WO2012028654A2

WO2012028654A2 - Cell based assay for measurement of biological activity of anti tshr autoimmune antibodies

Info

Publication number: WO2012028654A2
Application number: PCT/EP2011/065035
Authority: WO
Inventors: Ulrich Loos; Dimitrij Zeiger
Original assignee: Krelo Gmbh Medical Diagnostics
Priority date: 2010-08-31
Filing date: 2011-08-31
Publication date: 2012-03-08
Also published as: WO2012028654A3; EP2612145A2

Abstract

The invention relates to a bioassay for the determination of biological activity of stimulating autoimmune antibodies directed against the TSHR receptor comprising the steps of a) incubating TSHR expressing cells with a patient sample, b) detecting the intensity of a signal in the supernatant of the cells that is caused by a change of cAMP concentration in the cells resulting from TSHR stimulation, c) determining the stimulating activity of the anti TSHR autoimmune antibodies by comparing the signal intensity of step b) with the signal intensity obtained after incubation of a first reference sample with TSHR expressing cells; the invention also refers to a bioassay for the determination of the biological activity of blocking autoimmune antibodies directed against the thyroid stimulating hormone receptor (TSHR), comprising the steps of a) incubating TSHR expressing cells with a patient sample, b) detecting the intensity of a signal in the supernatant of the cells that is caused by a change of cAMP concentration in the cells resulting from TSHR stimulation, c) determining the blocking/stimulation activity of the anti TSHR auto antibodies by comparing the signal intensity of step b) with the signal intensities obtained after incubation of a first and a second reference sample with TSHR expressing cells, wherein the incubation sample of step a) and the second reference sample contain the same amount of a TSHR stimulating agent.

Description

Cell based assay for measurement of biological activity of anti TSH autoimmune antibodies TECHNICAL FIELD OF TH INVENTION

The invention refers to a bioassay for the determination of the biological activity of autoimmune antibodies directed against the thyroid stimulating hormone receptor (TSHR) . The invention further refers to a test kit for performing the aforementioned bioassay.

BACKGROUND OF THE INVENTION The thyroid stimulating hormone receptor (TSHR) is a seven transmembrane receptor coupling to the cAMP signaling cascade. Under physiological conditions thyroid stimulation hormone (TSH) secreted by the pituitary is controlling its activation. TSHR contains a large extracellular domain presenting also epitopes for stimulating as well as blocking TSHR auto immune antibodies, which are pathologically produced by the immune system.

Stimulating TSHR autoimmune antibodies (sTRAb) cause hyper- thyroidism (HT) in Graves' disease (GD) . For the measurement of these autoimmune antibodies in the routine clinical laboratory, antigen displacement in vitro assays are well established . In these known antigen displacement assays labeled TSH binds to the TSHR. In the same way TSHR autoimmune antibodies bind to the TSHR and cause a displacement of the labeled TSH. The reduction of the label signal bound to the receptor is a measure for the amount of autoimmune antibodies of a patient.

However, the antigen displacement assay cannot distinguish between stimulating and blocking autoimmune antibodies. The latter are causing the opposite clinical picture, namely hy- pothyroidism. A further disadvantage of the known displacement assay is that high concentrations of autoimmune antibodies can only be measured after dilution of the sample. Critical is also the possible occurrence of both stimulating and blocking autoimmune antibodies in the sample. They define the overall or predominant biological activity, either a stimulating or blocking one, in dependence of the concentration ratio of both.

In contrast to the displacement assays, cell based bioassays can distinguish between blocking and stimulating autoimmune antibodies. Presently, two established methods of bioassays are being used.

In the first generation bioassay the stimulation or blocking is measured by detecting the amount of cAMP in the cell.

For measuring stimulatory autoimmune antibodies (sTRAB) in such bioassays thyroid cell lines containing TSHR are exposed to serum containing TSHR autoimmune antibodies. The stimula- tion of the TSHR initiates the cAMP signaling cascade and leads to an increase in the concentration of cAMP . For determination of the increased amount of cAMP, cells are harvested and lysed, followed by an extraction of cAMP from the cell lysate. The accumulation of cAMP is then measured by a special ELISA. The results are expressed as stimulation index (SI) .

