DE19645729C1 - Receptor binding assay for detecting auto-antibodies to thyroid stimulating hormone receptor - Google Patents

Abstract

Receptor-binding assay for detecting autoantibodies (AAb) to thyroid stimulating hormone receptor (TSHR) comprises reacting, simultaneously or sequentially, the sample with a TSRH preparation (A) and \- 1 of: (i) competitor, and (ii) agent for separation of TSHR and any material bound to it from the liquid phase, with one of (i) and (ii) being marked or selectively markable. The complex formed by binding of AAb and/or competitor to (A) is separated and the amount of label in it used to detect the presence and/or amount of AAb. (A) is a recombinant TSH-fusion receptor (I) which, compared with functional TSHR, is extended by a peptide (II) that: (i) is marked or selectively markable, and/or (ii) is immobilised or immobilisable, by binding to a selective binding partner, without significant effect on the function of TSHR in the assay. Also claimed are: (1) (I); (2) vaccinia virus having inserted in its genome, at the site of the thymidine kinase gene, a DNA sequence encoding (I), and (3) a similar but more general receptor binding assay for a hormone or autoantibody (able under physiological conditions to interact with cell-membrane localised G-protein coupled glycoprotein receptor to induce or block hormone activity) in which the receptor preparation used is a recombinant fusion extended by (II).

Description

The present invention relates to a receptor binding assay, especially for the determination of TSH receptor autoantibodies in a biological sample, using recombinant Fusion receptors used to determine TSH receptor Autoantibodies using such an assay are required recombinant TSH fusion receptors, a vaccinia virus Vector for the production of such fusion receptors and one Reagent set (kit) for performing an inventive Receptor binding assays.

It is known biologically active substances, their presence and / or amount in a biological sample, one liquid or liquefied biological sample with the help of determination methods (assays) determine where specific binders for the to be determined Substance in combination with other reagents such as labeled Competitors, immobilization reagents, precipitation reagents  and / or labeling reagents are used. The The best known such determination methods are among them immunodiagnostic methods of determination (immunoassays), which use specific bindings of the antigen-antibody type will. Even if it is due to the nature of the individual determining or to be used as competitors antigen moles cool cannot be easy to develop determination methods that meet all requirements for routine use in the clinical practice, as it usually prepares no fundamental difficulty in making such assays design that at the end of the determination a reaction product, the Contains marker portions and thus information about the Presence and / or amount of the substance to be determined provides, is bound to a solid phase, e.g. B. to the Walls of a test tube. In particular, it is usually not particularly difficult as part of such an assay used specific antibody-type binders without legs of their important for the determination procedure specific binding ability directly or indirectly mark and / or immobilize.

The situation is different in the case of receptor binding assays, d. H. in the case of assays, which at least one of the specific binding partners Is receptor. Binding of biomolecules from peptide or of a proteinic nature, e.g. B. of hormones or car antibodies, on receptors is usually very complex Nature, and the formation of a specific bond between Receptor and biomolecule is much more sensitive to it structural changes in particular of the receptor than that the case with a common binding pair of antigen / antibody is. Attempts to immobilize and / or receptors mark, usually lead to structural changes, that severely impair the functionality of receptors. As a result, there are numerous basic assay types involved in Immunoassays using an antibody / antigen binding are available for carrying out receptor bine  application assays cannot be used in practice.

In the patent DE 43 28 070 C1 is a type of a receptor dungsassays, which works according to the coated tube technique, described in which the difficulty of producing labeled or immobilized functional receptor preparation tion is avoided by going to the solid phase Components of a competitive reaction system that binds a kind of "shadow" of the actual receptor represents reaction. For creating assays for the Clinical routine diagnostics has become the method disclosed principle, however, as too complicated and therefore not very practical proven. On the general statements in the above Patent specification on the problem of receptor binding assays in general and of those for the determination of TSH receptor Autoantibodies in particular are also expressly referred to taken.

EP-B-0 488 170 also discloses cell-free receptor binding tests known in which recombinant fusion receptors an amino terminal receptor protein and a carrier protein, especially the constant part (Fc) of a heavy chain Immunoglobulins, which are used by means of an antiserum or a monoclonal antibody to a solid phase are coupled. The receptors discussed do not belong to the Class of high molecular weight G protein coupled glycoprotein Receptors. Immobilization by binding a Carrier protein that is the Fc part of an immunoglobulin for Receptor binding assays that are used to determine autoantibodies should not be suitable, since the autoantibodies themselves too belong to the immunoglobulins and with the immobilization system can interact.

To the receptors that have so far been trying to immobilize them Functionality and / or marking so affected that they have not been immobilized or in directly it marks form for determination procedures from  Receptor binding assay type could be used especially the TSH receptor.

Receptor binding assays, in which TSH recipe Tor preparations are used to form the actual Chen focus of the present invention. That closes it however, does not rule out that the below presented cognitions nisse and advantageous results with modified TSH recipe gates have been obtained in accordance with the present invention, can be transferred directly to other cases, in particular in cases where receptors are used that belong to the same substance type as the TSH receptor.

The TSH receptor is located in the thyroid membrane ter receptor to which the pituitary gland releases Hormone TSH (thyroid stimulating hormone or thyreotropin) binds and thereby the distribution of the actual shield triggers gland hormones, especially thyroxine. The TSH recipe tor belongs to the G protein-coupled receptor family Glycoprotein receptors with a large amino terminal extra cellular domain, which also includes the LH / CG and FSH receptors belong. An explanation of the chemical structure of the TSH recipe tors, d. H. the sequence of the DNA coding for it and the deduced amino acid sequence of the receptor itself, succeeded at the end of 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; and WO-A-91/09137; WO-A-91/10735 and WO-A-91/03483; also Yuji Nagayama & Basil Rapoport, in: Molecular Endocrino logy, Vol. 6 No. 2, pp. 145-156 and the litera cited therein door). With the availability of that for the human TSH receptor The cDNA encoding was able to express the recombinant human TSH receptor in a variety of different Ex pression systems. It was found that a functional full length human TSH receptor apparently only in suckling animal cells can be expressed, both transiently as well as in the form of stable cell lines (cf. e.g. Loos, U. et al.  in: Eur. J. Biochem. 232, pp. 62-65 (1995); or Matsuba T. et al., J. Biochem. 118, pp. 265-270 (1995) and cited therein Literature).

