DE19949184C2 - A method for the determination of various types of autoantibodies against the TSH receptor by selective immunoprecipitation, fusion proteins for carrying out such a method and the use of labeled TSH receptor chimeras in such a method - Google Patents

Description

The present invention relates to a method for determining different types of autoantibodies against the TSH receptor that differ from each other in terms of their distinguish pathogenic character by selective immune precipitation, labeled fusion proteins of TSH receptor Chimeras to carry out such a procedure and the Use of labeled TSH receptor chimeras in the Be autoantibodies against the TSH receptor Immunoprecipitation.

The receptor for the hormone TSH (thyroid stimulating Hor mon), the TSH receptor (TSHR), plays a key role in the function and growth of thyroid cells. This Receptor is a member of a subfamily of G protein-kop pelten glycoprotein receptors, which also in particular still the receptors for the luteinizing hormone / chorionic gonadotrophin  (LH / CGR) and the follicle stimulating hormone (FSHR) includes. The receptors of this subfamily have a large one N-terminal extracellular domain responsible for the ligands Binding is essential and shown for them was that she is involved in the signal transmission. The Transmission of the TSHR signal is mainly through the Akti Vation of adenylate cyclase mediates, leading to an increase of the intracellular cAMP level.

Part of the great interest in the TSHR is on its role as the primary autoantigen in various thyroid auto Immune diseases attributed to the appearance of the car antibodies to the TSHR are accompanied (TSHR-Auto-Ab). To Such thyroid autoimmune diseases belong especially Graves 'disease (Graves' disease) Graves' disease), a condition that leads to hyperthyroidism Autoimmune disease which is among the most common human auto immune disorders ever heard, as well as the Hashimoto Thy reoiditis and idiopathic myxedema with a shield gland under-function can be connected.

The TSHR only comes in in the surface of thyroid cells very small quantities (in the order of 1,000-10,000 Receptors per cell). An explanation of the chemical Structure of the human TSH receptor (hTSHR), d. H. the sequence the DNA coding for it and the DNA derived from it Amino acid sequence of the receptor itself, succeeded in late 1989 (cf. Libert F. et al., Biochem. Biophys. Res. Commun. 165: 1250- 1255; Nagayama Y. et al., Biochem. Biophys. Res. Commun. 165: 1184-1190; see. also EP-A-0433509 and WO-A-91/09121; such as WO-A-91/09137; WO-A-91/10735 and WO-A-91/03483; furthermore Yuji Nagayama & Basil Rapoport, in: Molecular Endocrinology, Vol. 6 No. 2, pp. 145-156 and the literature cited therein). Since became the hTSHR polypeptide and selected partial peptides thereof expressed in a variety of systems, showing that a functional TSHR is made only in mammalian cells can be, both transient and in the form of a  stable cell line, e.g. B. using adenovirus and Vaccinia virus expression systems.

The traditional difficulty, the hTSHR without functionality to mark loss of activity and in a soluble or disper To provide formable form, the Ent Development of clinically usable direct immunoprecipitates tion assays for the detection of pathological hTSHR autoantibodies pern.

An immunoprecipitation assay for TSHR-Auto-Ab is known from the work of NG Morgenthaler at al., Exp Clin Endo crinol Diabetes 104 (1996) Suppl 4, pp. 56-59. In this assay, a preparation of an extracellular part of a recombinant TSH receptor labeled by ³⁵ S-methionine is used as a reagent for the precipitation of the TSHR-Auto-Ab present in a sample. The assay shows neither selectivity for stimulating (TSAb) nor for blocking (TBAb) TSHR-Auto-Ab, but also detects those TSHR-Auto-Ab that do not compete with bTSH. The production of the ³⁵ S-labeled receptor by in vitro translation is, however, extremely complex and expensive, and there are no measuring devices which are suitable for a routine clinical measurement of the radiation emitted by ³⁵ S. The method is therefore not suitable as a measuring method for routine clinical diagnosis.

From DE 198 01 154 or J. Endocrinol. 160: 239-245 (1999) known a method that enables a complete functional rhTSHR in pure, solubilized and radioactive to produce the marked shape. It is u. a. suggested such a radiolabelled rhTSHR to determine the Total amount of TSHR-Auto-Ab in a precipitation assay use, which has the advantage that it is not only such TSHR-Auto-Ab detects who compete with marked TSH (i.e. so-called "immunoglobulins inhibiting TSH binding" or TBII). The proposed procedure does not allow within the  A distinction is present in a TSHR-Auto-Ab sample after stimulating (TSAb), blocking (TBAb) and possibly neither stimulating nor blocking, "neutral" TSHR- Auto-Ab to meet. Also working with a radio In many cases, active labels are generally undesirable.

The method according to DE 198 01 154 is based on a method for the production of functional rhTSHR fusion products, the was described in DE 196 45 729 and WO 98/20343 and there was used to produce rhTSHR fusion proteins in addition to the TSHR sequence, relatively short peptide residues for one Immobilization or labeling of the TSHR or a biotiny had peptide residue. The production of fusions proteins with a functional TSHR and long proteinre most, e.g. B. Enyzmresten, has not been reported yet. Because on the Working methods disclosed in DE 196 45 729 and DE 198 01 154 also used in the context of the present application will be based on the content of the two publications Supplement the disclosure of the present application express Lich referred.

From WO 91/10735 fusion proteins are known which Sequence of the entire TSHR or significant parts of it in Contain linkage with β-galactosidase or ubiquitin. The Fusion proteins are made by transfection of E. coli corresponding cDNA constructs. Because of the named The prokaryotic expression system is not used functional TSHR fusion proteins obtained.

