JPH08228769A - Myeloma cell line expressing recombinant human thyroid stimulating hormone receptor - Google Patents

Abstract

(57) Abstract: A myeloma cell line expressing a recombinant human thyroid stimulating hormone receptor which facilitates efficient culture and further collection to prepare a membrane fraction, and a membrane fraction of the cell line To provide an immunochemical assay method for autoantibodies against human thyroid stimulating hormone receptor. SOLUTION: A recombinant human thyroid stimulating hormone is characterized by culturing a myeloma cell line such as SP56 expressing a recombinant human thyroid stimulating hormone receptor and a myeloma cell line expressing these recombinant human thyroid stimulating hormone receptors. A method for producing a membrane fraction containing a hormone receptor, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a myeloma cell line expressing a recombinant human thyroid stimulating hormone receptor,
The present invention relates to a method for producing a membrane fraction containing a recombinant human thyroid stimulating hormone receptor, a method for measuring an anti-human thyroid stimulating hormone receptor autoantibody, and a reagent kit for measuring an anti-human thyroid stimulating hormone receptor autoantibody.

[0002]

2. Description of the Related Art Human thyroid stimulating hormone receptor (Th
yroid Stimulating Hormone Receptor (TSHR) is a receptor for thyroid stimulating hormone (TSH) existing on the thyroid membrane and is secreted from the pituitary gland.
When SH binds to TSHR on the thyroid follicular cell membrane,
The thyroid gland secretes T3 and T4, which are hormones that regulate metabolic function. TSHR is a 7-transmembrane receptor with a molecular weight of about 87,000, and its extracellular domain has a molecular weight of about 4500.
0.

Graves' disease is the production of thyroid hormone,
It is hyperthyroidism due to hypersecretion. The cause is that a stimulant that stimulates thyroid hormone secretion is present in patient serum. Previous studies have revealed that autoantibodies to the thyroid component are present in patient sera, which cause the production and secretion of thyroid-stimulating hormone, which eventually leads to the destruction of thyroid tissue.

Antigens for diagnosing thyroid autoimmune diseases represented by Graves' disease include thyroid peroxidase, thyroglobulin, TSHR and the like. Among them, anti-TSH
It is known that R antibody is most strongly involved in the abnormality of thyroid function associated with disease.

As the anti-TSHR autoantibody assay method, the method developed by Smith is currently known (Endocr. Rev., 9, 106-120, 1988), and a reagent therefor is also commercially available. (Cosmic, etc.) It uses a thyroid membrane fraction of pig as a source of TSHR, ¹²⁵
The anti-TSHR autoantibody is detected by observing the competition between the I-labeled bovine TSH and the anti-TSHR autoantibody in the serum of the patient for the TSHR source.

[0006]

In the conventional method, since the cross-reaction of binding of porcine TSHR and anti-human TSHR autoantibody in human serum is used, human TSHR and human serum originally generated in the living body are used. It is questionable whether it correctly reflects the binding of the anti-human TSHR autoantibodies in it. In fact, since the amino acid sequences of human TSHR and porcine TSHR differ, porcine TSH in the conventional method was
It is also expected that the binding of R to anti-human TSHR autoantibodies is not completely identical to the binding of human TSHR to anti-human TSHR autoantibodies. However, it is impossible to use a human-derived thyroid membrane fraction derived from a living body as a TSHR source instead of the porcine thyroid membrane fraction due to ethical problems and the like.

In 1989, the gene for human TSHR was isolated, and a Chinese hamster oocyte (CHO cell) expressing recombinant human TSHR was reported (JP-A-5-5046).
83, JP-A-5-503849). On the human TSHR-expressing CHO cell membrane described in these documents, about 10 ⁵
The expression of individual TSHRs was confirmed and is significantly higher than the expression of several hundred per cell of the conventional porcine thyroid membrane fraction. Further, in these documents, at the same time, the above-mentioned assay method using the CHO cell membrane fraction as a thyroid membrane fraction is described.