In the publications by Basil Rapoport et al. in J Clin Endocrinol Metab, Vol. 81, pp. 2525-2533, 1996 and the one therein mentioned earlier publication of the same working group in J Biol Chem 270, pp. 1543-1549, 1995 experiments are wrote the extracellular domain of the TSH receptor (TSHR-ECD), extended by a 6His residue, which isolates the Expression product should facilitate in CHO cells or in Reach bacuolvirus / insect cell system. In CHO cells was an expressed protein in the soluble fraction of the Cells found, however, only very small amounts of this Proteins were functional, i. H. in the sense of an autoantibody were effective. Purification of the expressed protein over the 6His rest turned out not to be possible. In the Baculovi Rus / insect cell expression systems were similar unsatisfied results have been obtained.  

Autoantibodies can be formed against the TSH receptor which are responsible for various autoimmune diseases, taking so-called stimulating autoantibodies that lead to a Graves 'disease (English: Graves' disease) known shield cause hyperthyroidism, for clinical diagnostics are particularly important. One for determining such autoanti The well-known assay currently on the market is the TRAK-Assay® from B.R.A.H.M.S Diagnostica GmbH.

For the determination of TSH receptor autoantibodies after the conventional procedures so that the determining autoantibody from a serum sample in liquid Phase with a radiolabelled bovine TSH competitor around the binding sites of a detergent-solubilized porcine TSH receptor can compete (see Southgate, K. et al., Clin. Endocrinol. (Oxford) 20, 539-541 (1984); Matsuba T. et al., op. cit .; Product information on the TRAK-Assay® from the company B.R.A.H.M.S Diagnostica GmbH). To the receptor preparation tion-bound marked TSH will be determined after completion incubating the TSH receptor with a precipitation reagent and a subsequent centrifugation step from the liquid Phase separated. Determination of the receptor-bound marked TSH is done by measuring the bound in the sediment Radioactivity.

Due to the availability of recombinant human TSH receptor Preparations have not changed in this assay design. As far as those obtained in the different expression systems recombinant human TSH receptors functional at all Were receptors, the TSH receptor Preparations in a similar manner in solubilized form used like a conventional porcine TSH receptor preparation tion. In addition, the  Functionality of the expressed receptors also on the whole transformed cells measured.

The release of the recombinant human TSH receptors from the host cells, e.g. B. CHO or COS cells, with the help of Detergents proved to be due to the growth properties such cells as difficult and was of a partial accompanied proteolytic cleavage of the recombinant receptor, and the preparations obtained were also without Loss of functionality can be immobilized or marked like that previous TSH receptor obtained from biological material Preparations so that through the use of recombinant TSH recipes Tor preparations do not change the traditional Determination method were made possible. The need to all previously known methods a precipitation and Zen to carry out the centrifugation step, however, stresses the Routine determination of TSH receptor autoantibodies in clini everyday laboratory work, and it is also an obstacle to one Automation of such provisions, e.g. B. using automatic measuring systems known per se.

In the context of the present application, a "functio nalen receptor "understood a receptor that is responsible for the Perform a receptor binding assay required Has functionality, d. H. the property of both Competitor, usually TSH, as well as the car sought to bind antibodies. The ability to stimulate cAMP is in contrast, without major importance. If further from it it is said that the functionality of the TSH receptor an additional peptide residue and / or by immobilization or marking is not affected, it does not mean that properties such. B. binding affinities in everyone Terms, particularly in quantitative terms, with the corresponding properties of comparison receptors naturally Chen or recombinant origin must be identical. Variations in properties that are not critical the behavior of the receptor preparation that is important in practice  in one of the receptor binding assays described below impact are not to be regarded as "impairment".

It is an object of the present invention to have a receptor binding assay, in particular a receptor binding assay for Determination of TSH receptor autoantibodies to create that with a functional receptor, in particular TSH receptor, can be worked that can be immobilized and / or marked is, so that basic process types in which with immobilized ten or marked specific binders is worked, too can be used for receptor binding assays.

It is another object of the present invention that required functional receptor preparations and the To create means for their production.

Finally, it is also an object of the present invention To create reagent kits that are compatible with receptor embodying new types of determination methods, especially those in which the coated tube technology or Microtiter plate technology for the area of receptor binding assays is used.

These tasks are used in a receptor binding assay Determination of TSH receptor autoantibodies in a biological specimen, especially a human serum, and ent speaking of a general definition of such a receptor binding assays in the broadest sense according to the generic term of Claim 1, characterized in that the TSH receptor Preparation a preparation of a recombinant TSH fusion receptor, especially a recombinant human TSH-Fu sionsreceptor, used over the corresponding naturally occurring functional TSH receptor, in particular compared to the naturally occurring functional TSH receptor full length, around a terminal or as an insert NEN peptide residue is extended, this peptide residue (i) a Has marking or can be selectively marked and / or (ii)  by binding to an immobilized selective binding partner is immobilized or immobilizable without the Functionality of the TSH receptor is impaired.

Advantageous configurations of such a receptor binding assays can be found in subclaims 2 to 10 or for the person skilled in the art are evident from the following explanations gene.

The invention further relates to such according to the invention Receptor binding assays required recombinant TSH fusion receptors according to claim 11 or subclaims 12 and 13 and one for the production of such a recombinant Vacciniavi suitable for TSH fusion receptors in warm-blooded cells Rus vector according to claims 14 and 15.

The invention also relates in a generalized form Receptor binding assays according to claim 16 and the subclaims 17 and 18.

The present invention also relates to a reagent Ziensatz (Kit) according to claim 19, which as a component a solid phase, especially coated tubes, with one on it contains immobilized functional fusion receptor.

The invention is based on the knowledge that it is possible without loss of functionality at the C-terminus around a peptide residue extended functional receptors of the TSH receptor type produce and that such fusion receptors without functio Loss of quality in specific binders for the peptide residue can be bound, the immobilized or immobilizable or marked or markable.

The amino acid sequence referred to as "peptide residue" in the context of the present application, which extends a naturally occurring functional receptor, in particular a full-length receptor, in its sequence or in particular at its C-terminus, is primarily characterized in that a selective binding partner to this sequence is available, which binds to the sequence mentioned, on the one hand without impairing the receptor functionality and on the other hand with the remaining components of a receptor binding assay, ie in the case of a receptor binding assay for the determination of TSH receptor autoantibodies with the autoantibodies to be determined and / or the competitor , e.g. B. ¹²⁵ TSH to enter into interfering interactions.