The only one currently usable in clinical practice Assay for the determination of TSHR-Auto-Ab is therefore an indirect one working competitive assay in which the inhibition of Binding of labeled bTSH to a solubilized porcine TSHR from porcine thyroid or to one for "coated tube" Applications immobilized rhTSHR according to DE 196 51 093 or WO 98/26294 or DE 198 01 319 or WO 99/3678 or J. Clin. Endo CINOL. Metab., Vol. 84, No. 1, pp. 90-97 (1999) used for the measurement  becomes. It only detects the autoantibodies from the so-called TBII Type.

A direct immunoprecipitation assay would be used for diagnosis Graves' disease is an advantage in that it it z. B. could allow all autoantibodies against the TSHR evidence, including those who bind to the TSHR, however, the interaction of TSH or the competitor Do not disturb bTSH with the TSHR. Since it is now known that  TSHR-Auto-Ab also not in the case of Graves' disease patients only stimulating autoantibodies that cause hyperthyroidism call, include, but also non-pathogenic "neutral" autoantibodies and even blocking, it would be however, it is desirable to also have an assay available have, for a type of autoantibodies to be determined is selective and e.g. B. a distinction from pathogenic stimulating and blocking and "neutral" autoantibodies pern enables.

To differentiate between stimulating, blocking and neutral autoantibodies have so far been essentially organic assays used to determine whether the cAMP product tion of cells that have a natural Chen or recombinant TSHR contained by the presence of certain sera or autoantibodies is increased or whether caused by a given TSH concentration in the sample te cAMP production by the sera or autoantibodies is weakening. In the context of such investigations or Determination of those areas of the sequence of the extracellular Domain of the TSHR, to the TSH or the different types of Binding autoantibodies (TSAb, TBAb) has already been done worked with hTSHR chimeras in which certain hTSHR chimeras Partial sequences by corresponding sequences of other G-protein coupled receptors, especially rat LH CG receptors that were replaced. It showed up in cAMP-Stimu lation assays using cells containing these chimeras express that for the binding of stimulating TSHR auto From (TSAb) the amino acids of the N-terminus of the hTSHR are required, in particular amino acids of the Sequence 8-165 calculated from the initial methionine residue. For the Binding of blocking TSHR-Auto-Ab (TBAb) has been demonstrated amino acids from the C-terminus of the extracellular domain of hTHSR, especially amino acids of sequence 261-370, as required.

Related to the use of TSHR / LHCGR chimeras in  Bioassays deserve special mention in the work of Tahara K, Ban K, Minegishi T, Kohn LD. 1991, Biochem Biophys Res Commun. 179: 70-77; Tahara K, Ishikawa N, Yamamoto K, Hirai A, Ito K, Tamura Y, Yoshida S, Saito Y, Kohn LD. 1997, Thyroid 7: 867-877 and Grasso YZ, Kim MR, Falman C, Kohn LD, Tahara K, Gupta MK. 1999, Thyroid 9: 531-537. To those mentioned in the Works cited further references will become Ver directed deepening.

It is an object of the present invention to provide a differential diagnostic method for the selective determination of TSHR To create Auto-Ab with different pathogenic effects, that works on the principle of an immunoprecipitation assay and where it is preferably also possible to radio to dispense with active markings or detection methods.

It is also an object of the present invention for a New labeled hTSHR preparation suitable for such a method rate to create.

These tasks are accomplished by a method according to claim 1, the labeled rhTSHR chimeras according to claim 8 and, in whole general form, through the use of rhTSHR chimeras in Form of fusion proteins according to claim 11 solved.

Advantageous, currently preferred embodiments of the Objects of claims 1, 8 and 11 are in the sub claims 2 to 6, 9 and 10 and 12 reproduced.