For measurement of blocking TSHR autoimmune antibodies

(bTRAb) thyroid cells are exposed to a serum containing bTRAb in combination with TSH and in parallel to TSH alone. Incubation of the cells is also followed by the same procedure of cell lysis and cAMP extraction. The reduction of increase in cAMP concentration of the TSH plus serum containing TRAb in comparison to the TSH alone is a measure for the blocking activity determined as the inhibitory index. Second generation bioassays are also using host cells containing TSHR. These cells are transfected with a reporter gene, namely luciferase gene under the control of a cAMP response element (CRE) . In this gene set up an increase of cAMP concentration leads to an increase of luciferase transcrip- tion and consequently to an accumulation of luciferase in the cell .

The procedure of stimulation, cell harvest, lysis and extraction is similar to that of the first generation assay. In contrast to the first generation bioassay the cAMP increase is determined using a luciferase assay in the ceil lysate. The intensity of the luminescence is proportional to the concentration of luciferase and hence to the cAMP concentration. In each of the bioassays of the first and second generation an extraction/disruption step including the cell lysis is necessary. Due to this step the bioassays. are time consuming. A further disadvantage of these known methods is that the steps of cell lysis and extraction may lead to a reduced accuracy of the measurement and may increase the susceptibility to interferences with diverse cell components. Another critical disadvantage is that such assays are limited to low samples throughput only.

SUMMARY OF THE INVENTION

The problem underlying the invention is the provision of a precise, easy-to-perform and cost-efficient method to charac^¬ terize and quantify the biological activity of anti TSHR autoimmune antibodies in patient samples .

This problem is solved according to one embodiment of the invention with a bioassay for the determination of biological activity of stimulating autoimmune antibodies directed against the TSHR receptor comprising the steps of a) incubating TSHR expressing cells with a patient sample, b) detecting the intensity of a signal in the supernatant of the cells that is caused by a change of cAMP concentration in the cells resulting from TSHR stimulation, c) determining the stimulating activity of the anti TSHR autoimmune antibodies by comparing the signal intensity of step b) with the signal intensity obtained after incubation of a first reference sample with TSHR expressing cells.

The invention further refers to a bioassay for the determination biological activity of blocking autoimmune antibodies directed against the thyroid stimulating hormone receptor (TSHR) , comprising the steps of a) incubating TSHR expressing cells with a patient sample, b) detecting the intensity of a signal in the supernatant of the cells that is caused by a change of cAMP concentration in the cells resulting from TSHR stimulation, c) determining the blocking/stimulation activity of the anti TSHR auto antibodies by comparing the signal intensity of step b) with the signal intensities obtained after incubation of a first and a second reference sample with TSHR expressing cells,

wherein the incubation sample of step a) and the second reference sample contain the same amount of a TSHR stimulating agent.

The bioassay of the invention has the advantage that the signal is measured in the cell supernatant. The time consuming and complicated steps of cell harvest, cell lysis and extrac- tion are not necessary. Thus, the bioassay consists of only a few handling steps, allows a high throughput measurement of patient samples and therefore is appropriate for routine diagnostics also in automated systems. Further, even high concentrations of autoimmune antibody in sera can be measured without dilution of the sample. Advantageously, in the bioassay of the invention the intensity of the signal is proportional to the stimulating activity of TSH over a wide range. Furthermore the ability to measure high concentrations of TSHR autoimmune antibodies may lead to new diagnostic differentiation and clinical interpretation of the data . Fig. 1 shows as an example a schematic display of the mechanism of a preferred embodiment of the bioas- say .

Fig. 2 shows the stimulating activities of different concentrations of bovine TSH (bTSH) in serum.

Fig. 3 shows the reproducibility of the bioassay.

Fig. 4 shows a comparison of the clinical score with results from bioassay and values obtained by a competitive assay (in vitro) (TRAK^®-Assay ) in patient samples .

Fig. 5 shows an explanatory scheme of the determination of stimulating activity (A) and blocking activity (B) .

