum in a TSH radioligand receptor assay is considered to be an index of the prevalence of anti-TSH receptor autoantibodies in that serum. We have previously shown that the specific inhibition by Graves' serum of hCG-stimulated steroidogenesis by Leydig cells was at a site distal to receptor binding and second messenger activation. In this report, we have investigated whether the effect of Graves' serum upon Leydig cells is a property of the constitutive antibodies. Immunoglobulin-enriched fractions were obtained from Graves' and normal sera using three increasingly rigorous procedures; ammonium sulphate precipitation, caprylic acid treatment and Protein A or G-affinity purification. The TSH-BI was determined for untreated and extracted sera in two radioreceptor assays developed for use with serum, one using human thyroid membranes and the other using HeLa cells transfected with the human TSH receptor, and the results were compared with effects in the Leydig cell steroidogenesis bioassay. The specific inhibition of hCG-stimulated Leydig cell steroidogenesis by Graves' sera was not retained in the antibody fraction causing TSH-BI. Thus, the inhibitory factor appears not to be an antibody and we are now attempting to purify and identify the responsible factor from Graves' serum.

Topics: Ammonium Sulfate; Autoantibodies; Caprylates; Graves Disease; HeLa Cells; Humans; Immunoglobulin G; Leydig Cells; Male; Sepharose; Staphylococcal Protein A; Testosterone; Thyroid Gland; Thyrotropin

Thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) in fragments released from TSAb-IgG by protease digestion were examined. The unbound fraction (UF) and the bound fraction (BF) were separated using a protein A-Sepharose column after papain hydrolysis (more hydrolysis at pH 5.0 than pH 7.5) of TSAb-IgG. When both fractions were gel filtrated on a Sephadex G-100 column, the TS and TBI activity were found in both Fab fraction (Mr 50 kDa) and the retarded fraction (between Mr 50 and 20 kDa) in the UF, and also in the first fraction (undigested IgG, Mr 160 kDa), the second fraction (Fc with tracer amounts of Fab, Mr 50 kDa), and the retarded fraction (between Mr 50 and 20 kDa) of the BF. The biological activity in the second fraction was suggested as being derived from Fab, because the activity bound to the anti-F(ab')2 column but did not bind to the anti-Fc column. Anti-Tg and anti-TPO activities were found in Fab, but were not found in the retarded fraction that consisted of Mr 20-30 kDa. In pepsin hydrolysis the UF from the protein A column consisted of both F(ab')2 (Mr 100 kDa) and pF'c (CH3) (Mr 25 kDa), and the BF consisted of only the undigested IgG. The biological activities were found in both the F(ab')2 fraction and the retarded fraction (between Mr 100 and 25 kDa). Anti-Tg and anti-TPO activities were found in F(ab')2, but no activity was observed in the Mr 25-kDa fraction. The present study showed that the biological activity of TSAb is distributed in not only the Fab or F(ab')2 fragment, but also in thyroactive smaller components (TSC) (Mr 20-30 kDa) without antigen-binding activity such as anti-Tg and anti-TPO. We suggest that TSC may be released from the Fab fragment region of TSAb-IgG by protease hydrolysis.

Topics: Animals; Chromatography, Affinity; Chromatography, Gel; Endopeptidases; Graves Disease; Humans; Hydrolysis; Immunoglobulin Fab Fragments; Immunoglobulins, Thyroid-Stimulating; Iodide Peroxidase; Long-Acting Thyroid Stimulator; Papain; Pepsin A; Sepharose; Staphylococcal Protein A; Swine; Thyroglobulin; Thyrotropin

For further characterization immunoglobulins G (IgG) of 14 patients with Graves' disease were fractionated on Protein A bound Sepharose. Sufficiently enriched IgG-subclasses were obtained despite incomplete separation. TSH displacing activity (TDA) was not homogeneously distributed in the subclasses, however IgG1 was most predominant in TDA followed by IgG3, IgG2 being essentially inactive. In two subjects TDA in whole IgG gave positive results in subclasses after fractionation, thus providing evidence, that it would be theoretically possible to move sensitivity of the method close to 100% after subfractionation. TDA was not enriched together with thyroid cell antibodies. We conclude from these results that TDA-IgG is characterized by restricted heterogeneity notwithstanding the incomplete separation of the IgG-subclasses.

Topics: Autoantibodies; Chromatography, Ion Exchange; Graves Disease; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin G; Immunoglobulins, Thyroid-Stimulating; Nephelometry and Turbidimetry; Radioligand Assay; Sepharose; Thyroid Gland

Sera from a reference pool, 29 controls and 18 patients with Graves' disease were fractionated on sepharose CL-4B protein A. After a primary elution of the unbound fraction at normal pH, IgG subclasses 1, 2 and 4 were eluted using a discontinuous pH gradient. The eluate was monitored by UV-absorption at 280 nm and three peaks were observed within narrow pH ranges in all sera tested. From the reference pool (n = 10) peak A was eluted at pH 6.6 +/- 0.1, peak B at 4.2 +/- 0.03 and peak C at 3.8 +/- 0.01. This distribution was partially related to the IgG subclasses with IgG1 recovered in peak C, while IgG2 and IgG4 were distributed in both peak B and C. Thyroid adenylate cyclase stimulating antibodies (TSAb) and thyrotrophin binding inhibiting immunoglobulins (TBII) were measured in the pooled fractions under these peaks. In the reference pool total assay variations (SD) were 10-22 per cent. The 95 percentiles of the 29 control values were used as reference range. In the 18 patients TSAb were positive in 4 in fraction A, 9 in B and 12 in C. TBII were found in one in fraction A, none in B and 13 in C. No correlation between TSAb and TBII values were found before or after fractionation. In most cases TSAb activity was highest in fraction C, however, in 3 patients fraction A was the more active and in these patients TBII were only found in fraction C. It is concluded, that the present results indicate a heterogeneity of the thyroid stimulating immunoglobulins with separation of the TSAb and TBII activities in some patients.

Topics: Absorption; Adult; Aged; Chromatography, Affinity; Female; Graves Disease; Humans; Immunoglobulin G; Male; Middle Aged; Receptors, IgG; Receptors, Immunologic; Sepharose; Staphylococcal Protein A; Thyroid Gland; Thyrotropin; Ultraviolet Rays