In the preceding and the following text of this application, the reagents or analytes / biomolecules used are generally characterized by various abbreviations, which are always to be understood in the following meanings, unless something for the person skilled in the art results from the specific context other. The use of the special information is given for the sake of an exact description of the tests and measurements carried out, but does not mean that the results and conclusions described apply only to the special case described and cannot be generalized using the relevant specialist knowledge.
TSH = thyroid stimulating hormone (Thyreotropin). If the abbreviation TSH is used without further additives, it is not a specific product, but the binding or function of the hormone is discussed in general terms.
¹²⁵ I-bTSH = radioiodinated bTSH (bovine TSH) used as competitor. In connection with the description of assays from BRAHMS Diagnostica GmbH, ¹²⁵ I-bTSH stands in particular for a product as obtained according to DE 42 37 430 C1 or EP 0 668 775 B1 and part of the TRAK-Assay® from Fa. BRAHMS Diagnostica GmbH is.
TSHR = The TSH receptor, a glycoprotein receptor anchored in the thyroid membrane. If the abbreviation TSHR is used without further additions, it is not a specific product, but the function of the receptor or its binding participation is discussed in a general way.
rTSHR = genetically engineered (recombinant) polypeptide which has the amino acid sequence of a naturally occurring TSHR, in particular a human TSHR (hTSHR), at least to such an extent that it can be referred to as a "functional TSH receptor", which means that with respect to the binding of autoantibodies against the TSHR or hTSH to a significant extent like the naturally occurring, in particular human, TSHR behaves. If the abbreviation rTSHR is used without further additives, it is not a specific product, ie rTSHR can stand for the recombinant complete, more or less glycosylated polypeptide, a partial sequence of a sufficient length or a genetically engineered fusion product thereof (as is e.g. described in the international patent application PCT / EP 97/06121). Without further explanation, a product simply referred to as “rTSHR” or “rhTSHR” is available as a membrane preparation, from which the product may be used as a purified extract, if appropriate by suitable solubilization of membranes of the cells used for expression of the recombinant polypeptide using detergents was obtained.
WT rTSHR = "wild type TSHR" - an essentially complete rhTSHR for determining the total amount of TSHR-Auto-Ab (Total) in a sample.
Fusion-TSHR = rhTSHR with attached amino acid sequences that extend the polypeptide sequence of a functional TSHR.
TSHR-LUC = rhTSHR fusion protein extended around the protein residue of an enzyme, namely in all examples of a firefly luciferase enzyme (LUC).
TSHR / LH-CGR = "TSHR chimera", ie a rhTSHR, in which parts of the amino acid sequence by comparable sequences of another receptor with a different binding behavior towards TSHR-Auto-Ab, in particular non-binding sequences, by all examples Sequences of a rat LH-CG receptor (LH-CGR) are replaced.
TSHR / LH-CGR-LUC = rhTSHR fusion protein which contains a TSHR chimer and an enzyme residue which is a firefly luciferase residue in all examples.
TSHR-Auto-Ab = Detectable autoantibodies against the TSH receptor of any specificity in biological samples, especially human serum or plasma.
TSAb = TSHR-Auto-Ab stimulating the thyroid. Their detection is particularly important for the diagnosis of Graves' disease.
TBAb = The effect of TSH on the thyroid-blocking TSHR-Auto-Ab, the detection of which can be related to hypothyroidism.
TBII = In competitive assays based on a competition of labeled TSH with TSHR-Auto-Ab for the binding sites of a TSHR preparation, detectable TSHR-Auto-Ab. They can be TSAb and / or TBAb and / or neutral TSHR-Auto-Ab. The TRAK-Assay® from BRAHMS Diagno stica GmbH is a commercial assay (radioreceptor assay) for determining TSHR-Auto-Ab of the TBII type.

As part of the work described in this application have been described with the intention of selective immune precipitation assays for the determination of TSHR-Auto-Ab types  create new reagents in the form of fusion proteins create a firefly luciferase residue as an example contain for a detectable enzyme residue, and one recombinant functional human TSH receptor (rhTSHR), the also known as "wild type TSHR" (WT TSHR), as well as three different TSHR chimeras (chimeras A, B and C). The chimeras were constructed in such a way that, based on knowledge gained with Bioassays have been obtained using such chimeras were the hope that they would be for stimulative or blocking TSHR-Auto-Ab could be selective. Another Chimaera (Chimaera C) was one that was neither stimulant still blocking TSHR-Auto-Ab should react and therefore for so-called "neutral" autoantibodies without known ones pathogenic effect should be selective if sera actually also contain such neutral TSHR-Auto-Ab.

Various ver search series carried out in which it should be determined whether adding an enzyme protein residue to the functionality of the TSHR residue in the fusion product impaired and / or whether the enzyme activity through the integration into a fusion product of the attached enzyme residue is changed significantly. the Such investigations were also necessary because the previously known TSHR fusion products only ever shorter Peptide residues, but not long enzyme residues of protein Contained nature.

As will be shown in more detail below, the Functionality of the TSHR or the TSHR chimeras by the Adding an enzyme residue is not significantly changed, and also that the enzyme residue contained in the concrete Examples was a firefly luciferase residue, its effect retains and can be used for the usual detection reaction can.

After it was found that the TSHR or TSHR Chima ren-enzyme fusion products had the desired reactivities,  have been used in immunoprecipitation assays, taking sera from normal people and patients with thyroid diseases have been tested and the results obtained have been related to the results related to the previously known assays were obtained, namely once with a competitive assay for determining TSHR-Auto-Ab using TSH compete (TBII), and on the other hand with bioassays which measured the cAMP production of transformed cells has been.

The invention based on manufacturing and Application examples with reference to a figure yet explained in more detail.

Materials and methods

The concentration data mM refer to mmol / l.

materials

¹²⁵ I-bTSH (56 µCi / µg) and the commercial TRAK-Assay® kits used for the determination, as well as the protein A suspension used and the monoclonal mouse antibodies against the extracellular domain of the hTSHR, were from BRAHMS Diagnostica GmbH , Berlin provided. The plasmid pcDNA3-rLHR (B9) was developed by Dr. DL Segaloff (The UNivesity of Iowa, USA). The plasmid pSP-luc + NF was purchased from Promega (Heidelberg, Germany). Kits from Amersham (Braunschweig, Germany) were used as the ECL Western blot detection kit and cAMP-RIA kit. The DNA primers

were obtained from Interactiva (Ulm, Germany).  

cell culture

HeLa cells were grown in Dulbecco's modified Eagle's Medium supplemented with 10% fetal bovine serum. The cells were cultured in a 5% CO ₂ atmosphere at 37 ° C.

Construction of TSHR / LH-CGR chimeras

Three different chimeras of the human TSHR, in which Partial sequences by corresponding sequences of a rat LH-CGR were replaced as described in Biochem Biophys Res Commun. (1991), 179: 70-77. At one of the Chimeras, hereinafter referred to as "Chimeras A", were the TSHR amino acids 8-165 by the comparable amino acids 10-166 of the LH-CGR replaced; for "Chimera B" the TSHR Amino acids 261-370 by the comparable amino acids 261-329 a rat LH-CGR replaced; while in the case of "Chimera C" both amino acids 8-165 and amino acids 261-370 of TSHR were replaced by comparable LH-CGR amino acids.

