Reporter cell for TSH activity measurement

Abstract

The present invention addresses the problem of providing a new method for easily measuring the activity of thyroid stimulating hormone (TSH).　Provided is a reporter cell for measuring the activity of thyroid stimulating hormone (TSH). In the reporter cell, a TSH receptor gene that encodes a TSH receptor or an active fragment thereof and a reporter gene that encodes a reporter protein are introduced. The reporter gene is arranged under the control of a promoter of which the promoter activity is controlled depending on the activity of the TSH receptor or an active fragment thereof.

Description

Reporter cells for measuring TSH activity

[0001] The present invention relates to a reporter cell for measuring thyroid-stimulating hormone (TSH) activity, a TSH activity measurement kit, a method for measuring TSH activity, a method for determining hypothyroidism or hyperthyroidism, and the like.

[0002] The amount of thyroid hormone in the blood is regulated by the hypothalamus and pituitary gland in the brain. Thyrotropin-releasing hormone (TRH), secreted from the hypothalamus, stimulates the pituitary gland to promote the synthesis of thyroid-stimulating hormone (TSH). TSH, secreted from the pituitary gland, stimulates the thyroid gland to promote the production and secretion of thyroid hormones T4 and T3. Most of the thyroid hormones (T4 and T3) secreted into the blood exist bound to proteins, but the free thyroid hormones (mainly FT3), which are not bound to proteins, enter the cells of target tissues and bind to receptors in the cell nucleus to exert their hormonal effects. It is also known that thyroid hormones regulate TSH secretion from the pituitary gland by acting on the hypothalamus and pituitary gland.

[0003] Graves' disease and Hashimoto's disease are well-known as typical diseases that cause abnormalities in thyroid function. The former causes hyperthyroidism due to an increase in free thyroid hormones in the blood, while the latter causes hypothyroidism due to a decrease in free thyroid hormones.

[0004] Furthermore, a condition in which the free thyroid hormone (FT4 and FT3) levels in the blood are within the normal range but the TSH level in the blood is elevated is called subclinical hypothyroidism, and conversely, a condition in which the TSH level in the blood is decreased is called subclinical hyperthyroidism, which respectively represent mild hypothyroidism and mild hyperthyroidism. There are an extremely large number of people who fall into the state of subclinical hypothyroidism (FT4 and FT3 levels are normal, and TSH levels are above the upper limit of the normal range), with the Japan Endocrine Society estimating that this applies to 3.3-6.1% of "healthy" adults.

[0005] Many patients with subclinical hypothyroidism complain of symptoms of hypothyroidism (fatigue, constipation, sensitivity to cold, dry skin, depressed mood, irregular menstruation, etc.), and it is known that there may be a risk of progression of arteriosclerosis, elevated LDL cholesterol, cardiovascular disorders such as angina pectoris and myocardial infarction, non-alcoholic fatty liver disease (NAFLD), fatty liver, infertility, miscarriage and premature birth, and reduced IQ of the fetus.

[0006] As with hypothyroidism, thyroid hormone replacement therapy, such as levothyroxine, is an effective treatment for subclinical hypothyroidism. However, there is no international consensus on the TSH level at which replacement therapy should be initiated. It is generally recommended that replacement therapy be initiated in individuals with a TSH concentration of 10 μIU / mL or higher.

[0007] For patients with a TSH concentration below 10 μIU / mL, the decision on whether to administer thyroid hormone medication is difficult because the risk of side effects from excessive thyroid hormone administration may be higher than the therapeutic benefits. Generally, replacement therapy is often indicated for patients with a TSH concentration of approximately 7-8 μIU / mL, but the current situation is that the decision is made on an individual basis, taking into account risk factors such as hyperlipidemia.

[0008] Regarding the effectiveness of replacement therapy for subclinical hypothyroidism, a previously conducted randomized, double-blind, placebo-controlled, parallel-group comparative study (TRUST study) failed to demonstrate the effectiveness of levothyroxine in improving symptoms in elderly patients. However, only small-scale randomized controlled trials have been conducted on levothyroxine replacement therapy for subclinical hypothyroidism to date. Therefore, sufficient evidence regarding the efficacy and risks of replacement therapy has not yet been obtained (Non-Patent Documents 1-2).

[0009] Thyroid hormones are essential hormones that are extremely important for the growth and development of the fetus. Because the fetus cannot yet produce sufficient thyroid hormones on its own and the thyroid hormones in the mother's blood are the main source of thyroid hormones, it is strongly recommended that pregnant women avoid even mild hypothyroidism. Therefore, it is internationally recommended that pregnant women begin thyroid hormone replacement therapy when their TSH levels reach the high normal range of 4 μIU / mL or higher (or 2.5 μIU / mL or higher if the pregnant woman has Hashimoto's disease).

[0010] N Engl J Med. (2017) 376(26):2534-2544.JAMA. (2019) 322(20):1977-1986.

[0011] An object of the present invention is to provide a novel method for easily measuring thyroid-stimulating hormone (TSH) activity.

[0012] The most widely used method for quantifying blood TSH is the immunoassay based on an antigen-antibody reaction. This method measures the amount of TSH by sandwiching human TSH between two monoclonal antibodies that can bind to TSH. Currently, diagnosis and treatment guidelines are determined based on the amount of TSH measured by the immunoassay.

[0013] However, immunoassays only measure TSH levels and may not reflect TSH biological activity. This is because TSH is bound to a carbohydrate chain, and the structure of this chain may vary between individuals, potentially affecting biological activities such as thyroid-stimulating activity. Therefore, even if subjects have the same TSH levels measured by immunoassays, their actual TSH activity may vary significantly. For example, among subjects with TSH levels measured by immunoassays (approximately 7-8 μIU / mL), many do not require replacement therapy due to low TSH activity, and even among subjects with levels below approximately 7-8 μIU / mL, some may require replacement therapy. Therefore, conventional immunoassays may not accurately diagnose subclinical hypothyroidism.

[0014] To achieve the above object, the present inventors have prepared reporter cells for measuring TSH activity by introducing a TSH receptor gene and a luciferase gene downstream of a promoter activated downstream of the TSH receptor. When a serum sample containing TSH is reacted with these reporter cells, TSH binds to the TSH receptor on the cell surface, and luciferase is synthesized within the reporter cells depending on the degree of TSH receptor activation. The present inventors have found that TSH activity can be easily quantified by reacting this luciferase with luciferin and measuring the amount of luminescence using a luminometer.