To measure the stimulating activity of TSHR autoimmune antibodies it is sufficient to compare the signal strength from the patient sample with a first reference, which has in principal no TSHR stimulating activity. The first reference sam^¬ ple is therefore used to determine the background signal that is measured without stimulation. This background signal is then subtracted from the signal of the patient sample to obtain the signal strength of the stimulating activity in the patient sample. To measure blocking TSHR autoimmune antibodies (bTRAbs) a defined amount of a TSHR stimulating agent is added to the patient sample and to the second reference sample. When expos- ing the cells to the patient sample containing bTRAb in combination with a TSHR stimulating agent and in parallel to same amount of TSHR stimulating agent alone, the reduction of the measured signal strength in the patient sample is a meas- ure for the blocking activity in the patient sample. The procedure of determination of stimulating and blocking activity is summarized in Fig. 4.

According to the invention THSR can mean the wild type TSHR, described in "Molecular cloning, sequence and functional ex^¬ pression of the cDNA for the human thyrotropin receptor; Na- gayama,Y., Kaufman, K. D. , Seto,P. and Rapoport , B . ; Biochem. Biophys. Res. Commun. 165 (3), 1184-1190 (1989)", or a polypeptide construct of the TSHR that is fully functional. In particular, the TSHR construct can couple to the cAMP signaling pathway in the cell and contains the epitopes for TSHR blocking or stimulation.

A "patient sample" according to the invention comprises at least blood serum or plasma from a human who may have Graves' disease (hyperthyroidism) or hypothyroidism.

A "reference sample" is a sample that is used as a comparison to the patient sample.

The first reference sample is a sample that substantially (in principal) does not contain a TSHR stimulating agent. Pref^¬ erably, the first reference sample is a serum, because a serum should have the same amount of nonspecific signal as the patient sample. This first reference sample is also referred to as negative control sample. The second reference sample contains a defined amount of a TSHR stimulating agent and can be used as a positive control. Preferably, the second reference sample contains serum and the TSH stimulating agent. This second reference sample is also referred to as positive control sample.

A "TSHR stimulating agent" is any molecule that enables an activation of the TSHR receptor to start the cAMP signaling. In particular, TSHR stimulating agents are TSH, a functional fragment of TSH, a stimulating TSHR antibody (sTRAB), a func^¬ tional fragment of the antibody or thyreostimulin .

In the bioassay of the invention the TSHR expressing cells can be eukaryotic cells stably transfected with the TSHR gene. In principal, any eukaryotic cell line can be used.

Preferred cell lines are human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells. The cells used in the bioassay according to the invention can either stem from a freshly transfected cell line or from a frozen cell line (CryoCells) . The frozen cells are thawed before use in the bioassay .

According to one embodiment of the invention the signal caused by an increase of cAMP in the cell is provided by an increased production of a polypeptide that is secreted from the cell. Advantageously, this is a polypeptide that can be easily detected and the concentration of which can be easily determined. The transcription of the polypeptide can be controlled by a cAMP Response Element (CRE) . This is achieved by fusing the CRE gene to the gene encoding for the signal poly^¬ peptide. The fused genes of CRE and the signal polypeptide are introduced into the stably TSHR expressing cell by trans- fection .

The polypeptide can be an exoplasmatic protein. Alternatively, it can be a polypeptide that is normally anchored to the outer membrane of the cell, but the part that is responsible for membrane anchorage is deleted. Further the polypep^¬ tide may be a modified cytoplasamtic polypeptide, which is modified by addition of a secretion signal. Preferably, the signal measured in the supernatant is the chemoluminescence of the polypeptide. Preferably, the polypeptide is secreted alkaline phosphatase (SEAP) . SEAP is a modification of a human placental alkaline phosphatase, a protein normally anchored to the outer leaflet of the plasma membrane via a phosphatidylinositol-glycan moiety. In SEAP the sequence responsible for membrane anchorage is deleted.

A SEAP which is suited for the purpose of the invention is disclosed in EP 0 327 960 Al . The plasmid pCRE-SEAP contain- ing a SEAP under control of the CRE can be purchased at Clo- netech as part of the "Pathway profiling systems" Kit (Cat. No. 631910) .

For the detection of SEAP any known of alkaline phosphatase assay can be employed. McComb and Bowers, Clin. Chem. 18, 97- 104 (1972) describe optimum buffer conditions for measuring such activity using p-nitrophenylphosphate as a substrate of alkaline phosphatase. Accordingly, the stimulation of the TSHR receptor leads to an increase in cAMP in the cell and the transcription and translation of the SEAP is proportional to the increase in cAMP . According to the invention the SEAP is secreted out of the cell after translation. The amount of SEAP in the supernatant can be determined using an alkaline phosphatase assay which is commercially available.