However, since CHO cells are highly adherent cells, suspension culture is impossible. In order to collect cells in the culture of adherent cells, a method of treating with trypsin or other protease is usually used. However, when the CHO cells are treated with protease and collected, TSHR of the membrane fraction is cleaved, Therefore, it is difficult to prepare a membrane fraction that can be used for binding the anti-human TSHR autoantibody.

On the other hand, if the cells are collected by a mechanical method, a membrane fraction that can be used for binding anti-human TSHR autoantibodies can be prepared, but it is inefficient and a clinical field requiring a large amount of TSHR is required. Can not meet the requirements of.

[0010]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have conducted diligent research on the expression of TSHR, and as a result, efficiently cultivated cells and further recovered the membrane fraction. A myeloma cell line expressing recombinant human thyroid stimulating hormone receptor was established, and at the same time, an immunochemical assay for autoantibodies against the human thyroid stimulating hormone receptor was completed using the membrane fraction of the cell line. did.

That is, the present invention is a myeloma cell line expressing recombinant TSHR. The present invention also relates to recombinant TSH
A method for producing a membrane fraction containing recombinant TSHR, which comprises culturing a myeloma cell line expressing R. Then, the present invention is a method for measuring anti-human TSHR autoantibodies, which comprises a step of contacting a membrane fraction containing recombinant TSHR produced by culturing a myeloma cell line expressing recombinant TSHR with an analysis sample. Further, the present invention is a reagent kit used for measuring anti-human thyroid stimulating hormone receptor autoantibodies, which comprises a membrane fraction containing recombinant TSHR produced by culturing at least a myeloma cell line expressing recombinant TSHR. . Hereinafter, the present invention will be described in detail.

1. Myeloma Cell Line Expressing Recombinant TSHR The myeloma cell line expressing the recombinant TSHR of the present invention is produced by a conventional method using genetic engineering, that is, by introducing the following expression vector into the myeloma cell line. be able to.

The replicable expression vector that expresses the DNA sequence encoding the membrane TSHR used in the present invention, that is, capable of producing the membrane TSHR, has already been reported as TSH.
In addition to the DNA sequence encoding R, there is no limitation as long as it has a DNA sequence for expressing the DNA sequence, an origin of replication for replicating the vector DNA in the host, etc., and can transform the selected host. It can be appropriately selected and used. A promoter system is important as the DNA sequence for expressing the DNA sequence, and examples thereof include the SV40 promoter system and the adenovirus promoter system, which may be appropriately selected in relation to the host. Examples include SV40 promoter system / CHO cells (CHO-K1) and SV40 promoter system / dhfr-deficient CHO cells.

The DNA sequence encoding TSHR may be prepared by referring to known literature (eg, BBRC, 165, 1184, 1989) and the like.

As the myeloma cell line used in the present invention, the mouse myeloma cell line SP2 / O shown in the examples is used.
(Ig non-secreting mouse myeloma, CRL1581; ATCC)
Can be preferably exemplified.

The transformation of the myeloma cell line with the expression vector may be carried out by an ordinary method such as electroporation, and there is no particular limitation.

Culturing of myeloma cells can be carried out by a usual method. More specifically, for example, static culture using an ordinary culture device and mass culture using an appropriate cell culture device can be exemplified. Examples of the cell culture device include a hollow fiber type and a tank type, but a tank type is preferable from the viewpoint of not requiring an operation of peeling the cells from the carrier.

The DNA sequence encoding TSHR introduced into myeloma cells is expressed by the action of the promoter system, and TSHR-expressing myeloma cells appear on the membrane. The membrane fraction can be prepared from myeloma cells by, for example, homogenizing the cells with a homogenizer and then collecting the precipitate by centrifugation (Shinsei Chemistry Laboratory 9, Hormone I, Peptide Hormone, p369, Tokyo Kagaku Dojin). It can be carried out. The membrane fraction thus prepared is a membrane fraction containing recombinant TSHR.