"Peptide residue" is without limitation in a broad sense to understand short-chain typical "peptides" and includes Amino acid chains with peptide bond that correspond to the range of Assigned oligopeptides or polypeptides or even proteins can be. For example, the definition B. another avidin rest (512 amino acids, molecular weight 66,000) and one Streptavidinrest (molecular weight about 60,000) include that selective very strong bonds to biotin as selective Enter into loyalty partners, d. H. Peptides with up to about 600 Amino acid residues. However, the preferred peptide residues are shorter, these peptide residues, optionally in addition to Chen, also as a spacer to the actual receptor molecule serving amino acid sequences, the so-called FLAG epitope and / or a peptide residue from six histidine residues (6His residue) can include. The so-called FLAG epitope is a marker pep tid of structure N-Asp Tyr Lys Asp Asp Asp Asp Lys-C. One for the expression of this marker peptide of usable expressions vector as well as monoclonal antibodies, the FLAG-Epi recognize top, are commercially available (for Germany from Integra Biosciences GmbH). On the prospectus In addition to the IBI-FLAG® system, reference is made.

The 6His peptide residue (also referred to as "6His affinity tag "known) is also part of a known one commercial peptide marker and purification system. The 6His Peptide residue goes a very tight complex bond with metal atoms, in particular nickel atoms, and when this  Nickel atoms firmly chelated to a carrier resin are bound, a very tight binding of a protein or Peptide with the 6His peptide residue on the corresponding carrier resin is obtained. A preferred carrier resin is So-called Ni-NTA resin, which is sold (for Germany) from Qiagen GmbH and its structure and use is written in the patent EP-B-0 253 303. NTA thereby for a nitrilotries bindable to a carrier resin matrix acetic acid derivative. The use of metal chelate resins in the Immunodiagnostics for the production of solid phases such as B. coated test tubes (coated tubes) is not one of the usual uses of such resins.

As below and especially in the experimental part it was shown that it was in a suitable Expression system is possible, a recombinant TSH receptor to produce a peptide residue at its C-terminus, in special case a FLAG epitope and a terminal 6His Rest, is extended without being terminated by said Extension of the TSH receptor to a TSH fusion receptor its functionality is impaired.

An expression system has proven to be an expression system proven to be suitable for recombinant vaccinia viruses in Warmblood cells for the expression of the TSH fusion receptor be used. The use of vaccinia viruses as vectors has long been known (see, e.g., J.D. Watson et al., Recombined DNA, 2nd edition, Spektrum Akademischer Verlag GmbH, p. 206; D.R. Glick et al., Molecular Biotechnology, Spectrum Akademischer Verlag GmbH, pp. 234f .; Kieny M.P. et al., Nature, Vol. 312, pp. 163-166, 1984; Moss B., Science 252, pp. 1662-1667, 1991). The introduction of DNA sequences required for a desired encode the peptide or protein into the genome of the Vacciniavi rus using suitable plasmids is also fundamental known. In the context of the present invention, a Vaccinia virus vector, however, was first used Protein from the family of G protein-coupled glycosiders  receptors with a large N-terminal extracellular domain Express in animal cells. As animal cells HeLa cells were used, the large amounts of human TSH-Fu sionsreceptor generated. HeLa cells face the COS7 cells or CHO cells described in the literature Advantage on that they are quickly high density in both Carrier culture as well as grown in a suspension culture can be.

The production of the described in more detail in the experimental part recombinant TSH fusion receptor can be briefly as follows to summarize: The TSH receptor cDNA was inserted into the DNA of the Genome of the vaccinia virus in analogy to a previously known Operation (Kieny, M.P. et al., Nature, 312, pp. 163-166 1984)) was introduced. The foreign genes need for an effective one Transcriptional viral promoters. The recombination used Plasmid p7.5K131 contains the early / late promoter 7.5K des Vaccinia virus flanked by the vaccinia virus thymidine kinase (TK) sequences to recombine into the TK site of the to direct the viral genome. The recombination plasmid was constructed so that in the recombinant viruses obtained TSH receptor cDNA segment downstream of the 5'-untrans latent leader sequence of encephalomyocarditis virus (EMCV) is flanked. This sequence came from the pTM1 vector and increases the effectiveness of mRNA translation in cells using were infected with the vaccinia virus (Moss, B. et al., Nature, 348, pp. 19-92, (1990)). Recombinant virus clones with the EMCV leader Sequence generated much more TSH receptor than such without this sequence (data not shown). The recombinant TSH recipe tor also contained the FLAG octapeptide epitope, which is from commercially available monoclonal antibodies are recognized, and a 6His peptide at its carboxyl terminus.

The above recombination plasmid p7.5K131 is under the Name pAvB2 described in a doctoral thesis (Ruprecht- Karls University Heidelberg) from Mr. Albrecht von Brunn 1989 with the title "The surface antigen of  Hepatitis B virus as a carrier system for epitopes of a Merozoi surface molecule of the malaria pathogen Plasmodium falciparum - a new way of vaccine development? ". The use of the specific plasmid is not critical, and another vector can be used instead be that of a thymidine kinase gene and the so-called 7.5K promoter contains, e.g. B. with the vector pZVneo5529, which is described in C. Bouvier et al., European Journal of Pharmacology, Molecular Pharmacology Section 290 (1995), pp. 11-17.

Recombinant virus clones were analyzed by Western blot analysis TSH receptor expressed in infected HeLa cells identified.

The one with the help of the vaccinia virus vector in HeLa cells TSH fusion receptor produced was found to be complete functional in terms of TSH binding. Infected HeLa- Cells had high affinity TSH binding sites, the Binding affinity for that of the non-recombinant TSH recipe tors in human thyroid cells as well as in FRTL5 cells is remarkably similar. It also turned out that the TSH recipe from infected HeLa cells by the non-ionic Detergent Triton® X100 can be extracted effectively can. The fusion receptor preparation obtained by extraction tion is both in terms of binding TSH as well of Graves' disease autoantibodies active. The extension of the naturally occurring functional TSH receptor by one Peptide residue, in the present case the FLAG epitope and the 6His Peptide residue, did not affect the ability to bind the TSH hormones still bind autoantibodies, and the TSH fusion receptor was also stimulated with regard to TSH cAMP production fully functional.