[0015] The present inventors further used the reporter cells to measure TSH activity in serum obtained from patients with subclinical hypothyroidism. The surprising result was that approximately 7% of subjects with TSH concentrations of 10 μIU / mL or higher based on conventional immunoassays had actual TSH activity of less than 7 μIU / mL, and therefore did not require replacement therapy. The present invention is based on the above-mentioned research results and provides the following:

[0016] (1) A reporter cell for measuring thyroid-stimulating hormone (TSH) activity, into which a TSH receptor gene encoding a TSH receptor or an active fragment thereof and a reporter gene encoding a reporter protein have been introduced, the reporter gene being placed under the control of a promoter whose activity is controlled depending on the activity of the TSH receptor or an active fragment thereof. (2) The reporter cell according to (1), wherein the promoter comprises a cAMP response element. (3) The reporter cell according to (1), wherein the reporter protein is a luminescent protein, a fluorescent protein, or an enzyme protein. (4) The reporter cell according to (1), wherein the reporter cell is a mammalian cell. (5) The reporter cell according to (4), wherein the mammalian cell is an HEK293 cell, a HeLa cell, an NIH3T3 cell, a CHO cell, a COS cell, or a Vero cell. (6) A TSH activity measurement kit comprising the reporter cell according to (1). (7) The TSH activity measurement kit according to (6), further comprising a TSH standard solution containing a standard concentration of TSH. (8) The TSH activity measurement kit according to (6) or (7), wherein the reporter protein is a luminescent protein and further comprises a luminescent substrate for the luminescent protein. (9) A method for measuring the activity of thyroid-stimulating hormone (TSH) in a sample, comprising: a culturing step of culturing reporter cells in a medium containing the sample; and a reporter activity measurement step of measuring the activity of the reporter protein derived from the reporter cells after culturing, wherein the reporter cells have been introduced with a TSH receptor gene encoding a TSH receptor or an active fragment thereof, and a reporter gene encoding the reporter protein, and the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or the active fragment thereof. (10) The method according to (9), wherein the reporter protein is a luminescent protein, and the reporter activity measurement step comprises adding a luminescent substrate for the luminescent protein to the reporter cells and measuring the luminescence intensity of the luminescent substrate.(11) A method for diagnosing hypothyroidism or hyperthyroidism, comprising: a culturing step of culturing reporter cells in a medium containing a body fluid derived from a subject; a reporter activity measuring step of measuring the activity of a reporter protein derived from the reporter cells after culturing to obtain a reporter activity value; and a determining step of determining that the subject has overt hypothyroidism or subclinical hypothyroidism if the reporter activity value or a TSH activity value calculated based thereon is higher than an upper reference limit for TSH, and determining that the subject has overt hyperthyroidism or subclinical hyperthyroidism if the reporter activity value or the TSH activity value is lower than a lower reference limit for TSH, wherein a TSH receptor gene encoding a TSH receptor or an active fragment thereof, and a reporter gene encoding the reporter protein are introduced into the reporter cells, and the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or the active fragment thereof.(12) A method for diagnosing hypothyroidism or hyperthyroidism, comprising: a culturing step of culturing reporter cells in a medium containing a body fluid derived from a subject; a reporter activity measuring step of measuring the activity of a reporter protein derived from the reporter cells after culturing to obtain a reporter activity value; an FT4 / FT3 measuring step of measuring the concentrations of free thyroxine (FT4) and free triiodothyronine (FT3) in the body fluid derived from the subject to obtain an FT4 measurement value and an FT3 measurement value; and based on the reporter activity value or the TSH activity value calculated thereon, and the FT4 measurement value and the FT3 measurement value, (a) diagnosing the subject as having overt hypothyroidism when the reporter activity value or the TSH activity value is higher than the upper reference value of TSH and at least one of the FT4 measurement value and the FT3 measurement value is lower than its lower reference value, (b) determining that the subject has subclinical hypothyroidism when the reporter activity value or the TSH activity value is higher than the upper reference value for TSH and the FT4 measurement value and the FT3 measurement value are equal to or higher than the lower reference value and equal to or lower than the upper reference value, respectively; (c) determining that the subject has subclinical hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference value for TSH and the FT4 measurement value and the FT3 measurement value are equal to or higher than the lower reference value and equal to or lower than the upper reference value, respectively; and (d) determining that the subject has overt hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference value for TSH and at least one of the FT4 measurement value and the FT3 measurement value is higher than its upper reference value; wherein a TSH receptor gene encoding a TSH receptor or an active fragment thereof and a reporter gene encoding the reporter protein are introduced into the reporter cells; (13) The method according to (11) or (12), wherein the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or an active fragment thereof. (14) The method according to (13), wherein the TSH activity value is calculated based on the reporter activity value and a standard reporter activity value obtained based on a standard concentration of TSH in the determining step.(14) The method according to (11) or (12), wherein the reporter protein is a luminescent protein, and the reporter activity measuring step comprises adding a luminescent substrate of the luminescent protein to the reporter cells and measuring the luminescence intensity of the luminescent substrate. (15) The method according to (11) or (12), wherein the body fluid is blood, serum, or plasma. (16) The method according to (11) or (12), wherein the lower reference limit of TSH is 0.1 to 1.0 μIU / mL and / or the upper reference limit of TSH is 4.0 to 9.0 μIU / mL. (17) The method according to (11) or (12), wherein the lower reference limit of FT4 is 0.90 ng / dL, the upper reference limit of FT4 is 1.70 ng / dL, and / or the lower reference limit of FT3 is 2.30 pg / mL and the upper reference limit of FT3 is 4.00 pg / mL. (18) The method according to (11) or (12), wherein the subject is a pregnant woman or a woman wishing to become pregnant. (19) A TSH activity measurement kit according to (6) or (7), for use in the method according to (11) or (12). This specification incorporates the disclosure of Japanese Patent Application No. 2023-190104, from which the present application claims priority.

[0017] The reporter cells of the present invention provide a novel method for easily measuring thyroid-stimulating hormone (TSH) activity, and also provide a method for determining hypothyroidism or hyperthyroidism using the reporter cells of the present invention.

[0018] Figure 4A shows a schematic diagram of the thyroid hormone regulatory system. Figure 4B shows the relationship between overt hypothyroidism, subclinical hypothyroidism, subclinical hyperthyroidism, and overt hyperthyroidism and thyroid-stimulating hormone (TSH) and free thyroid hormones (FT3 and FT4) in the blood. Figure 4C shows a schematic diagram of reporter cells for measuring TSH activity. The reporter cells contain a TSH receptor gene and a luciferase gene downstream of a promoter activated downstream of the TSH receptor. Figure 4D shows the results of quantifying luciferase luminescence intensity using TSH standard solutions. Figure 4A shows the TSH concentration (μIU / mL) in each TSH standard solution, the luminescence intensity (RLU) of the reporter cells for each TSH standard solution, and the relative luminescence intensity (%), where the luminescence intensity at a TSH concentration of 0 μIU / mL is taken as 100%. Figure 4B shows a standard curve showing the relationship between TSH concentration and relative luminescence intensity. 1 shows the results of measuring the amount of TSH in serum obtained from subjects (n=284) by an immunological assay based on an antigen-antibody reaction (horizontal axis) and the results of measuring reporter activity using reporter cells (vertical axis). 2 shows the results of measuring TSH activity in serum obtained from healthy subjects (n=267), patients with subclinical hypothyroidism (n=89), and patients with overt hypothyroidism (n=121) by a reporter activity assay method based on reporter cells.

[0019] 1. Reporter Cells 1-1. Overview A first aspect of the present invention is a reporter cell for measuring thyroid-stimulating hormone (TSH) activity. A TSH receptor gene and a reporter gene are introduced into the reporter cell of the present invention. In the reporter cell of the present invention, the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or an active fragment thereof. Therefore, when TSH binds to the TSH receptor on the reporter cell, the expression level of the reporter gene increases. Therefore, TSH activity can be measured by measuring the activity of the reporter protein encoded by the reporter gene.