The plasmid pcDNA3-rLHR (B9) assumed that the sequence for the rat LH-CGR receptor contains. The DNA sequences for amino acids 10-165 as well 261-329 were encoded using the PCR technique of the primer pairs P1-P2 and P3-P4, the NsiI, AatII, BfrI and SacI restriction sites contained, duplicated, whereby NsiI / AatII or BfrI / SacI-PCR fragments were obtained. In parallel was a pTM1-TSHR-FLAG-6HIS plasmid which according to DE 196 45 729 or Exp Clin Endocrinol Diabetes. 5: 282-290 (1997) with PstI-AatII or BfrI-SacI restriction endonuclea digested. The fragments generated were removed and by the PCR fragments obtained from the rat LH-CG receptor replaced, making the cDNA sequences for the different TSHR / LH-CGR chimeras A, B and C were obtained.  

The vector pTM1-TSHR-FLAG-6HIS or the new one obtained therefrom Vectors with the TSHR / LH-CGR-DNA (pTM1-TSHR / LH-CGR) were labeled with ClaI linearized, and the "sticky" ends were used filled up by Klenow polymerase. That for expression certain fragment was extracted using BamHI cut and into the HindII / BamHI site of a vaccinia trans fervectors p7.5K131 subcloned, as in DE 196 45 729 or Exp Clin Endocrinol Diabetes. 5: 282-290 (1997) is.

Production of vectors for the expression of TSHR or TSHR / LH-CGR chimeras and the enzyme luciferase

Using the plasmid pSP-luc + NF, which the sequence of Firefly luciferase contains DNA sequences designed for the complete human "wild type" TSHR or for TSHR / LH-CGR- Encode chimeras associated with the cDNA required for the 550th Sequence of firefly luciferase comprising amino acids encode. For this, the sequence for the firefly luciferase cut from plasmid pSP-luc + NF with BstEII and EcoRI and to corresponding locations of the p7.5K131-TSHR- or p7.5K131-TSHR / LH- CGR vectors inserted, causing vaccinia recombination plasmids p7.5K131-TSHR-LUC or p7.5K131-TSHR / LH-CGR-LUC were obtained.

Generation of vaccinia virus recombinants

Using the further details in DE 196 45 729 or Exp Clin Endocrinol Diabetes. 5: 282-290 (1997) or in J Virol. 68: 8423-8427 (1994), the cDNA for the TSHR luciferase or TSHR / LH-CGR luciferase construct from p7.5K131-TSHR-LUC or p7.5K131-TSHR / LH-CGR-LUC plasmids in the Genome of the wild-type vaccinia virus strain Copenhagen under homologous Recombination incorporated into the thymidine kinase gene.  

Expression and extraction of fusion proteins TSHR-LUC or TSHR / LH-CGR-LUC as cell extract

Confluent HeLa cells grown in a 75 cm ² plate (approximately 20 x 10 ⁶ cells) were infected with one colony forming unit (cfu) of recombinant vaccinia virus per cell and incubated for 24 h at 37 ° C. Infected cells were scraped into a phosphate buffered saline (PBS) and washed four times with PBS with centrifugation at 2500 rpm. The cells obtained were lysed in 0.3 ml of a buffer A (20 mM Hepes-KOH; pH 7.5; 50 mM NaCl; 1% Triton X100; 10% glycerol) with freezing and thawing. The suspension obtained was centrifuged at 30,000 g for 1 hour, the supernatant (about 8 mg / ml total protein) was collected and stored at -70 ° C.

The supernatant (extract) obtained as described was in the Immunoprecipitation experiments as TSHR-LUC or TSHR / LH-CGR-LUC used.

Production of cell fractions

As described above, a monolayer of confluent HeLa cells grown in a 75 cm ² plate was infected with one plaque-forming unit of a recombinant vaccinia virus per cell and incubated at 37 ° C for 24 hours. Infected cells were washed with PBS, harvested and pelleted with centrifugation at 1200 rpm. The cell pellet obtained was resuspended in 0.3 ml of a buffer which contained 10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 10% glycerol and a mixture of proteases inhibitors. The suspension was then homogenized at 4 ° C. by 20 strokes in a glass / Teflon homogenizer and then centrifuged at 800 g for 15 min and then at 30,000 g for 1 h. The supernatant (the cytoplasmic fraction) was collected. The membrane pellet was worked up by homogenizing it at 4 ° C. by 20 strokes in a glass / Teflon homogenizer in 0.3 ml of 1% Triton X100 in the same buffer and then centrifuging at 30,000 g for 1 h. The supernatant (Triton X100 membrane extract) was collected and stored at -70 ° C.

Immunoprecipitation experiments

For the immunoprecipitation analyzes described below, 40 μl TSHR-LUC or TSHR / LH-CGR-LUC (2 × 10 ⁶ relative light units) were mixed with 10 μl serum and incubated overnight at 4 ° C. To precipitate the immune complexes formed, 25 μl of a 20% protein A suspension in buffer A were added, and the mixture was incubated with shaking at 4 ° C. for 1 hour. With centrifugation, the suspension was washed 4 times with 1 ml of buffer A (without Triton X100). Finally, the pellet was resuspended in 50 ul buffer A (without Triton X100) and the luciferase activity in the suspension obtained from the pellet was determined using a luminometer (Lumat 9501, Berthold, Wildbad, Germany) according to the usual determination protocol. The results obtained were expressed as bound relative light units.