HeLa cells infected with the recombinant vaccinia virus produced significant amounts of human TSH fusion receptor, in the order of 150,000 functional ones Receptor molecules per cell. This value is higher than that for  most of the previously known expression systems and comparable to that of Matsuba et al., J. Biochim. 118, pp. 265-270 (1995).

The one attached to the TSH receptor in the following examples Peptide residue can serve as a model for a markable and immobili sizable peptide residue can be viewed without the invention should be limited to the concrete rest mentioned. About the FLAG epitope can be the recombinant TSH fusion receptor Use of specific available monoclonal antibody immobilized, but also specifically marked. The TSH fusion receptor can also via the 6His peptide residue immobili without losing its functionality on a solid phase be and is therefore entitled to receptor binding assays Available to implement the procedural variants for such assays have not previously been used.

Thus, due to the availability of an immobilisable and / or markable functional TSH receptor and taking into account the competition and thus to a certain extent mutual substitution of TSH and TSH receptor antibodies, for example the following possibilities for realizing a receptor according to the invention binding assay.

• 1. The fusion receptor is over its peptide residue and one specific immobilized binding partner on a Solid phase, e.g. B. the walls of coated tubes, immobili siert, and the determination of TSH receptor autoantibodies takes place analogously to the previously known method by their Competition with marked TSH for the binding sites of the Receptor.
• 2. However, this reaction scheme can also be reversed, by immobilizing TSH on a solid phase and that immobilized TSH with the autoantibodies to be determined a TSH fusion labeled over the peptide residue  receptor can compete. The marking can, for example with the help of a labeled monoclonal antibody take place that binds specifically to the peptide residue. The Amount of the solid phase immobilized on the TSH bound label is inversely proportional for the concentration of autoantibodies in the sample.
• 3. As above under 1. with an immobilized TSH recipe worked, but not as a competitor labeled TSH used, but labeled monoclonal or polyclonal TSH receptor antibodies that are compatible with the determining autoantibodies around the binding sites of the immobilized receptor compete.
• 4. Analogous to the process variant described under 2. can also use a TSH receptor antibody instead of TSH be immobilized.
• 5. A TSH receptor immobilized on a solid phase can the TSH recipe available in a sample can be used to completely bind tor autoantibodies, which then then by a suitable labeling reagent, the responsive to antibodies, e.g. B. an antiserum or protein A in marked form, can be marked.
• 6. Another process variant is the inversion of Process variant described under 5. by first with the help of suitable specific or also non-specific binder the autoantibodies in the sample bound a solid phase and then with the help of a labeled TSH receptor.
• 7. The determination method can also be used for determination more specifically, having two receptor-binding epitopes Autoantibodies serve by hitting one on one Solid phase immobilized receptor and a soluble one labeled receptor works and the amount of labeling  on the solid phase, which determines the binding of the determining autoantibodies both through the immobilized as well as the marked receptor.

Other assay designs known per se, e.g. B. those with a gradual incubation and a difference measurement are the Known to those skilled in the art.

Suitable antibodies can be used in these assay designs monoclonal and polyclonal antibodies, in particular also human monoclonal and human polyclonal antibodies, as well recombinant antibody constructs, e.g. B. so-called single Chain antibodies can be used. It is also possible, too to use suitable antibody fragments, in particular also for receptor labeling.

The availability of the functional fusion according to the invention receptors also makes it possible to create new defined monoclonal Antibodies against the recombinant functional TSH fusion generate receptor that is against parts of the receptor sequence and adjacent parts of the attached peptide residue, e.g. B. the FLAG epi top and / or part of the 6His peptide, and the further new application due to its specificity open up opportunities and advantages for assay design.

Further information on the implementation and the advantages of the present invention arise for the expert from the following experimental part with reference to the Characters. The figures show:

Fig. 1 shows the time course of the TSH receptor expression in HeLa cells. In 24 well microtiter plates, HeLa monolayer cells were infected with one plaque-forming unit of the recombinant vaccinia virus per cell. 0, 2, 4, 6, 8, 14 and 24 h after the infection, the cells were removed, separated by means of an SDS page, and the TSH receptor was detected by Western blot analysis. The molecular size markers are given in kDa.

Fig. 2 shows the binding of ¹²⁵ I-TSH to extracts of infected HeLa cells as a function of the time of infection. The determination was made using the components of the TRAK-Assay® in a total volume of 100 µl according to the instructions for use.

Fig. 3 shows the distribution of the recombinant TSH fusion receptor on different cell fractions, which were obtained from infected HeLa cells. In the illustration, band 1 represents a marker extract of HeLa cells, band 2 represents the water-soluble cytoplasmic fraction, band 3 the membrane extract obtained using Triton® X100, band 4 the remaining insoluble protein which cannot be extracted with Triton® X100. The molecule size markers are again given in kDa.

Fig. 4 is a Scatchard plot of the ¹²⁵ I-TSH binding to with the recombinant vaccinia virus infected HeLa cells.

Fig. 5 shows the analysis of autoantibodies in unfractionated sera of Graves' disease patients compared with control sera from normal patients. The effect of six control sera and 13 sera from Graves' disease patients on the binding of ¹²⁵ I-TSH to the recombinant TSH receptor in the extract from infected HeLa cells was compared with the porcine TSH receptor (from the TRAK Assay® Kit) examined. The results are reported as a percentage of total binding, with 100% binding representing ¹²⁵ I-TSH binding in the presence of zero standard serum. The results are given as averages of duplicate determinations.

As obtained using the coupled to Ni-NTA agarose TSH fusion receptor and TRAK-Assay® Standards Fig. 6 is a standard curve.

FIG. 7 shows the measurement results for the determination of TSHR-auto antibody titer of Graves' disease patients and a control group using Ni-NTA-aga rose coupled recombinant TSH Fusionsrezep tor; and

Fig. 8 shows the measurement results obtained for normal patients and Graves' disease patients in a comparative graphical comparison.