2. Immunochemical Assay for Anti-TSHR Autoantibodies The immunochemical assay for anti-TSHR autoantibodies of the present invention aims to accurately and rapidly measure the physiological concentration of anti-TSHR autoantibodies contained in human serum. It is a thing.

In the present invention, specifically, the above-mentioned membrane fraction bound to the solid support may be bound with the anti-TSHR autoantibody in the analysis sample, and the bound anti-TSHR autoantibody may be measured.

In this case, as the solid support, in addition to a plate-like support such as a microtiter plate, a plastic support such as polystyrene or polypropylene, or a bead-like support such as an inorganic substance such as a metal ceramic can be used. .

There are two methods for immobilizing TSHR, that is, for binding to the solid support, a method of directly coating a membrane fraction of membrane-type TSHR-expressing cells and a method of binding via a anti-TSHR antibody. To be As a method of direct coating,
An example is a method in which a membrane fraction having a protein concentration of, for example, 1 mg / ml is added to a well of a plate in an appropriate amount (for example, about 100 μl) and left to stand overnight.

As a method for binding via an anti-TSHR antibody, for example, first, an anti-TSHR antibody is dissolved in a PBS solution to an appropriate concentration (eg, about 2 μg / ml), and then an appropriate amount (eg, 100 μg /
(about 1 ml / ml) and left to stand overnight, and then a membrane fraction having a protein concentration of, for example, about 1 mg / ml is added in an appropriate amount (for example, 10 ml).
A method of adding about 0 μl) and allowing to stand overnight can be exemplified.

The anti-TSHR autoantibody bound to such a solid support via TSHR can be measured by a method known per se, which is a sandwich method or a competition method. That is, in the case of the sandwich method, if a labeled anti-human immunoglobulin antibody is used, and the labeled anti-human immunoglobulin antibody is specifically bound to the anti-TSHR autoantibody bound to the solid support via TSHR, the label can be detected. good.

On the other hand, in the case of the competitive method, labeled TSH or labeled anti-TSHR antibody may be used. When the labeled TSH is used, the TSHR between the labeled TSH and the anti-TSHR autoantibody
Binding to is competitively occurring. In this case, non-human TSH such as bovine-derived TSH can be used as TSH, but it is particularly preferable to use human-derived TSH or immunologically identical TSH such as recombinant human TSH. Similarly, when a labeled anti-TSHR antibody is used, the labeled anti-T
Competition for TSHR occurs between SHR antibody and anti-TSHR autoantibody in serum (specimen).

Examples of the label used in the present invention include commonly used radioactive substances, fluorescent substances, luminescent substances, enzymes such as alkaline phosphatase and horseradish peroxidase, as well as biotin and the like, but they are easy to handle. From the viewpoints of the above, enzymes such as alkaline phosphatase and horseradish peroxidase are particularly preferable.

The reagent kit for use in the measurement of anti-TSHR autoantibodies provided by the present invention comprises at least recombinant TSHR produced by culturing a myeloma cell line expressing recombinant TSHR as described above. Although it has a membrane fraction, it may contain other reagents required by the sandwich method or the competitive method as described above.

3. Method for Producing Anti-TSHR Monoclonal Antibody The method for producing the anti-TSHR monoclonal antibody of the present invention is carried out by using the myeloma cell line of the present invention expressing human TSHR, which is preferably SP56, which has been described above, as an immunogen. It is characterized by immunizing animal species. SP56 is widely used as a fusion partner with mouse spleen cells during the production of ordinary monoclonal antibody SP2.
/ 0 has been transformed with an expression vector, and therefore when immunized with, for example, Balb / c mice, only the expressed human TSHR becomes a heterologous protein for Balb / c mice, and the desired anti-human TSHR is obtained.
It becomes possible to prepare a monoclonal antibody.

Examples of screening include a method using a cell sorter and a method using a microtiter plate. In any case, by using a negative control SP2 / 0 for the myeloma cell line of the present invention typified by SP56 used for immunization, it is possible to distinguish between specific binding and non-specific binding. Is.