[0020] 1-2. Definitions The following terms frequently used in this specification are defined below. "Thyroid stimulating hormone (often referred to as "TSH" herein)" is a glycoprotein hormone composed of an α subunit and a β subunit. TSH is secreted from the pituitary gland and stimulates the thyroid gland to secrete the thyroid hormones T4 and T3. In thyroid dysfunction accompanied by abnormal blood TSH concentrations, elevated TSH concentrations are referred to as hypothyroidism (overt or subclinical), while decreased TSH concentrations are referred to as hyperthyroidism (overt or subclinical). The source of TSH is not particularly limited herein, nor are the source species of the α and β subunits that make up TSH. A specific example of the TSH α subunit is the human α subunit consisting of the amino acid sequence set forth in SEQ ID NO: 1. A specific example of the TSH β subunit is the human β subunit consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0021] As used herein, the term "thyroid stimulating hormone receptor (TSHR; sometimes referred to herein as "TSH receptor")" refers to a G protein-coupled receptor capable of binding to TSH as a TSH receptor. TSH receptors are known to be expressed in thyroid epithelial cells. The origin of the TSH receptor is not particularly limited herein. Examples of TSH receptors include human TSH receptors, TSH receptor orthologs derived from non-human species, and mutant TSH receptors or TSH receptor orthologs. A specific example of a human TSH receptor is the human TSH receptor consisting of the amino acid sequence set forth in SEQ ID NO: 3. Examples of TSH receptor variants or orthologs include amino acid sequences in which one or several (e.g., 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3) amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 3, and amino acid sequences that share 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) amino acid identity with the amino acid sequence of SEQ ID NO: 3.

[0022] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues between two amino acid sequences relative to the total amino acid residues when the two amino acid sequences are aligned, with gaps introduced as necessary, to maximize the degree of amino acid identity between the two. Amino acid identity can be calculated using a protein search system such as BLAST or FASTA.

[0023] As used herein, the term "active fragment" of the TSH receptor refers to any fragment capable of regulating the transcriptional activity of a reporter gene in the reporter cells of the present invention in response to binding to TSH. For example, the fragment may be a polypeptide fragment that retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the activity of the full-length TSH receptor, or at least the activity of the full-length TSH receptor. The amino acid length of the active fragment is not particularly limited as long as it retains the activity of the TSH receptor. For example, the fragment may be a polypeptide consisting of at least 500, at least 600, or at least 700 consecutive amino acid residues, including the transmembrane domain, of the full-length amino acid sequence of the TSH receptor, or a polypeptide consisting of at most 750, at most 700, or at most 600 consecutive amino acid residues.

[0024] As used herein, the term "TSH receptor gene" refers to a gene encoding a TSH receptor. Examples of TSH receptor genes include genes encoding human TSH receptors, TSH receptor orthologs from non-human species, and mutant TSH receptors or TSH receptor orthologs. Specific examples of genes encoding human TSH receptors include genes encoding human TSH receptors consisting of the amino acid sequence set forth in SEQ ID NO: 3, such as the human TSH receptor gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4. Examples of genes encoding mutant or ortholog TSH receptors include nucleotide sequences that share 60% or more nucleotide identity with the nucleotide sequence set forth in SEQ ID NO: 4 (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

[0025] As used herein, "base identity" refers to the percentage (%) of identical bases between two base sequences relative to the total base sequence when the two base sequences are aligned and gaps are introduced as necessary to maximize the degree of base identity between the two.

[0026] As used herein, unless otherwise specified, the term "reporter cells" refers to cells that can exhibit a change in reporter activity, as described below, depending on the presence or amount of extracellular TSH.

[0027] As used herein, unless otherwise specified, "reporter activity" refers to an activity that enables the determination of the presence or absence of TSH and / or the measurement of the amount of TSH. More specifically, reporter activity refers to the activity of a reporter protein (described below) that is expressed in a reporter cell. The type of reporter activity is not particularly limited, and specific examples include the luminescence activity of a photoprotein, the fluorescence activity of a fluorescent protein, and the enzymatic activity of an enzyme protein.

[0028] As used herein, the term "reporter protein" refers to a polypeptide or peptide that is expressed by a reporter cell and enables determination of the presence or absence of extracellular TSH or measurement of the amount of extracellular TSH. There are no particular limitations on the type of reporter protein, and specific examples include luminescent proteins, fluorescent proteins, and enzyme proteins (e.g., chromoproteins).

[0029] The term "luminescent protein" refers to a substrate protein that can emit light without requiring excitation light or an enzyme that catalyzes the luminescence of the substrate protein. Examples include luciferin or aequorin as the substrate protein and luciferase (e.g., Renilla luciferase, Gaussia luciferase, firefly luciferase, and bacterial luciferase) as the enzyme.

[0030] A "fluorescent protein" refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either natural or non-natural. Furthermore, there are no particular limitations on the excitation wavelength or fluorescence wavelength. Specific examples include CFP, BFP, RFP, mCherry, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), and the like.

[0031] A "chromoprotein" is a protein, usually an enzyme, involved in the biosynthesis of a pigment or capable of chemical detection by a pigment when a substrate is provided. Specific examples of such enzymes include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucuronidase (GUS), and melanin-based pigment synthesis proteins.

[0032] In addition to the above examples, a tag peptide or epitope sequence that can be detected by an antibody or the like can also be used as the reporter protein.

[0033] As used herein, a "reporter gene" is a gene that encodes a reporter protein. Examples of reporter genes include luminescent protein genes, fluorescent protein genes, and enzyme genes (e.g., chromoprotein genes).

[0034] 1-3. Configuration The reporter cell for measuring thyroid-stimulating hormone (TSH) activity of this embodiment is introduced with a TSH receptor gene encoding a TSH receptor or an active fragment thereof, and a reporter gene encoding a reporter protein. In the reporter cell of this embodiment, the reporter gene is placed under the control of a promoter whose promoter activity is regulated depending on the activity of the TSH receptor or an active fragment thereof.

[0035] The type of reporter cell in this embodiment is not limited. The reporter cell may be either a prokaryotic cell or a eukaryotic cell. Examples of prokaryotic cells include bacterial cells such as Escherichia coli cells. Examples of eukaryotic cells include fungal cells (e.g., yeast cells), algae cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). The reporter cell is preferably a mammalian cell. Specific examples of mammalian cells include, but are not limited to, HEK293 cells, HeLa cells, NIH3T3 cells, CHO cells, COS cells, and Vero cells. The host cell may be either an adherent cell or a suspension cell, with adherent cells being more preferred.

[0036] As used herein, a "promoter whose activity is controlled depending on the activity of the TSH receptor or an active fragment thereof" refers to any promoter whose transcriptional activity (e.g., activation or inactivation) of a gene located downstream of the promoter changes in response to TSH binding to the TSH receptor or an active fragment thereof expressed by reporter cells, and the specific type of promoter is not limited. Examples of such promoters include promoters containing cAMP response elements. For example, a promoter combining one or more cAMP response elements with the minimal Herpes Simplex Virus (HSV) thymidine kinase gene promoter can be used.

[0037] As used herein, "cAMP response element (CRE)" refers to a nucleotide sequence that controls (e.g., promotes or suppresses) the transcriptional activity of a gene in response to an increase in intracellular cAMP concentration. Various nucleotide sequences are known as cAMP response elements, and any nucleotide sequence known in the art may be used. Specific examples of cAMP response elements include the nucleotide sequence TGACGTCA (SEQ ID NO: 5), and sequences in which one or several (e.g., 2 to 3) bases have been deleted, substituted, or added to the nucleotide sequence TGACGTCA (SEQ ID NO: 5), and which have cAMP response activity.

[0038] As used herein, the phrase "a reporter gene is placed under the control of a promoter" means that the reporter gene is placed in an expressible state in the downstream region of the promoter under the control of the promoter.