The measured chemoluminescent signal is given in "relative light units (RLU)". The quotient of the mean of RLU-values from patient samples and the mean of RLU-values with the first reference (e.g. serum without anti TSHR antibodies) is defined as stimulation unit (SU) .

Intra-assay variance is the variance measured in the same assay with the same lot of cells for different aliquots of a patient or reference sample. For example, a patient or refer- ence sample is measured in duplicates within one multiwall plate .

Inter-assay variance is the variance measured for different aliquots of a patient or reference sample with different lots of cells.

According to a further embodiment of the invention the cells are cultivated in a serum-free medium before incubation with the test samples. Serum-free means that the growth medium contains no sera such as fetal calf serum. In those sera TSH is present in low concentrations and would influence the measurements. Therefore, the use of TSH containing sera in the seeding medium makes an additional washing step necessary in order to remove the TSH of the serum. Using a serum free medium for cultivating avoids a washing step and consequently makes the procedure easier and faster. Generally, several measurements are taken from the samples to increase the accuracy. The bioassay of the invention can be carried out in multi-well plate. A multi-well plate offers the opportunity of measuring several samples in parallel. These can be samples from different patients, as well as reference samples including positive and negative control samples. In principal, plates with any number of wells can be used in the bioassay. In particular, the bioassay of the invention can be performed in a multi-well with at least 48 wells. Common multi-well plates that are preferred have 48 wells, 96 wells or 368 wells. Multi-well plates with at least 96 wells are preferred.

Advantageously, the samples are heated after step b) at a temperature in the range from 50 °C and 72 °C, in particular 60 °C to 70 °C. With the heat treatment (endogenous) phosphatases from the cells or the serum are deactivated. In contrast, the SEAP is heat resistant and remains active upon treatment in the defined temperature range. The chemolumines- cence from the endogenous phosphatases may vary from one sample to the other and cannot be distinguished from the

chemoluminescence of the SEAP. This would decrease the accuracy of the estimate of the blocking or stimulating activity. Preferably, the determination of the invention can be performed in the range from 0.01 to 100 mU/ml (TSH equivalent activity), in particular in the range from 0.1 to 10 mU/ml . As shown in Fig. 1 in the range from 0.01 to 100 mU/ml of TSH the measured SU value is proportional to the activity of TSH (in mU/ml) . This has the advantage that very high (100 mU/ml) and very low activities of stimulation or blocking can be measured. Therefore, a dilution of the sample is in principal not necessary. Fig. 1 also shows that the sensitivity of the assay is at the maximum in the range from 0.1 to 10 mU/ml, because in this range the slope of the dilution curve is the steepest.

As to a suited amount of the TSHR stimulating agent in the patient sample and in the second reference sample a range of 0.01 mU/ml to 100 mU/ml TSH equivalent activity is preferred. In particular, the patient sample and the second reference sample may contain 0.01 mU/ml, 0.1 mU/ml, 1 mU/ml, 10 mU/ml or 100 mU/ml of TSH equivalent activity. The concentration is given in TSH equivalent activity because the dilution curve for calibrating the assay is carried out with TSH. Fig. 1 shows schematically and as an example the determination of stimulatory autoimmune antibodies in line with the invention. A HEK cell line containing TSHR and containing the CRE-SEAP construct is exposed to a patient sample serum. The stimulating TSHR autoimmune antibodies in the patient sample bind to and stimulate the TSHR. The stimulation of the TSHR initiates the cAMP signaling cascade and leads to an increase in the concentration of cAMP . The increased amount of cAMP leads to an activation of the CRE and consequently to an increased transcription and translation of the SEAP. The SEAP is then secreted from the cell. Hence, the concentration of SEAP is directly proportional to the concentration of the stimulating antibodies in the patient sample. With the measurement of chemoluminescence caused by SEAP the biological activity of the stimulating autoimmune antibodies is deter- mined. Fig. 5 explains how the stimulating activity (A) and blocking activity (B) is derived from the measured signal.