The prepared anti-TSHR monoclonal antibody can be used in the above-mentioned immunochemical assay method for anti-TSHR autoantibodies.

The SP56 cell line of the present invention has been deposited under accession number FERM BP-5349.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, examples will be shown in order to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Isolation of human TSHR gene A series of genetic recombination procedures (m-R) in the following examples.
NA extraction, DNA restriction enzyme cleavage, etc.) can be performed by the method of Maniatis et al. (Molecular Cloning, Cold Spring H
arbor Laboratory, 1982), Harwin et al.'s method (DNA Cloning, aPractical Approach vol. 1, p49, Ox)
Ford, 1985). Also λ-gt
11 screening of a cDNA library with an antibody was performed using a commercially available vector (lambda Directi).
onal Cloning Vector, Pharm
Acia) was used.

First, human thyroid cells (excised thyroid tissue)
MRNA was extracted from the obtained cDNA and λ-gt11 (manufactured by Clontech) was used to prepare a cDNA library. Next, human-derived monoclonal antibody TRMo against TSHR
(See JBC 263, 16341, 1988), about 500,000 clones were screened, and finally one clone was obtained. Analysis of this cDNA revealed that it was identical to the sequence reported by Rapoport et al. (BBRC 165, 1184, 19).
1989).

Example 2 Construction of membrane-type TSHR expression vector Using the TSHR gene obtained in Example 1 as a starting material
A recombinant human membrane type TSHR expression vector was constructed.

The EcoRI fragment containing the entire TSHR gene was inserted into a commercially available phagemid vector pUC118 (manufactured by Takara Shuzo Co., Ltd.) to obtain p118-TSHR. p1
For 18-TSHR, oligonucleotide (5'-
CCGCCGGCCTCATGAATTCCACGGAC-3 ') and a commercially available kit (In Vitro Mutagenesis Ki
t, manufactured by Amersham), and an EcoRI site was newly introduced immediately before the start codon.

This EcoRI fragment (about 2.3 kb base pair) was first introduced into a commercially available vector (pBluescript,
After being introduced into Toyobo Co., Ltd., the fragment cut out with XhoI and NotI was used as an animal cell expression vector BCMGSne.
o (Eur. J. Immunol., 18: 97-104,
1988 and J. Exp. Med. , 169, pp. 13-25,
1989) to obtain a membrane type TSHR gene expression vector pBCMGS-hTSHR. The construction of the vector is shown in FIG.

Example 3 Establishment and selection of membrane type TSHR-expressing myeloma cell line pBCMGS-hTSHR (10 μm obtained in Example 2)
g) was introduced into 2 × 10 ⁷ SP2 / O cells (Ig non-secreting mouse myeloma, CRL1581; ATCC) by electroporation (Gene Pluser, B).
IORAD, 900V, 25 μF). After standing on ice for 10 minutes, the cells were collected and cultured in E-RDF medium (containing 10% FCS) for 24 hours. Then, the selective medium (ER
DF medium, 10% FCS, 600 μg / ml G418
After culturing for 3 weeks, the cells were monoclonalized by the limiting dilution method. Each clone was evaluated for the binding ability of labeled TSH by the method shown in Example 7.

Further, in order to confirm whether the binding of the labeled TSH is specific, the 5 clones having the binding of the labeled TSH were confirmed to have a large excess (10 ⁻⁶ M) of unlabeled TS.
H was made to coexist, and it was investigated whether the binding of labeled TSH was inhibited. The results are shown in Figure 2. As a result, it was found that the binding of the labeled TSH was completely inhibited by the addition of the unlabeled TSH, and it was shown that the labeled TSH specifically binds to the cell surface TSHR. The clone having the highest TSH binding amount (SP56) was selected as a membrane TSHR-expressing myeloma cell line. Note that SP56 is the consignment number F
Deposited as ERM BP-5349 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.