[0039] As used herein, the phrase "the TSH receptor gene and the reporter gene have been introduced into" a reporter cell means that the TSH receptor gene and the reporter gene are contained in a gene expression vector introduced into the reporter cell and / or are integrated into the genome of the reporter cell. For example, both the TSH receptor gene and the reporter gene may be contained in the same gene expression vector, or the TSH receptor gene and the reporter gene may be contained in two or more different gene expression vectors.

[0040] As used herein, the term "gene expression vector" refers to a vector that contains a gene of interest, such as a reporter gene or a TSH receptor gene, in an expressible state and includes an expression unit capable of regulating its expression. The gene expression vector may be a plasmid vector or a viral vector. The plasmid vector may be a commercially available expression vector for mammalian cells, such as Promega's pSI vector, or a shuttle vector capable of replicating between mammalian cells and bacteria, such as Escherichia coli. Viral vectors that can be used include retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated viral (AAV) vectors, simian viral vectors, vaccinia viral vectors, Sendai viral vectors, Epstein-Barr viral (EBV) vectors, and HSV vectors. In addition to the gene of interest and a promoter, the gene expression vector may also contain, as necessary, components such as a marker gene, intron, enhancer, terminator, replication origin, and / or poly(A) signal.

[0041] In addition to the TSH receptor gene and reporter gene, the reporter cell of this embodiment can contain a gene encoding a signal transduction factor that regulates promoter activity depending on the activity of the TSH receptor or an active fragment thereof. For example, a G protein that can be activated by the TSH receptor or an active fragment thereof can be Gα sA gene encoding a protein (e.g., the human GNAS gene consisting of the base sequence shown in SEQ ID NO: 5), an adenylate cyclase gene, a protein kinase A gene (PKA gene), and / or a cAMP response element binding protein gene (CREB gene) may be introduced into the reporter cells.

[0042] 1-4. Effects In the reporter cells of the present invention, the transcription level of the reporter gene is controlled depending on the presence or amount of extracellular TSH. For example, the higher the presence or amount of extracellular TSH, the higher the transcription level of the reporter gene, and vice versa. Therefore, the presence of TSH can be determined and the amount of TSH can be measured by assessing TSH activity through measuring the reporter activity of the reporter protein encoded by the reporter gene.

[0043] 2. TSH Activity Measurement Kit 2-1. Overview A second aspect of the present invention is a TSH activity measurement kit. The TSH activity measurement kit of this aspect includes the reporter cell of the first aspect. With the TSH activity measurement kit of this aspect, the presence or amount of TSH in any sample can be quantified as reporter activity to evaluate TSH activity. The TSH activity measurement kit of this aspect can also be used in the method of the third or fourth aspect described below.

[0044] 2-2. Configuration The TSH activity measurement kit of this embodiment includes the reporter cell of the first embodiment. The configuration of the reporter cell is as described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0045] In one embodiment, the TSH activity measurement kit of this aspect further comprises a TSH standard solution in addition to the reporter cells. As used herein, the term "TSH standard solution" refers to a liquid (e.g., an aqueous solution) containing a standard concentration of TSH. The standard concentration of TSH is not particularly limited and may be, for example, 0 μIU / mL to 1000 μIU / mL, 1 μIU / mL to 500 μIU / mL, 5 μIU / mL to 200 μIU / mL, 10 μIU / mL to 100 μIU / mL, or 20 μIU / mL to 50 μIU / mL. By contacting a TSH standard solution containing a standard concentration of TSH with the reporter cells of the present invention and measuring the reference reporter activity, the presence or amount of TSH in the sample can be evaluated from the reporter activity obtained for the sample to be measured.

[0046] The TSH activity measurement kit of this embodiment may also contain a substrate for the reporter protein. The substrate for the reporter protein may be selected appropriately depending on the type of reporter protein. For example, if the reporter protein is a luminescent protein such as luciferase, the substrate may be a luminescent substrate such as luciferin. Furthermore, if the reporter protein is a chromogenic protein, the substrate may be a chromogenic substrate.

[0047] The TSH activity measurement kit of this embodiment may also include a medium for culturing reporter cells, an agent such as an antibiotic to be added to the medium, and / or an instruction manual.

[0048] 3. TSH Activity Measurement Method 3-1. Overview A third aspect of the present invention is a method for measuring the activity of thyroid-stimulating hormone (TSH) in a sample (hereinafter referred to as the "TSH activity measurement method"). The measurement method of the present invention allows the presence or amount of TSH in a sample to be measured accurately and simply.

[0049] The method for measuring TSH activity in this embodiment includes a culturing step and a reporter activity measurement step as essential steps, and a TSH standard solution measurement step as an optional step. Each step will be specifically described below.

[0050] (1) Culturing step The "culturing step" is a step of culturing reporter cells in a medium containing a sample. The specific configuration of the reporter cells is as described in the first embodiment.

[0051] As used herein, the term "sample" is not particularly limited as long as it contains or may contain TSH, but typically refers to a liquid sample. Examples of liquid samples include liquids isolated from living organisms, such as body fluids isolated from a subject. The sample can be diluted as necessary, for example, when the reporter activity exceeds the measurable range in the reporter activity measurement step described below.

[0052] Examples of "body fluids" used herein include blood (including serum, plasma, and interstitial fluid), lymph, cerebrospinal fluid, interstitial fluid, pleural effusion, sputum, tears, nasal discharge, saliva, urine, ascites, and extracts of various tissues or cells. Blood may be serum or plasma prepared from blood. For example, blood, serum, plasma, cerebrospinal fluid, urine, saliva, and ascites are preferred because they are relatively easy to collect. Body fluids may be derived from healthy subjects or patients with any disease. For example, they may be derived from patients with thyroid dysfunction or patients suspected of having thyroid dysfunction. Inactivated serum may be used.

[0053] The type of medium used in this step is not limited as long as it does not inhibit the binding of TSH to the TSH receptor and is capable of maintaining the reporter cells as viable cells. For example, the medium may be any medium commonly used in cell culture and known in the art. The medium may be any of basal medium, serum-free medium, low-serum medium, and serum-added medium, but typically may be a basal medium, such as a standard cell culture medium. The term "standard cell culture medium" as used herein refers to a versatile basal medium primarily used for culturing various types of mammalian cells. Specific examples include Eagle Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F10 (F10), Ham's Nutrient Mixture F12 (F12), M199, High Performance Medium 199, Roswell Park Memorial Institute-1640 (RPMI-1640), and Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F12 (DMEM / F12). Specific compositions of standard cell culture media are known in the art and can be prepared based on the compositions described in appropriate literature (e.g., Kaech S. and Banker G., 2006, Nat. Protoc., 1(5): 2406-2415).

[0054] Culture conditions for this step can be any culture conditions known in the art that are suited to the type and origin of the reporter cells and that allow inducible reporter gene expression. Typically, culture is performed in 5% CO2 at 30°C to 40°C, preferably 35°C to 38°C (e.g., 37°C).

[0055] The culture time in this step varies depending on the types of reporter cells and reporter proteins, etc., but to achieve the above-mentioned objective of this step, it is 1 to 72 hours, 2 to 36 hours, or 3 to 24 hours, and preferably 2 to 6 hours.

[0056] (2) Reporter Activity Measurement Step The "reporter activity measurement step" is a step of measuring reporter activity based on a reporter protein. In this step, reporter activity is detected and quantified, thereby enabling quantitative analysis of TSH activity.