Binding of ¹²⁵ I-TSH to TSHR-LUC or TSHR / LH-CGR-LUC

The binding of ¹²⁵ I-TSH to the various solubilized luciferase fusion proteins was tested in a total volume of 200 µl, which was composed of 50 µl fusion protein solution (cell extract), 50 µl zero standard serum and 100 µl of a ¹²⁵ I TSH solution (about 20,000 cpm). The reaction mixture was incubated for 2 hours at room temperature, after which the complexes of ¹²⁵ I-TSH / fusion proteins were precipitated by adding 2 ml of polyethylene glycol. After centrifugation at 2000 g for 10 min, the gamma radiation in the pellet was measured.

The extent of non-specific binding was determined in the presence of 10 ^-7 M TSH and was <5% of the total binding.

Determination of immunoglobulins that inhibit TSH binding (TBII)

Determination of the TSHR-Auto-Ab by type of the TSH binding inhibitory immunoglobulins (TBII) was determined using Test tubes that have antibody affini on their walls have immobilized rhTSHR (TRAK Assay® from B.R.A.H.M.S Diagnostica GmbH; see. also J Clin Endocrinol Metab. 84: 90-97, 1999), according to the manufacturer's instructions guided. The results were, using a standard curve (displacement curve with standard sera 0, 1, 2, 4, 16, 40 IU / l from the TRAK Assay®), in international units / liter (IU / l). The amount of autoantibodies in the test serum is proportional to the measured value (IU / l).

Bioassay for the detection of stimulating TSHR-Auto-Ab (TSAb)

Detection of TSAb was performed in unfractionated sera using CHO cells that stably express the TSHR. The determination of extracellular cAMP in the supernatant was carried out using a commercial cAMP-RIA kit in accordance with the manufacturer's instructions. The stimulation index (SI) was determined in accordance with the publication Vlase H et al. in Endocrinology (1995), 136: 4415-4423 calculated as follows:

SI (%) = 100 × (cAMP patient / cAMP negative control sample).

Bioassay for the detection of blocking TSHR-Auto-Ab (TBAb)

Bovine TSH (10 mU / ml) was added to CHO cells stably expressing the hTSHR with either control or test serum. The respective cAMP production was measured and compared as recently described (Wallaschofski and Paschke, Clin Endocrinol. 50: 365-372, 1999). The determination of extracellular cAMP in the supernatant was carried out using a commercial cAMP-RIA kit in accordance with the manufacturer's instructions. The inhibition index (II) was calculated as:

II (%) = 100 × [1 - (cAMP patient TSH / cAMP negative control TSH)].

Western blotting

Proteins were subjected to polyacrylic gel electrophoresis (PAGE) in 8% SDS subjected and in the usual way on nitrocellulose membranes transfer. The membranes were monocloc overnight at 4 ° C nal mouse antibodies against amino acids 378-388 of extracellular domain of the TSHR in a dilution of 1:20 added. Bound antibodies were then taken under Use a Western blotting kit for enhanced chemistry luminescence detected.

Using the above materials and using the Techniques described have been described below Experiments carried out, the results of which refer to the Figures are explained in more detail.

The figures show:

. Figure 1 shows the binding of TSH to various expressed rhTSHR-products, namely curves in the form of displacement, the inhibition of binding of ¹²⁵ I-bTSH by increasing amounts of unlabeled wild-type TSH to TSHR (WT; ∎); Show TSHR-LUC (o) as well as on chimera A-LUC (▲). The results are expressed as percent of total binding in the absence of unlabeled TSH, which were 4512 cpm, 2793 cpm and 2856 cpm for WTTSHR, TSHR-LUC and Chimera A-LUC, respectively. Each value is the mean ± 2 SD of double measurements in two separate experiments.

Figure 2 shows the ability of control sera and sera from Graves' disease patients to immunoprecipitate WTTSHR-LUC compared to the ability of these sera to inhibit ¹²⁵ I-bTSH binding to an hrTSHR preparation in a conventional competitive TRAK assay. The immunoprecipitation activities of the sera are expressed as bound RLU (relative light units), the inhibition activities are expressed as international units / liter (IU / l), as explained in more detail in the text. The horizontal dotted line is the line for two standard deviations (SD) above the mean for normal people (30737 RLU).

Figure 3 shows the ability of control sera and sera from Graves' disease patients to immunoprecipitate chimeric C-LUC compared to the ability of these sera to inhibit ¹²⁵ I-bTSH binding to an hrTSHR preparation in a conventional competitive TRAK Assay® beers. The immunoprecipitation activities of the sera are expressed as bound RLU (relative light units), the inhibition activities are expressed as international units / per liter (IU / l), as explained in more detail in the text. The horizontal dotted line is the line for two standard deviations (SD) above the mean for normal people (38980 RLU).

Figure 4 shows the difference in immunoprecipitation of TSHR-LUC and chimeric C-LUC (WT-C) with sera from normal subjects and Graves' disease patients compared to the ability of these sera to bind ¹²⁵ I-TSH to a rhTSHR preparation to inhibit. The immunoprecipitation activities of the sera are expressed as values of bound RLU, the inhibition activities, as explained in the text, are expressed in IU / l. The horizontal dotted line indicates the value of two standard deviations (SD) above the mean for normal patients (3384 RLU).

Fig. 5, the difference of the Immunpräzipitatbildung of TSHR-LUC and chimera A-LUC (WT-A) in sera from normal patients (n = 62) and sera of Graves' disease patients (n = 63) compared to the ability of these sera, in stimulate cAMP synthesis using a cAMP bioassay. The immunoprecipitation activities of the sera are expressed as bound RLU, the cAMP-stimulating activities are expressed as a stimulation index (SI). The horizontal dotted line is the line for two standard deviations (SD) above the mean for normal patients (7000 RLU). The vertical dotted line shows the SI cutoff for normal sera (200%).