Example 4 Preparation of SP56 Cell Membrane Fraction About 4 × 10 ⁸ SP56 was added to 20 ml of buffer solution (15 m).
M Tris-HCl buffer, pH 7.5, 2 mM MgC
l ₂ , 0.3 mM EDTA, 1 mM EGTA, 1 m
M PMSF) and suspended on ice for 5 minutes. After that, it was homogenized with a Teflon homogenizer. The obtained homogenate is centrifuged (15000 × g, 15 minutes).
The pellets were collected. The collected pellets are porcine-derived T
Assay buffer 10 attached to a commercially available TBII measurement kit (TRAB, manufactured by Cosmic) using SHR
suspended in ml.

TBII means Thyrotropin binding inhibito immunoglobulin (Thyrotropin
Binding Inhibition Immunogloburin).

Example 5 Large-scale culture of SP56 cells in suspension A large-scale culture of SP56 was tried using a tank-type large-scale culture device (Axellon, manufactured by Kirin Brewery Co., Ltd.).
6 × 10 8 SP56 was added to E-RDF medium (10% FC
S, containing 600 μg / ml G418) was inoculated into 3 L (1 × 10 ⁵ cells / ml). Cultivated for 3 days, cell number is 1
When the medium reaches × 10 ⁶ cells / ml, perfusion of the medium (3
L / day) started. By continuing perfusion culture for another 3 days, the cell number reached 1 × 10 ⁷ cells / ml. In addition,
During the culture, the temperature was 37 ° C, the pH was 7.5, and the dissolved oxygen was 5
It was controlled to be ppm.

After completion of the culture, the cells were centrifuged and separated from the medium. Finally, 3 × 10 ¹⁰ from the 3 L culture tank.
Individual cells could be isolated. A membrane fraction was prepared from the obtained cells by the method described in Example 4, and a membrane fraction having a protein amount of about 3450 mg could be isolated. This membrane fraction amount corresponds to about 15,000 doses when used for immunoassay. That is, SP
It was shown that 56 can be used to prepare the membrane fraction required for a significant number of TBII measurements.

Example 6 ¹²⁵ I Labeling Reaction of TSH 5 μl of 5 μg bovine TSH (activity ratio 20 U / mg)
Of 0.1M borate buffer, pH 8.5,
Bolton-Hunter re dried with nitrogen gas
agent (manufactured by Amersham, 148 TBq / m
(mol) was added to start the TSH labeling reaction. After reacting on ice for 15 minutes, 0.1 M containing 0.2 M glycine
0.5 ml of M borate buffer pH 8.0 was added to separate unreacted reagents. Then commercially available filler (Tosoh Corporation)
Manufactured by G3000SWXL) and subjected to gel filtration to obtain a labeled T
The SH was purified.

Example 7 TSH binding assay to SP2 / 0 cells SP2 / 0 cells were centrifuged and separated from the medium. The obtained cells were washed once with Modified Hanks buffer (made to be isotonic by adding 280 mM sucrose instead of NaCl in Hanks buffer). 8000 cpm of labeled TSH was added thereto and reacted at 37 ° C. for 1 hour. After the reaction, the cells were collected by centrifugation, washed again with Modified Hanks buffer, and centrifuged to collect cells. Then, the radioactivity of the cells was measured with a γ-counter. Further, in order to measure the expression number of TSHR per cell, the amount of labeled TSH added was 650 to 37,000.
After changing to cpm, the binding amount of labeled TSH was measured.
As a result of Scatchard plot of the obtained values, 1.1 × 10 ⁵ TSHR were expressed per cell, and the Kd (dissociation constant) with bovine TSH was 2.2 × 10 5.
^Turned out to be ^-10 . The Scatchard plot obtained in this example is shown in FIG.

Example 8 Examination of Assay Conditions Using SP56 Cell Membrane Fraction The amount of labeled TSH bound when the amount of SP56 cell membrane fraction obtained in Example 4 was changed was measured. FIG. 4 shows the results.