[0057] The measurement method in this step varies depending on the type of reporter activity contained in the reporter protein, but can be easily determined by one skilled in the art. When the reporter protein is a luminescent protein, the luminescence activity in the reporter cells and / or culture supernatant can be measured by adding a luminescent substrate (e.g., luciferin) for the luminescent protein. When the reporter protein is a fluorescent protein, the fluorescence activity in the reporter cells and / or culture supernatant can be measured based on the excitation wavelength and fluorescence wavelength appropriate for the type of fluorescent protein. When the reporter protein is an enzyme such as HRP, the enzyme activity in the reporter cells and / or culture supernatant can be measured by adding a chromogenic substrate (e.g., HRP substrates such as TMB, 4-CN, and DAB, AP substrates such as BCIP / NBT, and LacZ substrates such as X-Gal).

[0058] (3) TSH Standard Solution Measurement Step The "TSH standard solution measurement step" refers to a step of performing the incubation step and the reporter activity measurement step using a TSH standard solution containing a standard concentration of TSH as a sample to obtain measured values ​​of reporter activity for a standard concentration of TSH. By comparing the measured reporter activity for a standard concentration of TSH obtained in this step with the measured value obtained for a target sample in the reporter activity measurement step (2) above, the presence of TSH in the target sample can be determined and the amount of TSH can be quantified. For example, by measuring reporter activity for multiple standard concentrations of TSH in this step to create a standard curve, the presence of TSH in the target sample can be determined from the measured value obtained for the target sample in the reporter activity measurement step (2) above, and the amount or concentration of TSH can be estimated.

[0059] According to the TSH activity measurement method of this embodiment, TSH activity in a sample can be evaluated with extremely high sensitivity and reliability. For example, compared to the conventional ELISA method, the sensitivity and / or accuracy of the TSH activity measurement method of this embodiment are overwhelmingly higher.

[0060] 4. Method for determining hypothyroidism or hyperthyroidism 4-1. Overview A fourth aspect of the present invention is a method for determining hypothyroidism or hyperthyroidism. According to the method of the present invention, hypothyroidism or hyperthyroidism in a subject can be determined by evaluating the activity of TSH in a body fluid derived from the subject.

[0061] 4-2. Definition As used herein, "hypothyroidism" refers to a condition in which the amount of thyroid hormone synthesis is lower than normal. Hypothyroidism in which laboratory test values ​​show thyroid hormone levels lower than the normal range and TSH levels higher than the normal range is referred to as "overt hypothyroidism," while hypothyroidism in which thyroid hormone levels are within the normal range and TSH levels higher than the normal range is referred to as "subclinical hypothyroidism." Patients with subclinical hypothyroidism are known to experience the same symptoms as those of overt hypothyroidism (fatigue, constipation, sensitivity to cold, dry skin, depression, irregular menstruation, etc.), as well as the risk of progression of arteriosclerosis, elevated LDL cholesterol, cardiovascular disorders such as angina pectoris and myocardial infarction, nonalcoholic fatty liver disease (NAFLD), fatty liver, infertility, miscarriage and premature birth, and reduced fetal IQ.

[0062] As used herein, "hyperthyroidism" refers to a condition in which the amount of thyroid hormone synthesis is higher than normal. Hyperthyroidism in which laboratory test values ​​show thyroid hormone levels higher than the normal range and TSH levels lower than the normal range is referred to as "overt hyperthyroidism," while thyroid hormone levels within the normal range and TSH levels lower than the normal range are referred to as "subclinical hyperthyroidism." Examples of overt hyperthyroidism include Graves' disease, painless thyroiditis, subacute thyroiditis, and thyroid nodules.

[0063] As used herein, the term "subject" refers to a living organism to which the present method of assessment is applied. Examples of such an organism include humans (particularly referred to as "subject"), livestock animals (e.g., cows, horses, sheep, goats, pigs), racehorses, pets (e.g., dogs, cats, rabbits), laboratory animals (e.g., mice, rats, guinea pigs, monkeys, marmosets), and wild and zoo animals (e.g., chimpanzees, wildcats, squirrels). Examples of such an organism include humans or mammals that have or may develop thyroid dysfunction, such as hypothyroidism or hyperthyroidism. The subject may also be, for example, a pregnant woman or a woman seeking pregnancy. The subject may also be a patient with hypothyroidism after thyroid cancer surgery (e.g., total or hemithyroidectomy), a patient with microthyroid carcinoma, or a patient in whom TSH measurements obtained by conventional immunoassays are inconsistent with the trends of FT4 and FT3 measurements (e.g., cases with macro-TSH, anti-T4 antibodies, or anti-T3 antibodies).

[0064] As used herein, unless otherwise specified, the term "healthy individual" refers to an individual who is not suffering from a specific disease such as thyroid dysfunction, and preferably an individual who is not suffering from any disease.

[0065] As used herein, the term "control" refers to a healthy individual, i.e., an individual not suffering from a specific disease such as thyroid dysfunction, preferably an individual not suffering from any disease. The control may be one individual or multiple individuals.

[0066] The term "determination" as used herein can be replaced with "determination support" or "aid in determination."

[0067] The determination method of this embodiment includes, as essential steps, a culturing step, a reporter activity measurement step, and a determination step, and, as optional steps, an FT4 / FT3 measurement step and / or a TSH standard solution measurement step. Each step will be described in detail below.

[0068] (1) Culturing step The "culturing step" is a step of culturing reporter cells in a medium containing a body fluid derived from a subject. The specific configuration of this step is the same as that of the culturing step in the third embodiment.

[0069] (2) Reporter activity measurement step The "reporter activity measurement step" is a step of measuring the activity of a reporter protein derived from the reporter cells after the culture step to obtain a reporter activity value. The specific configuration of this step is as described for the reporter activity measurement step in the third embodiment.

[0070] (3) FT4 / FT3 Measurement Step The "FT4 / FT3 measurement step" is a step of measuring the concentrations of free thyroxine (FT4) and / or free triiodothyronine (FT3) in a body fluid derived from a subject. The concentrations of FT4 and FT3 can be measured by methods known in the art. For example, immunological assays such as ELISA and RIA (radioimmunoassay) using antibodies against FT4 and FT3, or mass spectrometry such as LC-MS / MS can be used. Measured FT4 and FT3 values ​​can be obtained by this step.

[0071] (4) Determination step In one embodiment of this aspect, the "determination step" is a step of determining that the subject has overt hypothyroidism or subclinical hypothyroidism when the reporter activity value or the TSH activity value calculated based on the reporter activity value is higher than the upper reference limit of TSH, and determining that the subject has overt hyperthyroidism or subclinical hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference limit of TSH.

[0072] In another embodiment of this aspect, the "determination step" comprises the steps of: (a) determining that the subject has overt hypothyroidism when the reporter activity value or the TSH activity value calculated based thereon, and the FT4 measurement value and the FT3 measurement value, if the reporter activity value or the TSH activity value is higher than the upper reference limit for TSH and at least one of the FT4 measurement value and the FT3 measurement value is lower than its lower reference limit; (b) determining that the subject has subclinical hypothyroidism when the reporter activity value or the TSH activity value is higher than the upper reference limit for TSH and the FT4 measurement value and the FT3 measurement value are equal to or higher than their respective lower reference limits and equal to or lower than their respective upper reference limits; (c) determining that the subject has subclinical hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference limit for TSH and the FT4 measurement value and the FT3 measurement value are equal to or higher than their respective lower reference limits and equal to or lower than their respective upper reference limits; (d) determining that the subject has overt hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference limit of TSH and at least one of the FT4 measurement value and the FT3 measurement value is higher than the upper reference limit thereof.