Figure 6 shows the difference in immunoprecipitation of chimeras A and C (A - C) by sera from Graves' disease patients (n = 63) compared to the ability of these sera to inhibit cAMP synthesis in a cAMP bioassay. The immunoprecipitation activities of the sera are expressed as bound RLU, the cAMP-blocking activities are expressed as an inhibition index (II). The horizontal dotted line shows the value of two standard deviations (SD) above the mean for normal people (835 RLU). The vertical dotted line shows the II cutoff for sera from normal people (10%).

Below are the different measurements using closer to the new TSHR-LUC or TSHR / LH-CGR-LUC fusion products described.

Reactivity of TSHR-LUC or TSHR / LH-CGR-LUC fusion products with ¹²⁵ I-TSH

In order to examine whether the luciferase fusion products show a binding behavior towards TSH which corresponds to expectations, the lysates of HeLa cells containing the corresponding fusion products and which had been infected with a suitable recombinant vaccinia virus according to the above experiments were ¹²⁵ I -TSH, and the degree of binding of ¹²⁵ I-TSH was measured as a function of the amount of unlabeled TSH present simultaneously in the reaction solution. The results of the experiments are shown in Fig. 1, in which the value for 100% represents the total amount of bound ¹²⁵ I-TSH in the absence of unlabeled TSH. It was shown that there were no fundamental differences in the displacement curves which could be seen for the fusion proteins TSH-LUC and the chimeras A-LUC, in which the TSHR amino acids 8-165 were replaced. The absolute value for the maximum TSH binding was about 1.6 times higher for the wild-type TSHR than for the LUC fusion products. If one takes into account that when expressing wild-type TSHR in HeLa cells a receptor density of approximately 150,000 receptors per cell is obtained, it can be calculated that such HeLa cells approximately two thirds of this amount, ie approximately 100,000 receptor-luciferase fusion molecules per Cell generated.

The other chimeric fusion products, namely chimeras B-LUC and chimeras C-LUC, in which TSHR residues 261-370 and TSHR residues 8-165 and 261-370 were replaced by rat LH-CGR residues, were unable to specifically bind ¹²⁵ I-TSH. These results are in agreement with what could be expected from literature data (Biochem Biophys Res Commun. 179: 70-77 (1991), J Clin Endocrinol Metab. 81: 1758-1767 (1996)).

It should be explicitly pointed out that all fusion products had the enzymatic activity of firefly luciferase. The luminescence intensity of cell lysates, which contained the fusion products TSHR-LUC or TSHR / LH-CGR-LUC, was largely identical by measuring about 10 ⁹ RLU per ml of lysate.

Like the experiments described above for the production of cell membrane branch fractions was in all cases more than 90% of the Localized luciferase activity in the membrane fraction. Just about  10% of the luciferase activity was in the water-soluble Cytoplasmic fraction found. The luciferase measurement data showed just like Western blotting experiments that all fusion products in HeLa cells according to the technique described with about same efficacy were expressed.

Detection of TSHR-Auto-Ab in sera from Graves' disease patients using the TSHR-LUC fusion products as well as Use of the various TSHR / LH-CGR-LUC fusion products

TSHR-LUC and TSHR / LH-CGR-LUC fusion products were used for the detection of autoantibodies in sera from Graves' disease by immunoprecipitation analysis according to the general procedure described above. If one assumes that the epitopes for TSAb are located in the region of amino acids 25-165 of the TSHR, and the epitopes for TBAb in the region of amino acids 261-370, the results of the immunoprecipitation analysis can be used to determine the relative amounts of the different types of Use autoantibodies in sera from Graves' disease patients. The following should apply:

• a) the binding of fusion products containing WT TSHR (WT), should be proportional to the total amount of TSHR-Auto-Ab in the sera his,
• b) the difference in the values for the binding of TSHR-LUC and Chimeras A-LUC (WT-A) (or between chimeras B-LUC and C-LUC, d. H. B-C) by the test sera should be the amount of TSAb in these Correspond to sera,
• c) the difference between the binding values for TSHR-LUC and chimeras B-LUC (WT-B) (or between chimeras A-LUC and C-LUC, i.e. H. A-C) should reflect the amount of TBAb in sera,
• d) the extent of binding to chimeric C-LUC that is neither the Epitopes for TSAb still contains for TBAb, the amount should reflect neutral autoantibodies in the test sera.

It was shown that the fusion products of both TSHR and the three TSHR / LH-CGR chimeras have a clear interaction with the autoantibodies present in sera from Graves' disease patients  showed. Table 1 shows the mean bond values, those using sera from 62 control subjects and 74 Graves' disease patients were obtained.

Table 1

The fusion products of chimeras A and C (which are not epitopes for Have TSAb) interacted with pathological autoantibodies worse than WT-TSHR products in all 74 cases. unexpected it was shown that the fusion product of chimera B (the contains no epitopes for TBAb), the pathological autoantibody in all cases more effective than fusion products of the WT TSHR. This may be due to one for TSAb binding advantageous change in the receptor structure attributed.