Experimental part materials

Bovines ¹²⁵ I-TSH (56 µCi / µg), unlabeled TSH and the TRAK-Assay®-Kit were provided by BRAHMS Diagnostica GmbH. The pTM1 vector was obtained from doctors RE Rhoads and B. Joshi (Louisiana State University Medical Center, Shreveport, USA), the p7.5K131 recombination vector was obtained from Dr. T. Mertens (Ulm University, Germany). The BIOTRAK® cAMP [ ¹²⁵ I] assay kit and the ECL® western blot detection kit were obtained from Amersham. The monoclonal mouse antibodies NCL-TSHR against the extracellular domain of the TSH receptor were obtained from Novocastra Laboratories Ltd. based. Protease inhibitor cocktail tablets were obtained from Boehringer Mannheim. The DNA primers P1 to P5 were obtained from Eurogentec. They had the following nucleotide sequences:

If no sources of supply are specified for individual reagents became commercially available products used where the sources of supply for the rework availability have no meaning. As far as further below for individual reaction steps no special reaction conditions gene specified, was among those known in the art Conditions worked, e.g. B. in the for each Restriction enzymes indicate optimal temperatures and reak times.

Cell culture

HeLa cells were grown in Eagle's medium modified by Dulbecco) supplemented with 10% fetal calf serum containing 50 U / ml penicillin and 50 µg / ml streptomycin. The cells were cultured at 37 ° C under a 5% CO ₂ atmosphere.

Production of one around the FLAG octapeptide epitope and one 6His peptide sequence of an extended TSH receptor 1. Insertion of sequences for the FLAG epitope and one His peptide sequence into a TSH receptor cDNA sequence

A complete human TSH receptor cDNA (Libert et al., Biochem. Biophys. Res. Commun. 165: 1250-1255, (1989)) subcloned in the pSVL vector according to a previously described technique (pSVL-TSHR plasmid). P1 and P2 primers were used to the FLAG-Octa recognized by commercially available antibodies peptide epitope (N-Asp Tyr Lys Asp Asp Asp Asp Lys-C; cf. Brochure IBI FLAG® SYSTEM from INTEGRA Biosciences) on the C-Ter minus the TSH receptor. The 0.5 kb Bgl II-Bam HI fragment of the TSH receptor cDNA was made in a conventional manner  by PCR (Polymerase Chain Reaction) using thermostable Taq DNA polymerase reproduced. The P1 primer contains a Bgl II restriction site that contains P2 primers the FLAG epitope sequence (underlined) followed by one Stop codon and a Bam HI restriction site. The PCR fragment was cut with Bgl II and Bam HI and used to make the same fragment in the TSH receptor cDNA in the pSVL-TSH plas to replace mid. The construct obtained received the Designation pSVL-TSHR-FLAG.

P1 and P3 primers were then used to make the 6His peptide directly after the FLAG epitope sequence at the C-terminus of the TSH recipe introduce tors. The 0.5 kb Bgl II-Bam HI fragment of the TSH receptor cDNA in the pSVL-TSHR-FLAG plasmid was designed according to the PCR technique reproduced. The P1 primer contains a Bgl II restriction tion site, the P3 primer contains the sequence for the 6His peptide followed by a stop codon and a Bam HI restriction station. The PCR fragment was mixed with Bgl II and Bam HI cut and used the corresponding fragment Replace in the TSH receptor cDNA in the pSVL-TSHR-FLAG plasmid. The vector obtained was called pSVL-TSHR-FLAG-6His. The nucleotide sequence of the extended cDNA was determined after Sanger procedure confirmed.

2. Construction of a pTM1-TSHR-FLAG-6His expression plasmid

The TSHR-FLAG-6His DNA segment was inserted into the pTM1 vector (cf. Moss et al., Nature, 348, pp. 19-92, (1990)) downstream of the untranslated encephalomyocarditis virus (EMCV) lea dersequenz, the efficiency of mRNA translation he high, advertises. Contains the Nco I interface in the pTM1 vector the translation initiation codon, which is responsible for the insertion of the 5 'end of the cDNA coding for the protein can be used should.

P4 and P5 primers were used to interface for the Rca I restrictionase in the start codon region of the TSH receptor  Generate cDNA in the pSVL-TSHR-FLAG plasmid. This interface is com with the Nco I restriction site in the pTM1 plasmid patible. The 0.7 kb Ava I-Afl III fragment of the TSH receptor cDNA was amplified by PCR, the pSVL-TSHR-FLAG-6His Plasmid served as a template. The P4 primer contains Ava I and Rca I restriction sites, the P5 primer contains the Afl III restriction site. The PCR fragment was treated with Ava I and Afl III cut and used the same fragment in the TSH receptor cDNA in the pSVL-TSHR-FLAG-6His plasmid replace. The extended 2.3 kb TSH recipe was made from this plasmid tor cDNA with the sequences for the FLAG epitope and the 6His peptide with Rca I and Bam HI released and in pTM1-Plas mid at the Nco I and Bam HI interfaces downstream subcloned from the EMCV leader sequence. The received The construct received the designation pTM1-TSHR-FLAG-6His. The Nucleotide sequence was confirmed using the Sanger method.

3. Construction of a vaccinia recombination plasmid p7.5K131-TSHR-FLAG-6His

The pTM1-TSHR-FLAG-6His plasmid was cut by cutting with the Cla I restrictase linearized and blunt to form Padded ends with Klenow fragment. After a Bam HI spal tion was the TSHR-FLAG-6His DNA segment, the upstream flanked by the untranslated EMCV leader sequence was isolated and at Hind II and Bam HI sites in subcloned the vaccinia recombination vector p7.5K131. The The construct obtained received the designation p7.5K131-TSHR-FLAG-6His.

4. Generation of vaccinia virus recombinants

The TSH receptor cDNA under control of the 7.5K early / late Promotors was created by homologous recombination in the thymidinki nose (TK) gene of the genome of the wild-type vaccinia virus strain Copenhagen incorporated (see Kieny, M.P. et al., Nature 312, Pp. 163-166, (1984)). In short, were for this purpose  confluent CV1 cells with the temperature sensitive vaccinia virus ts7 infected and with vaccinia virus DNA (strain Copenha gen) and the p7.5K131-TSHR-FLAG-6His construct according to the Calcium phosphate method transfected. The viruses became two Harvested days later, the TK-negative recombinants were made the virus offspring in two passes of a plaque cleaning via TK⁻ 143B cells in the presence of 0.1 mg / ml bromodeoxyuri din selected. Recombinant virus plaques were isolated, and positive clones were found after expression in infected HeLa Identify cells by Western blot analysis on TSH receptor graces.