For the determination of stimulating autoimmune antibodies the assay is performed with at least the two experimental compositions shown in Fig. 5 A. The left hand^' sample in Fig. 5 A contains cells transfected with TSHR and CRE-SEAP and the patient sample, the right hand sample contains the same cells and a negative serum without TSHR stimulating antibodies . With both samples the bioassay of the invention is performed and the signal strength is determined in each sample. The signal of the right hand sample is the background signal while the signal of the left hand sample is composed of the TSHR stimulation signal and the background signal. The back- ground signal determined in the right hand sample is subtracted from the signal of the left hand sample. The difference is proportional to the stimulating activity in the patient sample. For the determination of the blocking activity shown in Fig. 5 B three compositions are used. The signal on the right is again the background signal that is present without TSHR activation. Here, a composition identical to the right hand sample of the Fig 5 A is used. The signal in the middle of Fig. 5 B is the results from the bioassay with a composition containing cells, negative serum and 1 mU/ml of TSH. The signal on the left of Fig. 5 B results from a bioassay with a composition containing the cells, a patient sample and 1 mU/ml TSH. Because the same amount of TSH is used in the com- positions on the left and in the middle of Fig. 5 B both in principal should result in the same signal strength. However, the blocking activity leads to a decrease of the signal strength in the sample on the left of Fig. 5 B. The differ^¬ ence between the signal on the left and in the middle of Fig 5 B is therefore a measure for the blocking activity in the patient sample.

The invention is further illustrated by the following non limiting examples.

EXAMPLES

Example 1 - Production and selection of stabile bioactive cell clone

HEK 293 is a known and commercially available cell line (human embryonic kidney -293) originally established from pri- mary embryonal kidney. HEK 293 cells are adherent fibroblas- toid cells growing preferentially as monolayer

{Characteristics of a human cell line transformed by DNA from human adenovirus type 5; .Graham et al., J. Gen. Virol. 36: 59-72, 1977). A genetical construct was designed consisting of TSHR-wt sequence cloned in pIRESneo, an established vector for the stable expression of recombinant protein in mammalian cells (Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein; Rees, S., et al . (1996) BioTechniques 20:102-104).

HEK 293 cells were transfected in defined ratio (1:5) in with TSHR-wt in pIRESneo in combination with another construct consisting of a reporter gene secreted alkaline phosphatase (SEAP) under the control of a cAMP-responsive element (CRE) . Cell - cultivation was performed in 48 multi-well plates up to the confluence of 80 %. After subsequent 20 h incubation with a defined amount of TSH the quantification of the stable alkaline phosphatase in the supernatant was performed using a chemoluminescence assay. The signal values were given as stimulation units which are calculated as: SU = luminescence response of sample / light response of negative control (sample without TSH) .

The optimal cell clone was selected upon maximal response to TSH and was used for establishment of the bioassay.

Example 2 - Assay procedure

2a) Detection of the TSHR stimulating antibodies

30,000-70,000 HEK293 cells transfected with TSHRwt in pIRES- neo and pCRE-SEAP (CryoCells) were thawed and seeded in each well of 96 well-plates (NUNC, 96F Maxisorp white)—in 100 μΐ serum-free medium: RPMI 1640 + G418 + Penicillin/Streptomycin (medium and all additive components: Biochrom) . After 2 h incubation (37°C, 5 %C0₂, 92 % humidity) 20-50 μΐ of a patient samples were added in each well of the same 96-well-plate (NUNC, 96F Maxisorp white) as duplicates. The micro titer plate (MTP) with cells and samples was incubated for 18 - 22h (37 °C, 5 %C0₂, 92 % humidity) .

After the incubation, the MTP with cells and samples was heated to 65 °C for 30-45 min to inactivate the endogenous alkaline phosphatase in the samples. Afterwards the substrate for the chemiluminescence reaction (AP-juice from pjk 1:4- 1:10 in substrate buffer: 0,1M diethanolamine , 1 mM MgCl₂, pH9.5) was added, the plate was incubated for another 30-45 min (light protected) and measured in a plate-luminometer (BERTHOLD, CentroLIA LB961), measure time 2s per well. Values were given in relative light units (RLU) . The quotient of the mean of RLU-values from patient samples and the mean of RLU-values of wells with reference (negative serum without anti TSHR antibodies) is defined as stimulation unit (SU) .