It was also shown that sera from normal individuals bind to all fusion products, d. H. WT-TSHR and all chimeras, with about showed the same effectiveness, with differences in all Cases ranged from 10 to 15%. The in Table 1 The results shown allow us to calculate that the mean Content of the different autoantibody types for the 74 ver measured sera from Graves' disease patients about 59% TSAb, about 7% TBAb and about 35% neutral TSHR-Auto-Ab, given as Percentage based on the total amount of TSHR-Auto-Ab. There was a positive correlation of binding values for each Combination of fusion products if their immunoprecipitation behavior compared in 74 sera from Graves' disease patients (r in the range from 0.57 to 0.91; p <0.001, see table 2).  

Table 2

The total amount of TSHR-Auto-Ab in the test sera was determined using the wild-type TSHR fused with luciferase. The specificity in a TSHR-LUC immunoprecipitation using 62 control sera and 74 sera from patients with Graves' disease of various severity is shown in FIG. 2. Only two of the 62 normal sera showed an immunoprecipitation activity with the TSHR-containing fusion product that was higher than a value that corresponded to the mean for normal persons plus two standard deviations (30740 RLU). The binding values for all other control sera were in the range from 10,000 to 30,000 RLU. The separately determined level of non-specific binding of TSHR-LUC by the protein A suspension in the absence of serum was only about 1,000 RLU. In contrast to the control sera, 73 out of 74 sera from Graves' disease patients were measured as positive in the immunoprecipitation assay. In these cases the degree of binding ranged from 30,000 to 240,000 RLU.

The data shown indicate a high specificity of the TSHR-LUC or TSHR / LH-CGR-LUC immunoprecipitation assays. At a direct comparison, a positive correlation (r = 0.61; p <0.001) between those with the immunoprecipitation assay results obtained and the results of a TBII measurement of the 74 sera from Graves' disease patients with the competitive TRAK Get Assay®. Control experiments showed that sera from Patients suffering from another autoimmune disease (sera of ten patients with juvenile diabetes mellitus) TSHR fusion products precipitated only with a very low effectiveness. The precipitation activities were comparable to those which were obtained for normal people by the binding values  ranged from 10,000 to 23,000 RLU.

Detection of "neutral" TSHR-Auto-Ab in sera from Morbus Graves patients using TSHR / LH-CGR-LUC chimeras

The amount of "neutral TSHR-Auto-Ab" in the test sera was determined using the chimeric C-LUC fusion product. It was shown that such neutral TSHR-Auto-Ab could be detected in all 62 sera of the control subjects and in all 74 sera from Graves' disease patients. The binding values for the control sera ranged from 10,000 to 50,000 RLU, while for the patient sera ranged from 10,000 to 110,000 RLU. Most of the patient sera (57 out of 74) contain approximately the same amount of neutral TSHR-Auto-Ab as the control sera. The relative amount of neutral TSHR-Auto-Ab in the patient sera is in the range of 10 to 75% of the total amount of TSHR-Auto-Ab, whereas practically all TSHR-Auto-Ab in the control sera belong to the neutral TSHR-Auto-Ab ( 90-100%). Fig. 4 shows the differences of the Präzipitationsgrads with fusion products of the wild type TSHR and chimera C with the same sera. The values obtained give the sum of TSAb and TBAb (without neutral antibodies) in the sera examined. In this case, the difference between control sera and patient sera is more pronounced, since all 74 patient sera in the present assay were clearly positive. On the other hand, only two of the control sera were considered positive, which indicates that some normal people may contain a certain amount of both stimulating and blocking TSHR-Auto-Ab. The correlation between the results obtained with this assay and the TRAK Assay® was positive (r = 0.56; p <0.001; n = 74).

Detection of TSAb in sera from Graves' disease patients Use of TSHR / LH-CGR-LUC chimeras

The amount of TSAb (stimulating TSHR-Auto-Ab) in patient sera was calculated based on the difference between the binding effectiveness for the fusion products with WT-TSHR and chimera A. Fig. 6 shows the comparison for the stimulation index (SI) of 62 control sera and 63 patient sera, determined in a conventional cAMP bioassay, compared with the difference in the binding effectiveness of the fusion products with the WT-TSHR and the chimera A (WT-A) in these serums. In the immunoprecipitation assay, 59 out of 62 control sera were negative (binding efficacy <7000 RLU), while three sera can be regarded as weakly positive (binding efficacy of 8400, 7340 and 7160 RLU). Two of the normal sera, which are indicated by an arrow in FIG. 5, were positive in the cAMP bioassay (SI = 2930 or 3080%; SI cutoff for normal persons is in the range of 200%). However, according to the immunoprecipitation assay and the clinical evaluation, these two patients were not Graves' disease patients. In the sera from Graves' disease patients, 40 out of 63 sera (63%) were positive in the cAMP bioassay (SI ≧ 200%), while in the immunoprecipitation assay 61 out of 63 sera (97%) were positive (WT-A). There was a positive correlation between the results obtained in the cAMP bioassay and those obtained in the immunoprecipitation assay (WT-A) (r = 0.43; p <0.001; n = 63). If only those sera were considered that also reacted positively in the cAMP bioassay (SI SI 200%), the correlation became much better (r = 0.61; p <0.001; n = 40).