For this purpose, the proteins were subjected to SDS gel electrophoresis on a Subjected to 8% dissolution polyacrylamide gel (PAG) (see Laemmli, U.K., Nature, 227, pp. 680-685 (1970)). The unraveled Proteins were transferred to a nitrocellulose membrane, nonspecific binding sites were resolved within 1 h Incubation with TBS / 5% dry milk / 0.02% Tween® 20 solution saturated. The membrane was passed through at 4 ° C overnight Implementation with the monoclonal NCL-TSHR antibody as a probe treated for the TSH receptor, which was diluted 1:20. Bound antibodies were raised using the ECL® Western Detected blot detection kits.

A larger amount of the recombinant virus was found in HeLa cells manufactured.

5. Expression of the extended TSH receptor polypeptide in HeLa cells and generation of cell fractions

HeLa monolayer cells in 75 cm ² dishes (approximately 15.10 ⁶ cells) were infected with one plaque-forming unit of recombinant vaccinia virus per cell and incubated at 37 ° C. for 24 h. Infected cells were washed with PBS, harvested and pelleted 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 further homogenized by 20 cycles in a glass Teflon homogenizer and then centrifuged at 800 g for 15 min and at 30,000 g for 1 h. The supernatant (water soluble cytoplasmic fraction) was collected. The membrane pellet was extracted with 0.3 ml of 1% Triton® X100 in the same buffer and ultracentrifuged at 30,000 g for 1 h. The supernatant (Triton® X100 membrane extract) was collected, the pellet (insoluble protein) was resuspended in 0.3 ml of an SDS electrophoresis sample buffer. The amount of the TSH receptor in 10 ul of each fraction was estimated by Western blot analysis.

The kinetics of TSH receptor production in HeLa cells after infection with the recombinant vaccinia virus with a multiplicity of the infection of 1 is shown in FIG. 1. The cells were collected at times between 0 and 24 h after infection, separated by SDS gel electrophoresis and labeled with commercially available monoclonal NCL-TSHR antibodies against the TSH receptor. The recombinant protein appeared 6 hours after infection and the amount of protein increased up to 24 hours after infection, at which point the cells are still relatively healthy. Long-term incubation resulted in cell detachment and death. Western blot analysis of the infected cells shows various forms of the receptor with molecular weights of 230 to 50 kDa, the main fraction having a molecular weight of approximately 95 kDa. The result corresponds to the data known from the literature (Ban T. et al., Endocrinology 131, pp. 815-829 (1992)). In the case of infection of HeLa cells with the wild-type vaccinia virus, no signal was detectable (results not shown).

The distribution of the TSH receptor generated to different fractions obtained from infected HeLa cells is shown in FIG. 3. Only a small amount of the receptor can be detected in the water-soluble cytoplasmic fraction (band 2). The main part of the TSH receptor is in the membrane fraction from which it can be extracted effectively with Triton® X100 (band 3). Part of the receptor in the membrane fraction was solubilized only by SDS (band 4). This can be a non-extractable TSH receptor that is firmly integrated in the membrane structure.

To test whether there is a dependency on the yield of the TSH recipe tors consists of the intensity of the infection, the TSH binding activity of cells infected with an Multiplicity of 1 and 10 infected were measured. An increase in infection multiplicity from 1 to 10 increased the final yield of the functional TSH receptor up to 24 hours after the infection is not.

Binding assay for ¹²⁵ I-TSH and Graves' autoantibodies

HeLa cells in a 75 cm ² dish (approximately 15.10 ⁶ cells) were infected with 1 or 10 plaque-forming unit (s) of the recombinant vaccinia virus per cell and incubated at 37 ° C. for 24 h. Infected cells were washed with PBS, harvested and pelleted at 1200 rpm. The cells obtained were lysed in 0.3 ml of buffer A (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% Triton® X100, 10% glycerol) to which protease inhibitors were added. The suspension was centrifuged at 30,000 g for 1 hour and the supernatant provided the TSH receptor preparation.

The assay was carried out using components of the TRAK-Assay® kit in a total volume of 100 .mu.l, which consists of 25 .mu.l solution of the recombinant TSH receptor (about 200 .mu.g total protein), 25 .mu.l either zero standard serum or test serum and 50 ul of a ¹²⁵ I TSH solution (20,000 cpm). Incubation was carried out for 2 hours in accordance with the manufacturer's instructions. The reaction was stopped by adding 1 ml of a precipitant according to the instructions for use. The tubes were centrifuged at 2000 g for 10 min and the supernatant was removed. The gamma radiation in the pellet was measured.

Results are expressed either as bound radioactivity (in cpm - counts per minute) or as a percentage of total binding, calculated according to [(counts in the presence of the test serum) / (counts in the presence of the zero standard serum)]. 100% . The nonspecific binding was determined in the presence of ⁷ M 10⁻ TSH and was about 10% of total binding.

The TSH receptor generated in the HeLa cells proved to be fully functional with regard to the binding of TSH. Figure 2 shows the binding of ¹²⁵ I-labeled TSH with extracts from infected HeLa cells as a function of the time of infection. There is a clear connection between the TSH receptor expression in HeLa cells and the TSH-binding activity of the cell extracts ( FIGS. 1 and 2).

Scatchard analysis of TSH binding to infected HeLa cells

HeLa cells in 24-well microtiter plates (approximately 300,000 cells / well) were infected with one plaque-forming unit per cell of either the recombinant vaccinia virus or the wild-type vaccinia virus and incubated at 37 ° C. for 24 hours. Infected cells were incubated for 3 hours at 37 ° C. in 0.2 ml of modified Hank's buffer without NaCl, the isotonicity of the solution being ensured by 280 mM sucrose supplemented with 0.25% calf serum albumin. Different amounts of (a) ¹²⁵ I-labeled TSH or (b) a mixture of ¹²⁵ I-TSH (25,000 cpm) with varying concentrations of unlabeled TSH were added to this buffer. At the end of the incubation period, the cells were quickly washed with 1 ml of the same buffer and solubilized with 1 ml of 1N NaOH and the radioactivity was measured in a gamma counter. The specific TSH binding was defined as the difference between the total binding of ¹²⁵ I-TSH in the absence and in the presence of ⁷ M 10⁻ TSH. The non-specific binding was approximately 2% of the total binding. The dissociation constant for TSH binding was calculated according to the Scatchard method (Scatchard, G., Ann. NY Acad. Sci. 51, 600-672, (1949)). Control studies related to the binding of ¹²⁵ I-TSH to HeLa cells infected with the wild-type vaccinia virus.