[0073] In the determination step of any of the above embodiments, the upper reference limit for TSH (upper reference limit for TSH concentration or TSH activity) and the lower reference limit for TSH (lower reference limit for TSH concentration or TSH activity) refer to boundary values ​​for determining hypothyroidism and hyperthyroidism in a subject, respectively. For example, the upper reference limit and lower reference limit for TSH may be the upper and lower limits of the normal range for TSH concentration or TSH activity, respectively, or may be values ​​based on the body fluids of patients with hypothyroidism and / or hyperthyroidism and / or healthy individuals.

[0074] For example, the upper reference limit for TSH concentration or TSH activity may be 4.0 μIU / mL to 12.0 μIU / mL, 5.0 μIU / mL to 10.0 μIU / mL, 6.0 μIU / mL to 9.0 μIU / mL, or 7.0 μIU / mL to 8.0 μIU / mL, e.g., 7.0 μIU / mL or 8.0 μIU / mL. The lower reference limit for TSH concentration or TSH activity may be 0.1 μIU / mL to 2.0 μIU / mL, 0.2 μIU / mL to 1.5 μIU / mL, 0.3 μIU / mL to 1.0 μIU / mL, 0.4 μIU / mL to 0.8 μIU / mL, or 0.5 μIU / mL to 0.6 μIU / mL, e.g., 0.5 μIU / mL or 0.6 μIU / mL. Alternatively, based on the results of Example 4 described below, the upper reference limit for TSH concentration or TSH activity may be 4.0 μIU / mL to 10.0 μIU / mL, 5.0 μIU / mL to 9.0 μIU / mL, or 6.0 μIU / mL to 8.0 μIU / mL, for example, 7.0 μIU / mL or 8.0 μIU / mL, and the lower reference limit for TSH concentration or TSH activity may be 0.01 μIU / mL to 2.0 μIU / mL, 0.05 μIU / mL to 1.5 μIU / mL, or 0.1 μIU / mL to 1.0 μIU / mL, for example, 0.1 μIU / mL or 1 μIU / mL. Note that, throughout this specification, the TSH concentration corresponding to the amount of TSH in a given volume and the TSH activity indicating the biological activity of TSH in a given volume are both expressed in units of "μIU / mL." The TSH concentration or TSH activity value in μIU / mL can be determined, for example, by purchasing a TSH standard from the National Institute for Biological Standards and Control (NIBSC) and measuring and comparing the standard measurement value.

[0075] The upper and lower limits of the reference range can be determined based on the normal range of TSH activity measured in the body fluid of a control subject, such as a healthy individual. Alternatively, the upper and lower limits can be calculated based on the prevalence of hyperthyroidism and hypothyroidism and the sensitivity and specificity calculated from the receiver operating characteristic curve (ROC curve).

[0076] Furthermore, the upper reference limit for TSH concentration or activity can be 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more the TSH activity or the average value thereof in healthy subjects not suffering from hyperthyroidism or hypothyroidism, or either of these values. The lower reference limit for TSH concentration or activity can be 0.8 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less the TSH activity or the average value thereof in healthy subjects not suffering from hyperthyroidism or hypothyroidism, or either of these values.

[0077] Alternatively, the measured values ​​obtained from the control group can be classified by percentile, and the percentile values ​​used for the classification can be used as the upper and lower reference limits for the TSH concentration or TSH activity. For example, if the 95th percentile of the TSH activity values ​​obtained from the control group is used as the upper reference limit, and the subject's TSH activity value is equal to or higher than the 95th percentile, the subject can be diagnosed as having hypothyroidism (overt hypothyroidism or subclinical hypothyroidism). Similarly, if the 5th percentile of the TSH activity values ​​obtained from the control group is used as the lower reference limit, and the subject's TSH activity value is equal to or lower than the 5th percentile, the subject can be diagnosed as having hyperthyroidism (overt hyperthyroidism or subclinical hyperthyroidism).

[0078] The TSH activity of the control healthy subject differs from that of the subject, and does not necessarily need to be measured each time. For example, if the amount of sample, measurement method, and measurement conditions are kept constant, the TSH activity of the control healthy subject measured previously can be reused.

[0079] In one embodiment, the TSH activity value in the determination step can be calculated based on the reporter activity value obtained in the reporter activity measurement step and a standard reporter activity value obtained for a TSH standard solution of a standard concentration.

[0080] In any of the above determination steps, the upper reference limits for the FT4 and FT3 measurements are boundary limits for determining whether a subject has subclinical hyperthyroidism or overt hyperthyroidism, and the lower reference limits for the FT4 and FT3 measurements are boundary limits for determining whether a subject has subclinical hypothyroidism or overt hypothyroidism. For example, the upper and lower reference limits for the FT4 and FT3 measurements may be the upper and lower limits of the normal range for the FT4 and FT3 measurements, respectively, or may be values ​​based on measurements of a control subject such as a healthy individual.

[0081] For example, the upper reference limit for FT4 measurements may be 1 ng / dL to 3 ng / dL, 1.25 ng / dL to 2 ng / dL, or 1.5 ng / dL to 1.8 ng / dL, e.g., 1.7 ng / dL. The lower reference limit for FT4 measurements may be 0.3 ng / dL to 1.5 ng / dL, 0.5 ng / dL to 1.2 ng / dL, or 0.7 ng / dL to 1.0 ng / dL, e.g., 0.9 ng / dL. The upper reference limit for FT3 measurements may be 2.5 pg / mL to 6.0 pg / mL, 3.0 pg / mL to 5.0 pg / mL, or 3.5 pg / mL to 4.5 pg / mL, e.g., 4.0 pg / mL. The lower reference limit for FT3 measurements may also be 1.0 pg / mL to 3.5 pg / mL, 1.5 pg / mL to 3.0 pg / mL, or 2.0 pg / mL to 2.5 pg / mL, for example, 2.3 pg / mL.

[0082] For values ​​based on measurements of a control subject, upper and lower reference limits corresponding to the normal ranges of FT4 and FT3 measurements can be determined based on the FT4 and FT3 measurement ranges measured in body fluids derived from the control subject. For example, they can be calculated based on the prevalence of overt or subclinical hyperthyroidism or overt or subclinical hypothyroidism and the sensitivity and specificity calculated from a receiver operating characteristic curve (ROC).

[0083] Alternatively, as in the above-mentioned method, the upper reference limits for FT4 and FT3 measurements can be set to 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more the FT4 and FT3 measurements or their average values ​​in healthy subjects, or either of them. Furthermore, the lower reference limits for FT4 and FT3 measurements can be set to 0.8 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less the FT4 and FT3 measurements or their average values ​​in healthy subjects, or either of them.

[0084] Furthermore, the measurement values ​​obtained from the control group can be classified by percentile, and the percentile values ​​used for the classification can be used as the upper and lower reference limits for the FT4 and FT3 measurement values.

[0085] The accuracy of the determination method of the present invention can be improved by combining it with other determination methods (for example, diagnosis of symptoms of thyroid dysfunction, medical interview by a doctor, genetic diagnosis, etc.).

[0086] Based on the results of the determination method of the present invention, it is possible to perform thyroid hormone replacement therapy and to determine a treatment plan for thyroid dysfunction (e.g., the type, dosage, and administration interval of thyroid hormone such as levothyroxine).

[0087] Based on the measurement method or assessment method of the present invention, thyroid hormone replacement therapy can be appropriately initiated for patients with subclinical hypothyroidism (e.g., pregnant women and those hoping to become pregnant). Furthermore, in patients with subclinical hyperthyroidism diagnosed based on TSH measurements obtained by immunological assay, when no causative disease such as Graves' disease or functional nodules is detected, subclinical hyperthyroidism can be accurately diagnosed by the method of the present invention.