2b) Detection of the TSHR blocking antibodies

For the measurement of TSHR blocking antibodies the assay de^¬ scribed in example 2a is modified in the following way. In the patient samples (30 μΐ) an amount 1 mU/ml of TSH as com- petitor was added. As reference, cells in a different well were incubated with a negative serum and an amount of 1 mU/ml TSH. The blocking activity of antibodies in samples is derived from the signal difference of the patient samples and the reference.

2c) Detection of the TSHR blocking antibodies

In a different set up for the measurement of TSHR blocking antibodies the assay described in example 2a is modified in the following way. Stimulating monoclonal or polyclonal anti TSHR antibodies with defined biological activity correspond^¬ ing to 0.1 - 10 mU/ml TSH were added as competitor to the pa^¬ tient sample (total 30 μΐ) . As reference, cells in different wells were incubated with the same patient samples without additives and samples with serial dilutions of the competitor in a negative serum. The blocking activity of antibodies in samples is derived from the signal difference of the patient samples with competitor and the reference samples.

Example 3 - Relation of the measured signal and the concen- tration of TSH

For this experiment the assay procedure described in Example 2a is modified. The measurements were performed with parame- ters according to Example 2a. Instead of a patient sample, negative sera with different specific concentrations of TSH were incubated with the cells in the different wells. The concentrations of the TSH used in the experiment were 0.01 mU/ml, 0.1 mU/ml, 1 mU/ml, 10 mU/ml, and 100 mU/ml . The experiment was performed three times with three different lots of cryo-conserved cells (CryoCells) .

The results of the experiment are shown in Fig. 1. The SU values show clear dependency from the TSH concentration over the whole range of 0.01 TSH to 10 mU/ml TSH. No saturation is seen within the range.

The inter-assay variance for all five concentration data points was in the range of CV = 0.6 - 10 %. This shows the very good reproducibility of the assay.

Example 4 - Reproducibility of the Bioassay

The measurements were performed with parameters according to Example 2a.

In order to determine the reproducibility of the assay 5 representative samples A to E with a broad range of TSHR stimulating activity were measured in independent assays with 5 different lots of cryo-conserved cells. Each sample was measured with each cell line in duplicates. Sample A) is a Graves disease negative serum without anti TSHR antibodies, sample B) is a pool of Graves disease positive sera, sample C) is sample from a Graves disease patient, sample D) is a sample from another Graves disease patient, sample E) is a plasma sample from a Graves disease patient and sample F) is a posi- tive control sample with a high concentration of bTSH (about 10 mU/ml) .

The SU values measured in all sample / cell line lot combinations are shown as data in the diagram of Fig. 2. The results demonstrate an intra-assay variance and an inter-assay variability below 10% over the whole range of measured SU values. This means that the assay produces accurate reproducible SU values for patient sera (samples B, C) and plasma (sample D) . Sample E further shows that even at higher concentrations of TSHR stimulating agent (in. this case TSH) , the assay leads to highly reproducible results . Example 5 - Comparison of the clinical score of patients with the SU values of samples from the same patients determined by the bioassay according to the invention.

The assay was carried out according to Example 2a with the patient samples listed in the first column of Table 1. Each sample was measured in duplicates (3-5 assays) . A mean value of all measurements of a sample was determined which is listed in the second column of Table 1. This mean SU value was then compared with the clinical score. The clinical score is a value from 0 to 3 indicating the diagnosed severity of the disease. The diagnoses of the patients were obtained from patient records. The clinical scores are explained in Table 2. A value of 0 stands for Graves' disease negative patient. The values 1, 2 and 3 stand for moderate, distinct and severe symptoms of Grave's disease, respectively.

Table 1

When comparing the mean SU value with the clinical score the cut-off SU value for a Graves' disease positive prediction is 1.4. Below 1.4 the clinical score is 0. In the range from 1.4 to 1.9 the prediction is ambiguous. In this so-called grey zone the values can either correspond to clinical score of 0 or 1. A mean SU value in the bioassay in the range of 1.9 to 3.0 corresponds to a clinical score of 1, a SU value in the range of 5.0 to 20 corresponds to a clinical score of 1 and a SU value above 20 corresponds to a clinical score of 3. Con^¬ sequently, bioassay prediction scores of 0 to 3 are related to the individual ranges. These values are summarized in Table 2.