The interaction of HeLa cells infected with the recombinant virus with ¹²⁵ I-labeled TSH led to a specific and saturable binding of the hormone. The data obtained by the Scatchard method are plotted in FIG. 4. The fact that the Scatchard curve in Fig. 4 is a straight line indicates the existence of only one type of binding site. A dissociation constant of approximately 2.10 ^-10 M can be calculated from the slope of the curve. HeLa cells infected with the wild-type vaccinia virus showed no detectable specific TSH binding (data not shown). Based on the Avogadro number, a number of around 150,000 high-affinity TSH receptors per cell is calculated.

Interaction of the TSH receptor with Graves' disease-Autoanti bodies

The recombinant TSH receptor obtained by expression of the recombinant vaccinia virus was also examined for its ability to interact with TSH receptor autoantibodies in sera from patients with Graves' disease. The presence of such autoantibodies can be demonstrated by their ability to inhibit the binding of ¹²⁵ I-TSH to the TSH receptor. The amount of autoantibodies in the examined serum is inversely proportional to the measured radioactivity. Sera from six healthy individuals and from 13 Graves' disease patients with different autoantibody levels were measured with the TRAK-Assay®-Kit. The results are shown in FIG. 5. They show that the extended TSH fusion receptor obtained by expression of the recombinant vaccinia virus in HeLa cells is specifically recognized by Graves' disease autoantibodies. In the autoantibody-free control sera, a 95-106% binding of ¹²⁵ I-TSH was obtained, while only 30-75% corresponding binding was obtained when measuring sera from Graves' disease patients. The results showed a significant positive correlation to the values obtained with the TRAK-Assay® Kit.

Intracellular cAMP measurements

HeLa monolayer layers in microtiter plates with 24 ver Wells were made with one plaque-forming unit per cell from either the recombinant vaccinia virus or the wild-type Vaccinia virus infected and incubated at 37 ° C for 24 h. Infected Cells were kept in modified Hank's buffer without NaCl for 1 h (cf. above) and then another hour in the same Buffer, the 1 mM isobutylmethylxanthine as well as given Contained concentrations of TSH. Cellular cAMP was 65% Ethanol extracted and using the BIOTRAK® cAMP assay Kits measured according to the manufacturer's instructions. Control examinations concerned the measurements of the TSH-ab pending cAMP production in HeLa cells using the wild-type Vaccinia virus were infected. It turned out that the Exposure to TSH is a maximum of about 6 times dose-dependent Increased cAMP production. With the wild type Vaccinia virus infected HeLa cells did not respond. Consequently the recombinant TSH fusion receptor also proved to be functional in terms of stimulation of the intracellular cAMP production.

Production of an immobilized functional recombinant TSH fusion receptor

HeLa cells infected with the recombinant vaccinia virus were harvested after a 24-hour incubation at 37 ° C. as described above. The cell pellet obtained (about 2.10 ⁸ cells) was then resuspended in 3 ml buffer (10 mM Tris pH 7.6; 50 mM NaCl; 1% Triton® X-100; 10% glycerol) and frozen at -80 ° C. After thawing, the solubilized membrane fraction was enriched by centrifugation (15 min at 1000 g and 30 min at 30,000 g) and separated from insoluble constituents.

In parallel, 200 ul Ni-NTA agarose (obtained from. Qiagen GmbH) with 200 µl of the same buffer and in packed a small open pillar.

The centrifugation freed from insoluble constituents was applied to the column and overnight at 4 ° C recirculated to the TSH fusion receptor via its terminal 6His peptide residue to couple to the Ni-NTA agarose.

The loaded Ni-NTA agarose was then treated with 3 ml Whole blood saturated (hemoglobin binds non-specifically to Ni-NTA-Aga rose) to remove the remaining Ni-NTA binding sites satiate. After removing the column supernatant, it was like this treated Ni-NTA agarose with the buffer described on a Total volume brought to 1.5 ml and in portions of 25 µl distributed on Eppendorf test tubes.

Determination of TSH receptor autoantibodies using of the immobilized TSH fusion receptor

In the previously described Eppendorf test tubes, each with 25 µl Ni-NTA agarose with the attached TSH fusion receptor, 25 µl of a serum or 25 µl of the standard solutions from the TRAK-Assay® were added for measurement purposes, with the content of the Test tube mixed and then incubated for 20 min at room temperature with constant shaking. 50 μl of tracer ( ^125- TSH from the TRAK-Assay®) were then added to each test tube, and incubation was continued at 4 ° C. overnight while shaking continuously.

Then the tubes were briefly at room temperature centrifuged (1 min), 80 μl of the supernatant were removed, and the centrifugation pellet was resuspended in 350 ul buffer  dated. After renewed centrifugation, 320 µl of the The supernatant was pipetted off again, and the resulting washed Pellet was measured directly in a gamma counter.

When the standards from the TRAK-Assay® were measured, the results shown in Table 1 were obtained. The associated standard curve is shown in Fig. 6.

The patient sera were measured in duplicate in each case, mean values being formed from the individual measured values, which are shown in Table 2. For comparison, the same sera were measured with the conventional TRAK-Assay®. The results obtained are also listed in Table 2. Fig. 7 shows the correlation of the measured values obtained according to the conventional TRAK-Assay® to the measured values obtained using the immobilized recombinant TSH fusion receptor.

Fig. 8 shows the results of the measurements of sera from normal patients and Graves' disease patients in a graphi rule representation. The group of Graves' disease can be clearly distinguished from the group of normal patients.

It can thus be seen that for the first time the Determination of TSH receptor autoantibodies as used for Graves' disease are characteristic, and using a functional immobilized TSH receptor.  