[0088] Furthermore, in patients with hypothyroidism after thyroid cancer surgery (e.g., total or hemithyroidectomy), replacement therapy with thyroid hormones such as LT4 may be administered to prevent recurrence of thyroid cancer, and the method of the present invention can accurately monitor whether TSH activity is controlled at a desirable level. Furthermore, for microthyroid cancer, a follow-up observation method may be adopted without immediate surgery, and in this case, it is necessary that the TSH activity level is not high. By appropriately measuring TSH activity using the method of the present invention, it becomes possible to appropriately determine whether to perform surgery or follow-up observation for microthyroid cancer.

[0089] Furthermore, in cases where TSH measurements obtained by conventional immunoassays are inconsistent with the trends of FT4 and FT3 measurements (such as cases with macro-TSH, anti-T4 antibodies, or anti-T3 antibodies), the presence of measurement interferences is suspected. Even in such cases, accurate measurement of TSH activity using the method of the present invention allows for more appropriate evaluation of the case.

[0090] Example 1: Preparation of reporter cells for measuring TSH (Objective) To prepare reporter cells for measuring the activity of thyroid stimulating hormone (TSH).

[0091] (Methods and Results) (1) Preparation of a Vector for Introducing the TSH Receptor Gene A gene expression vector for expressing the TSH receptor gene in HEK293 cells (hereinafter referred to as the "vector for introducing the TSH receptor gene") was prepared by inserting the TSH receptor gene consisting of the nucleotide sequence shown in SEQ ID NO: 4, which encodes the human thyroid-stimulating hormone receptor (TSH receptor) consisting of the amino acid sequence shown in SEQ ID NO: 3, into the vector pcDNA3.1Zeo(+) (Thermo Fisher Scientific, #V86020).

[0092] (2) Preparation of reporter cells The TSH receptor gene transfer vector prepared in (1) above was transfected into HEK293 cells transfected with pNL[NlucP / CRE / Hygro] (Promega, #CS186804) by the following method.

[0093] NanoLuc-transfected HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), and the cell number was adjusted to 2.5 × 10 5 The concentration was adjusted to cells / mL, and 2 mL of the mixture was dispensed into a 6-well plate and cultured at 37°C and 5% CO2.

[0094] 3 μL of the TSH receptor gene transfer vector and 15 μL of PLUS REAGENT (Thermo Fisher Scientific, #11514015) were added to 100 μL of MEM, mixed, and then left to stand at room temperature for 15 minutes. 100 μL of MEM containing 4% Lipofectamin 2000 Transfection reagent (Thermo Fisher Scientific, #11668027) was then added. After mixing and leaving to stand at room temperature for 15 minutes, 800 μL of MEM was added and mixed.

[0095] After culturing, the medium of the NanoLuc-transfected HEK293 cells was removed by suction, and 1 mL of MEM containing the TSH receptor gene transfer vector and Lipofectamin 2000 Transfection reagent was added and cultured at 37°C and 5% CO for 4 hours. The cells were further expanded and stored in cell preservation medium (Cell Banker) in liquid nitrogen.

[0096] Hereinafter, the NanoLuc-transfected HEK293 cells into which the TSH receptor gene transfer vector prepared in this example has been introduced will be referred to as "TSH measurement reporter cells" or simply "reporter cells."

[0097] Example 2: Measurement of TSH activity using a TSH standard solution (Purpose) The exemplary principle of measuring TSH activity using the reporter cells prepared in Example 1 is believed to be as follows. In reporter cells, the TSH receptor expressed from a vector for introducing the TSH receptor gene is expressed on the cell surface. When TSH binds to the TSH receptor, the TSH receptor is activated, which activates adenylate cyclase via G protein. The resulting cAMP stimulates transcription of the luciferase gene, resulting in the expression of luciferase protein in the reporter cells. The amount of luciferase protein can be measured by quantifying the luminescence intensity of luciferin, which is based on luciferase activity. Therefore, the amount of TSH can be quantified as luciferase activity.

[0098] In this example, a TSH standard solution containing a standard concentration of TSH is added to reporter cells, the luminescence intensity based on luciferase activity is measured, and a standard curve showing the relationship between TSH concentration and relative luminescence intensity is prepared.

[0099] (Method and Results) 1 mL of the reporter cell suspension prepared in Example 1 was thawed in a 37°C water bath, then added to a 15 mL tube containing 9 mL of cell washing solution / diluent. The mixture was gently mixed and then centrifuged at 4°C and 1000 RPM for 5 minutes. The cell washing solution / diluent used was DMEM (No Glucose) with L-Gln, without Sodium Pyruvate, liquid (Nacalai Tesque, #09891-25) containing 1% penicillin-streptomycin (10,000 U / mL; Thermo Fisher Scientific, #15140-122). After removing the supernatant, 1 mL of cell washing solution / diluent was added and gently mixed. 3 × 10 cells were collected. 5 A cell suspension was prepared at 100 cells / mL.

[0100] A 40 μL standard solution containing TSH at standard concentrations (0 μIU / mL, 1 μIU / mL, 4 μIU / mL, 16 μIU / mL, 32 μIU / mL, or 128 μIU / mL) prepared using a TSH preparation (WHO International Standard Thyroid Stimulating Hormone, Human, for Immunoassay; NIBSC code 81 / 565) purchased from the National Institute for Biological Standards and Control (NIBSC) was dispensed into wells of a chemiluminescence microplate. 40 μL of the cell suspension prepared above was added to each well and gently mixed. The plate was then covered with a plate cover and allowed to stand at 37°C under 5% CO2 for 4 hours. 80 μL of Nano-Glo® Luminescent Substrate (Promega, Nano-Glo® Luciferase Assay System, #N1130) was dispensed into each well and shaken for 5 minutes at room temperature (20°C to 25°C). Luciferase activity was then measured by measuring the luminescence intensity of all wells for 0.5 seconds using a chemiluminescence microplate reader (Berthold Technology, TristarLB941).

[0101] The ratio (%) of the luminescence intensity of each TSH standard solution to the average luminescence intensity of the 0 μIU / mL standard solution was calculated and used as the relative luminescence intensity of each TSH standard solution. The results are shown in Figure 4A. Furthermore, a standard curve was constructed by plotting the TSH concentration of each standard solution on the horizontal axis and the relative luminescence intensity of each standard solution on the vertical axis (Figure 4B). The resulting standard curve demonstrated that TSH activity could be quantified over a range of at least 0.5 μIU / mL to 128 μIU / mL.

[0102] Example 3: Comparison of TSH activity measurement using reporter cells with immunological assay (Objective) To compare the results of measuring TSH activity in serum samples using reporter cells with the results of measuring the same serum samples using an immunological assay based on an antigen-antibody reaction.

[0103] (Methods and Results) (1) Serum Samples TSH was measured in serum obtained from 284 patients, including those with overt and subclinical hypothyroidism. The serum samples were inactivated by placing them in a thermostatic chamber set at 56°C for 30 minutes, then returned to room temperature (20°C to 25°C) and diluted 2-fold with cell washing solution / diluent before use in the following measurements.