Table 2

Clini¬

Bioassay : Bioassay : cal Clinical diagnosis

range [SU] prediction score score

Graves' Disease nega¬

0 0,5 - 1,9 0

tive

Graves' Disease posi¬

1 tive, moderate symp1,9 - 3,0 1

toms

Graves' Disease posi¬

2 tive, distinct symp5,0 - 20,0 2

toms

Graves' Disease posi¬

3 >20 3

tive, severe symptoms The results of the bioassay of the invention were then compared with the results of the TRAK^®-Assay.

The LUMItest TRAK human (TRAK-Assay) is an in vitro test kit for the detection of the anti TSHR antibodies. The quantita^¬ tive determination is based on the displacement of labeled TSH from the immobilized TSHR by the anti TSHR antibodies in patient sera. See also literature: Second generation assay for TSH-receptor antibodies has superior diagnostic sensitiv- ity for Graves' disease. S . Costagliola at. al . , J Clin Endo^¬ crinol Metab 84(1); 90-97 (1999) .

Accordingly, the samples of Table 1 were measured again with the TRAK assay. In order to facilitate the comparison the SU values of the patient samples determined with the bioassay, grouped according to the clinical are represented in the left panel of Fig. 3 and the IU/L values of the patient samples determined with the TRAK-Assay, also grouped according to the clinical are represented in the left panel of Fig. 3.

These data show a principle good correlation of the biologi^¬ cal activity of anti TSHR antibodies in the bioassay according to the invention with the TRAK quantification of anti TSHR antibodies.

Claims

A bioassay for the determination of biological activity of stimulating autoimmune antibodies directed against the thyroid stimulating hormone receptor (TSHR) , comprising the steps of

a) incubating TSHR expressing cells with a patient sample ,

b) detecting the intensity of a signal in the supernatant of the cells that is caused by a change of cAMP concentration in the cells resulting from TSHR stimulation,

c) determining the stimulation activity of the anti TSHR autoimmune antibodies by comparing the signal intensity of step b) with the signal intensity obtained after incubation of a first reference sample (control) with TSHR expressing cells.

A bioassay for the determination of biological activity of blocking autoimmune antibodies directed against the TSHR, comprising the steps of

a) incubating TSHR expressing cells with a patient sample ,

c) determining the blocking or stimulation activity of the TSHR autoimmune antibodies by comparing the signal intensity of step b) with

intensities obtained after incubatio: and a second reference sample with TSHR express^¬ ing cells,

The bioassay according to claim 1 or 2, wherein the TSHR expressing cells are eukaryotic cells stably transfected with the TSHR gene, in particular human embryonic kidney (HE ) cells or Chinese hamster ovary (CHO) cells.

The bioassay according to any of claims 1 to 3, wherein the TSHR stimulating agent is the thyroid stimulating hormone (TSH) , a functional fragment of TSH, a TSHR stimulating autoimmune antibody, a functional fragment of o a TSHR stimulating autoimmune antibody or thyreostimu- lin .

The bioassay according to any of claims 1 to 4, wherein the signal caused by an increase in cAMP concentration in the cell is presented by the increased production of a polypeptide that is secreted from the cell.

The bioassay according to claim 5, wherein the signal measured in the supernatant is the chemoluminescence of the polypeptide.

The bioassay according to claim 5 or 6, wherein the polypeptide is secreted alkaline phosphatase (SEAP) .

The bioassay according to any of claims 1 to 7 , wherein before incubation with the samples the cells are cultivated in serum-free media.

9. The bioassay according to any of claims 1 to 8, wherein the bioassay is carried out in multi-well plate.

10. The bioassay according to claim 9, wherein the multi-well has at least 48 wells, in particular at least 96 wells.

11. The bioassay according to any of claims 1 to 10, wherein the samples are heated after step b) at a temperature in the range from 50 °C and 72 °C.

The bioassay according to any of claims 1 to 11, wherein the intensity of the signal is proportional to the stimulating activity of TSH in a range from 0.01 to 100 mU/ml, in particular in the range from 0.1 to 10 mU/ml.

A test kit for the determination of autoimmune antibodies directed against the thyroid stimulating hormone receptor (TSHR) , comprising TSHR expressing cells transfected with CRE-SEAP and detection means for measuring chemolumines- cence .