From FIG. 4, it was decided to use a fraction containing 230 μg of protein per measurement in order to obtain a sufficient signal. Next, the bond obtained here is labeled TS
To confirm the specific binding between H and TSHR in the cell membrane fraction, a large excess (10 ⁻⁶ M) of unlabeled TS was prepared.
It was investigated whether H was allowed to coexist and binding of labeled TSH was inhibited.

The results are shown in FIG. From FIG. 5, labeled TSH
It can be seen that the binding of the is completely inhibited by the addition of unlabeled TSH. From the above, it is clear that the labeled TSH specifically binds to TSHR in the cell membrane fraction, and the assay method using labeled TSH is T
It was shown that it can be used for the measurement of autoantibodies against SHR.

Example 9 Measurement of TBII Using SP56 Cell Membrane Fraction 53 serum samples from Graves' disease were tested. First, the TBII of 53 samples was measured using a commercially available TBII measurement kit (TRAb kit, manufactured by Cosmic). afterwards,
TBI of 53 samples using the amount of membrane fraction shown in Example 8
I was measured. TBI obtained from each measurement method
The correlation of the values of I is shown in FIG. From this result, r = 0.7
A correlation of 26 was obtained. Furthermore, of the 28 specimens that were determined to be negative (points below 10% on the vertical axis) in the conventional kit, 25 in this method using the membrane fraction of SP56.
The sample was determined to be positive (dots in black in FIG. 6).

From the above, TBII using the SP56 membrane fraction is more sensitive than conventional TBII, and the assay system for TBII originally uses anti-TSHR in serum.
It is shown that using human TSHR recognized by autoantibodies is more suitable than using porcine thyroid membrane fraction containing porcine TSHR.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to produce a large amount of recombinant TSHR, particularly recombinant human TSHR, which is effective for diagnosing autoimmune diseases and the like. Moreover, various effects are achieved in the present invention in which a DNA sequence encoding TSHR is expressed in myeloma cells. That is, it becomes possible to express a large amount of TSHR by using myeloma cells. Further, since myeloma cells are not adherent cells, suspension culture can be performed relatively easily, and the culture operation can be performed using a simple device or the like.

Moreover, no special skill is required in this culturing operation, and a larger amount of culturing is possible as compared with adherent cells. Since myeloma cells are adherent cells, protease treatment with trypsin or the like is not necessary when the expressed TSHR is recovered, and TSHR having a transmembrane region is not cleaved. Therefore, anti-T
In terms of binding to SHR autoantibodies, T with higher reliability than TSHR damaged by protease treatment
It is also possible to manufacture SHR.

As shown in the examples, the TSHR produced according to the present invention enables a more accurate measurement as compared with the TSHR derived from pig which has been conventionally used for the measurement of anti-TSHR autoantibodies. To do.

[Brief description of drawings]

FIG. 1 is a membrane-type TSHR gene expression vector BCM.
It is a figure which shows the structure of GS-hTSHR. CMV in the figure
-P indicates a cytomegalovirus promoter, TS
HR indicates TSHR, BPV indicates bovine papilloma virus, Amp indicates a DNA sequence encoding β-lactamase, and poly A indicates a polyadenylate attachment signal. Further, Neo in the figure represents a neomycin resistance gene.

FIG. 2 is a myeloma cell line (SP51, SP56, SP6) expressing five recombinant TSHRs of the present invention.
FIG. 3 shows comparison of binding activity of labeled TSH to 3, SP80 and SP91) and a normal myeloma cell line SP2 / 0. In the figure, + (plus) indicates the amount of labeled TSH bound in the presence of unlabeled TSH, and- (minus) indicates unlabeled TSH.
The binding amount of labeled TSH in the absence of SH is shown.

FIG. 3 is a Scatchard plot, where the horizontal axis shows the amount of labeled TSH bound and the vertical axis shows the ratio of bound labeled TSH to unbound labeled TSH.