Detection of TBAb in sera from Graves' disease patients Use of TSHR / LH-CGR-LUC chimeras

TBAb were determined in the patient sera based on the difference in the binding behavior of the LUC fusion products of chimeras A and C. Fig. 6 shows the comparison of the inhibition index (II) of 62 control sera, and 63 patient sera (as determined in the cAMP bioassay) with the difference of the binding behavior of the fusion products with the chimeras A and C (AC). 58 out of 62 control sera were negative in the cAMP bioassay (II <10%) and all control sera were negative in the immunoprecipitation assay (binding effectiveness <835 RLU). Using the cAMP bioassay, 31 of 63 patient sera (49%) were found to have TBAb activity, while 45 of 63 patient sera (71%) were found to be TBAb positive in the immunoprecipitation assay (AC). Nine immunoprecipitation-positive Basedow sera were negative in the cAMP bioassay, and conversely, nine cAMP bioassay-positive sera were negative in the immunoprecipitation assay. There was a weak positive correlation between the results in the cAMP bioassay and those from the (AC) immunoprecipitation assay (r = 0.25; p <0.05; n = 63). If only those patient sera were taken into account that were measured as TBAb-positive in the cAMP bioassay (II ≧ 10%), however, a significant correlation was obtained (r = 0.57; p <0.001; n = 31). There was no correlation between the II values of different patient sera and the ability of these sera to precipitate TSHR or TSHR / LH-CGR fusion products (data are not shown).

The data shown show a high sensitivity and specificity act of the immunoprecipitation assay according to the invention Use of enzyme-labeled fusion products that TSHR / LH-CGR- Chimeras or the wild type TSHR contained. This proves that assay according to the invention as a promising instrument for Diagnosis and investigation of the outbreak, remission and Recurrence of Graves' disease. After the previous one Trials using fusion products with fireflies Luciferase as an enzyme residue was successful it can be assumed that corresponding assays also with others Enzyme residues, which are better especially from a cost perspective suitable for a practical clinical application and the com components of known enzyme assay detection systems to let. In particular, one has to think of enzyme residues that are selected from horseradish peroxidase, alkaline phosphates se, β-galactosidase or chloramphenicol acetyltransferase.

Claims (12)

1. A method for the differential diagnostic determination of autoantibodies (TSHR-Auto-Ab) formed against the TSH receptor (TSHR) in a serum or plasma sample from a patient who is suffering from an autoimmune thyroid disease or is suspected of having such a disease , characterized in that one
in the sample, the amount of at least one type of autoantibody (TSHR-Auto-Ab) selected from (i) stimulating autoantibodies (TSAb), (ii) blocking autoantibodies (TBAb) and / or (iii) neither stimulating the non-pathogenic TSHR-Auto-Ab is still blocking, determined
by in a reaction mixture a portion of the sample in the liquid phase with a solubilized and directly labeled binding reagent in the form of a labeled rTSHR chimera in which the essential sequences for the binding of stimulating and / or blocking autoantibodies (TSAb and / or TBAb) TSHR are replaced by corresponding sequences of another receptor from the class of G protein-coupled receptors which do not bind the respective type of autoantibodies,
and converting the complexes formed from autoantibody rTSHR chimera in the reaction into a precipitate, separating this from the liquid phase of the reaction mixture and determining the amount of the label in the precipitate. 2. The method according to claim 1, characterized in that one also the quantity of all to obtain a reference value the TSH receptor binding autoantibodies (TSHR-Auto-Ab) in the Sample determined by using a further subset of the sample a solubilized and directly labeled full rhTSHR The preparation implements the complexes formed during the implementation Autoantibody rTSHR converted into a precipitate, this from the  separates the liquid phase of the reaction mixture and for recovery of the reference value determines the amount of the label in the precipitate. 3. The method according to claim 1, characterized in that the solubilized and directly labeled rTSHR chimera as a fusion protein is used in which the sequence of the rTSHR chimera while maintaining the desired functionality with a radio actively labeled peptide residue or an enzyme that is linked Is part of a detection system for enzymatic analysis. 4. The method according to claim 3, characterized in that the Enzyme in the rTSHR-chimeric fusion protein is selected from Luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase or chloramphenicol acetyltransferase. 5. The method according to any one of claims 1 to 4, characterized characterized in that the autoantibody and labeled Binding reagent formed complexes by precipitation with a selective precipitant for human antibodies in a precipitate be transferred. 6. The method according to claim 5, characterized in that the Precipitant is Protein A. 7. The method according to any one of claims 1 to 6, characterized records that as a directly labeled rTSHR chimeric fusion uses proteins that are an enzyme for chemiluminescent Detection reaction and hTSHR chimeric sequences included in which are amino acids 8-165 and / or 261-370 of the complete human TSHR by corresponding sequences of a rat LH / CG Receptor are replaced. 8. Fusion proteins that contain the sequence of an rTSHR chimera in the Partial sequences of the complete rTSHR by comparable Sequences of a related G protein-coupled receptor are replaced in combination with an enzyme that is part of a Detection system for enzymatic analysis is included  where the rTSHR part sequences their functionality at least in Regarding the binding of the different types of TSHR-Auto have maintained antibodies (TSHR-Auto-Ab). 9. Fusion proteins according to claim 8, which are used as enzyme luciferase, Horseradish peroxidase, alkaline phosphatase, β-galactosidase or chloramphenicol acetyltransferase. 10. Fusion proteins according to claim 8 or 9, the sequence contain an rTSHR chimera which is a TSHR-LH-CGR chimera, in the stimulating and / or blocking for the binding TSHR autoantibodies important partial sequences of the complete rTSHR are replaced by LH-CGR partial sequences. 11. Use of rhTSHR-LH-CGR chimeras in solubilized and directly marked form for the selective determination of stimulate the, blocking and / or neither stimulating nor blocking acting, non-pathogenic autoantibodies against the TSHR in Patient samples. 12. Use according to claim 11, wherein the directly marked rhTSHR-LH-CGR chimeras linked to enzymes in the form of Fusion proteins are used.