Standard curve using a Ni-NTA agarose-coupled recombinant TSH fusion receptor

Standard curve using a Ni-NTA agarose-coupled recombinant TSH fusion receptor

Comparative determination of TSH receptor autoantibodies using the TRAK-Assay® as well as using of the immobilized recombinant TSH fusion receptor

Comparative determination of TSH receptor autoantibodies using the TRAK-Assay® as well as using of the immobilized recombinant TSH fusion receptor

Claims (20)

1. receptor binding assay for the determination of TSH receptor autoantibodies in a biological sample, in which the sample in a reaction mixture simultaneously or successively with a TSH receptor preparation and at least one of (i) a competitor and (ii) an agent for the separation of the TSH receptor and the constituents of the reaction mixture bound thereto is implemented from the liquid phase, one of the constituents of the reaction mixture being marked or selectively markable, and in which the autoantibody to be determined is bound by and / or the competitor separates the complex formed on the TSH receptor preparation from the liquid reaction mixture and calculates back from the amount of labeling in the complex mentioned to the presence and / or amount of the autoantibodies to be determined, characterized in that a TSH receptor preparation Preparation of a recombinant TSH fusion receptor used is extended by a peptide residue compared to a functional TSH receptor, this peptide residue (i) having a label or being selectively labeled and / or (ii) being immobilized or immobilizable by binding to a selective binding partner without the torbindassay assay for the receptor required functionality of the TSH receptor is significantly impaired. 2. Receptor binding assay according to claim 1, characterized records that the peptide residue is up to 600 amino acids in length acids, in particular from 6 to 200 amino acids.   3. Receptor binding assay according to claim 2, characterized indicates that the peptide residue is a C-terminal peptide residue. 4. receptor binding assay according to one of claims 1 to 3, characterized in that the selective binding partner Metal chelate resin, an antibody or biotin is or contains. 5. Receptor binding assay according to claim 1 or 2, characterized characterized in that the peptide residue at least one pepti contains the third section, which is selected from the so-called FLAG octapeptide and a 6His peptide, and that the immobili a selective antibody against the FLAG octapeptide or react with the 6His peptide metal chelate resin. 6. Receptor binding assay according to claim 5, characterized records that the metal chelate resin is Ni-NTA agarose. 7. receptor binding assay according to one of claims 1 to 6, characterized in that the selective binding partner a micro solid phase bound or as a coating of a Test tube or a microtiter plate. 8. receptor binding assay according to one of claims 1 to 7, characterized in that the recombinant TSH fusion recipe by expression of the cDNA of a functional human TSH recipe tors, which is a DNA Se coding for the peptide residue extended and into the genome of a recombinant vacci niavirus was inserted in with the recombinant vaccinia virus infected warm-blooded cells was produced. 9. receptor binding assay according to claim 8, characterized records that the warm-blooded cells are HeLa cells. 10. receptor binding assay according to one of claims 1 to 9, characterized in that the autoantibodies to be determined  are receptor stimulating autoantibodies whose occurrence in a human serum is characteristic of Graves' disease. 11. Recombinant TSH fusion receptor, the amino acid sequence of a functional human TSH receptor, the is extended by a peptide residue that is labeled or labeled bar and / or immobilized or immobilizable without the binding ability of the TSH receptor to TSH or TSH receptor autoantibodies is affected, and by Expression of the cDNA of a functional human TSH receptor, which ver by a DNA sequence coding for the peptide residue is elongated and into the genome of a recombinant vaccinia virus is inserted in with the recombinant vaccinia virus infi graced warm-blooded cells, especially HeLa cells was put. 12. Recombinant TSH fusion receptor according to claim 11, characterized in that it has a at its C-terminus Peptide residue having at least the FLAG octapeptide and / or contains a 6His peptide residue. 13. Recombinant TSH fusion receptor according to claim 12, characterized in that at its C-terminus it is only about FLAG octapeptide and an adjacent terminal 6His pep tidrest is extended. 14. Vaccinia virus, in its genome at the site of the thymidine kinase gene is expressively inserted a DNA sequence which is suitable for the human TSH receptor and a peptide extending this rest coded. 15. vaccinia virus according to claim 14, characterized in that the DNA coding for the peptide residue for the FLAG octa encoded peptide and a C-terminal 6His peptide residue. 16. Receptor binding assay for the determination of physiological effective analytes in the form of hormones or autoantibodies,  which in an organism through interaction with a cell membrane-bound G protein-coupled glycoprotein receptor trigger the normal physiological effects of hormones or block them in a biological sample, in which the sample containing the analyte to be determined holds, in a reaction mixture simultaneously or sequentially other with a preparation of the receptor in question and at least one of (i) a competitor and (ii) one Reacts binders for the analyte or the receptor, one of the constituents of the reaction mixture mentioned or is selectively markable, and in which the by binding the Analytes and / or the competitor to the receptor preparation separated complexes separated from the rest of the reaction mixture and from the amount of label in said complex the presence and / or amount of the analyte to be determined in the sample back, characterized in that one as a receptor preparation functional foot made by recombination processes sion receptor preparation used from a fusion receptor exists or contains such a fusion receptor, compared to the naturally occurring receptor by a C-th minimal peptide residue is extended, the peptide residue (i) has a marking or can be selectively marked and / or (ii) by binding to an immobilized selective bin partner is immobilized or immobilizable without the essential receptor function for the receptor binding assay nality is impaired. 17. Receptor binding assay according to claim 16, characterized records that the receptor is selected from recombinant FSH or LH / CG receptors and the peptide residue up to about 60 Amino acids. 18. Receptor binding assay according to claim 17, that the peptide residue contains at least one peptide residue section, the ex is selected from the FLAG octapeptide and a 6His peptide, and  that the immobilized selective binding partner is a selective ver antibodies against the FLAG octapeptide or with the 6His peptide-reacting metal chelate resin, especially Ni NTA agarose. 19. Reagent set for performing a receptor binding assays according to one of claims 1 to 10 or 16 to 18, there characterized in that, in addition to other usual com components of a corresponding set of reagents, test tubes comprises whose walls are coated with a metal chelate resin are functional to which via a suitable peptide residue recombinant fusion receptor, especially a recombinant TSH fusion receptor according to one of claims 11 to 13, is bound. 20. Reagent set according to claim 19, characterized that the metal chelate resin is Ni-NTA agarose and the peptide residue contains a terminal 6His peptide residue.