FIG. 4 is a recombinant human TS produced according to the present invention.
Labeled TS for membrane fraction containing HR (SP56 membrane fraction)
It is a figure which shows the change of the binding amount of H. The horizontal axis represents the amount of SP56 membrane fraction, and the vertical axis represents the amount of bound labeled TSH.

FIG. 5: Recombinant human TS produced according to the present invention
It is a figure which shows the binding inhibition of the labeled TSH by the unlabeled TSH using the membrane fraction containing HR (SP56 membrane fraction). In the figure, + (plus) indicates the binding amount of labeled TSH in the presence of unlabeled TSH, and- (minus) indicates the binding amount of labeled TSH in the absence of unlabeled TSH. Note that SP2 / 0 means
Fig. 2 shows the results for a membrane fraction prepared from myeloma cells SP2 / 0 that did not express recombinant human TSHR used as a control.

FIG. 6 shows a commercially available TBII assay kit or a membrane fraction containing recombinant TSHR produced by the present invention (SP5).
It is a figure which shows the correlation of the measurement result of the anti-TSHR autoantibody in a sample obtained using 6 membrane fractions. Horizontal axis is SP56
The result using the membrane fraction is shown, and the vertical axis shows the result using the commercially available measurement kit. In the figure, black circles indicate serum samples that were determined to be negative by a commercially available kit and were determined to be positive in the measurement using the SP56 membrane fraction containing recombinant human TSHR produced by the present invention, and open circles. Indicates that the determination was positive or negative in both measurements. In addition, the judgment of positive or negative with a commercially available kit is
Cutoff value according to the manual attached to the kit (in the figure,
The broken line parallel to the horizontal axis) was followed. In addition, in the figure, the broken line parallel to the horizontal axis is the cutoff value determined based on the measurement results of 20 healthy people.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 G01N 33/564 Z G01N 33/564 9162-4B C12N 15/00 A // (C12N 5/10 ZNA C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (11)

[Claims] 1. A myeloma cell line expressing a recombinant human thyroid stimulating hormone receptor. 2. The cell line of claim 1, which is the SP56 cell line. 3. A method for producing a membrane fraction containing a recombinant human thyroid stimulating hormone receptor, which comprises culturing a myeloma cell line expressing the recombinant human thyroid stimulating hormone receptor. 4. The method according to claim 2, wherein the myeloma cell line expressing the recombinant human thyroid stimulating hormone receptor is the SP56 cell line. 5. An anti-human thyroid stimulator comprising a step of bringing a membrane fraction containing a recombinant human thyroid stimulating hormone receptor produced by culturing a myeloma cell line expressing the recombinant human thyroid stimulating hormone receptor into contact with an analysis sample. Method for measuring hormone receptor autoantibody. 6. The method according to claim 5, wherein the myeloma cell line expressing the recombinant human thyroid stimulating hormone receptor is the SP56 cell line. 7. A method for producing an anti-human thyroid stimulating hormone receptor monoclonal antibody, which comprises a step of immunizing an animal species with a myeloma cell line expressing a recombinant human thyroid stimulating hormone receptor. 8. The method according to claim 7, wherein the myeloma cell line expressing the recombinant human thyroid stimulating hormone receptor is the SP56 cell line. 9. A reagent kit for use in measuring anti-human thyroid stimulating hormone receptor autoantibodies, which comprises a membrane fraction containing at least a recombinant human thyroid stimulating hormone receptor. 10. A method for measuring an anti-human thyroid stimulating hormone autoantibody comprising a conjugate of an anti-human thyroid stimulating hormone receptor monoclonal antibody produced by the method according to claim 7 or 8 and a water-insoluble carrier. Reagent kit. 11. An anti-human thyroid stimulating hormone receptor comprising a step of contacting a sample with a conjugate of an anti-human thyroid stimulating hormone receptor monoclonal antibody produced by the method according to claim 7 or 8 and a water-insoluble carrier. Method for measuring autoantibodies.