[0104] (2) Measurement of TSH activity using reporter cells Using the same method as in Example 2, the serum samples from (1) above were dispensed into wells of a chemiluminescence microplate, and 40 μL of a cell suspension containing reporter cells was added to each well and gently mixed. After that, the plate was covered with a plate cover and allowed to stand at 37°C and 5% CO2 for 4 hours. 80 μL of Nano-Glo® luminescent substrate was dispensed into each well, and the plate was shaken for 5 minutes at room temperature (20°C to 25°C). Luciferase activity was measured by measuring the luminescence intensity of all wells using a chemiluminescence microplate reader.

[0105] (3) Quantification of TSH by Immunoassay TSH in the serum samples obtained in (1) above was quantified by immunoassay using the thyroid-stimulating hormone kit, Eclusis (registered trademark) TSH Reagent (Cobas), according to the attached instructions.

[0106] (4) Comparison of quantitative results The measured values ​​obtained in (2) and (3) above were logarithmically transformed and plotted on the vertical and horizontal axes, respectively, as shown in Figure 5. An overall correlation between the two (correlation coefficient r = 0.874) was observed, but the correlation was strong in the high-concentration range (TSH activity range of 10 to 700 μIU / mL). On the other hand, the correlation was weak in the low-concentration range (TSH activity range of 0.1 to 10 μIU / mL). These results demonstrate that TSH concentrations measured by immunoassay do not accurately reflect TSH activity, especially in the low-concentration range.

[0107] Example 4: Measurement of TSH activity in serum samples from patients with subclinical and overt hypothyroidism (Objective) TSH activity in serum samples from patients with subclinical and overt hypothyroidism is measured using reporter cells.

[0108] (Methods and Results) Serum TSH activity was measured using reporter cells from euthyroid subjects (267 subjects), subclinical hypothyroid patients (89 subjects), and overt hypothyroid patients (121 subjects), classified based on physician diagnosis.

[0109] Inactivated serum samples were prepared in the same manner as in "(1) Serum Samples" in Example 3, and luciferase activity was measured in the same manner as in "(2) Measurement of TSH Activity Using Reporter Cells" in Example 3. Simultaneously, luciferase activity was measured for TSH standard solutions in the same manner as in Example 2 to create a standard curve, and TSH activity values ​​(μIU / mL) for each serum sample were calculated based on the luciferase activity and the standard curve.

[0110] The TSH activity values ​​for each group were plotted and shown in Figure 6. Significant differences in TSH activity were detected between healthy subjects and patients with subclinical and overt hypothyroidism (nonparametric comparisons were performed for all pairs, and significant differences were detected using the Steel-Dwass test). These results demonstrate that luciferase activity measurement using the reporter cells of the present invention makes it possible to distinguish between healthy subjects and patients with subclinical hypothyroidism, which was difficult to distinguish using conventional techniques. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A reporter cell for measuring the activity of thyroid stimulating hormone (TSH), into which a TSH receptor gene encoding a TSH receptor or an active fragment thereof, and a reporter gene encoding a reporter protein have been introduced, and the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or an active fragment thereof.

2. The reporter cell of claim 1, wherein the promoter comprises a cAMP response element.

3. The reporter cell according to claim 1, wherein the reporter protein is a luminescent protein, a fluorescent protein, or an enzyme protein.

4. The reporter cell of claim 1, which is a mammalian cell.

5. The reporter cell of claim 4, wherein the mammalian cell is a HEK293 cell, a HeLa cell, an NIH3T3 cell, a CHO cell, a COS cell, or a Vero cell.

6. A TSH activity measurement kit comprising the reporter cell according to claim 1.

7. The TSH activity measurement kit according to claim 6, further comprising a TSH standard solution containing a standard concentration of TSH.

8. The TSH activity measurement kit according to claim 6 or 7, wherein the reporter protein is a luminescent protein and further comprises a luminescent substrate for the luminescent protein.

9. A method for measuring the activity of thyroid stimulating hormone (TSH) in a sample, comprising: a culturing step of culturing reporter cells in a medium containing the sample; and a reporter activity measuring step of measuring the activity of a reporter protein derived from the reporter cells after culturing, wherein the reporter cells are introduced with a TSH receptor gene encoding a TSH receptor or an active fragment thereof, and a reporter gene encoding the reporter protein, and the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or an active fragment thereof.

10. The method according to claim 9, wherein the reporter protein is a luminescent protein, and the reporter activity measuring step comprises adding a luminescent substrate of the luminescent protein to the reporter cells and measuring the luminescence intensity of the luminescent substrate.

11. A method for determining hypothyroidism or hyperthyroidism, comprising: a culturing step of culturing reporter cells in a medium containing a body fluid derived from a subject; a reporter activity measuring step of measuring the activity of a reporter protein derived from the reporter cells after culturing to obtain a reporter activity value; and a determining step of determining that the subject has overt hypothyroidism or subclinical hypothyroidism if the reporter activity value or a TSH activity value calculated based thereon is higher than an upper reference value for TSH, and determining that the subject has overt hyperthyroidism or subclinical hyperthyroidism if the reporter activity value or the TSH activity value is lower than a lower reference value for TSH, wherein a TSH receptor gene encoding a TSH receptor or an active fragment thereof, and a reporter gene encoding the reporter protein are introduced into the reporter cells, and the reporter gene is placed under the control of a promoter whose promoter activity is controlled depending on the activity of the TSH receptor or the active fragment thereof.

12. A method for determining hypothyroidism or hyperthyroidism, comprising: a culturing step of culturing reporter cells in a medium containing a body fluid derived from a subject; a reporter activity measuring step of measuring the activity of a reporter protein derived from the reporter cells after culturing to obtain a reporter activity value; an FT4 / FT3 measuring step of measuring the concentrations of free thyroxine (FT4) and free triiodothyronine (FT3) in the body fluid derived from the subject to obtain FT4 measurement values ​​and FT3 measurement values; and based on the reporter activity value or the TSH activity value calculated based thereon, and the FT4 measurement values ​​and the FT3 measurement values, the method comprises the steps of: (a) determining that the subject has overt hypothyroidism if the reporter activity value or the TSH activity value is higher than the upper reference value of TSH and at least one of the FT4 measurement values ​​and the FT3 measurement values ​​is lower than its lower reference value; (b) determining that the subject has subclinical hypothyroidism when the reporter activity value or the TSH activity value is higher than the upper reference value of TSH and the FT4 measurement value and the FT3 measurement value are equal to or higher than the lower reference value and equal to or lower than the upper reference value, respectively; (c) determining that the subject has subclinical hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference value of TSH and the FT4 measurement value and the FT3 measurement value are equal to or higher than the lower reference value and equal to or lower than the upper reference value, respectively; and (d) determining that the subject has overt hyperthyroidism when the reporter activity value or the TSH activity value is lower than the lower reference value of TSH and at least one of the FT4 measurement value and the FT3 measurement value is higher than its upper reference value, wherein a TSH receptor gene encoding a TSH receptor or an active fragment thereof and a reporter gene encoding the reporter protein are introduced into the reporter cell, The method as described above, wherein the reporter gene is placed under the control of a promoter whose activity is regulated depending on the activity of the TSH receptor or an active fragment thereof.

13. The method according to claim 11 or 12, wherein in the determination step, the TSH activity value is calculated based on the reporter activity value and a standard reporter activity value obtained based on a standard concentration of TSH.

14. The method according to claim 11 or 12, wherein the reporter protein is a luminescent protein, and the reporter activity measuring step comprises adding a luminescent substrate of the luminescent protein to the reporter cell and measuring the luminescence intensity of the luminescent substrate.

15. The method of claim 11 or 12, wherein the body fluid is blood, serum, or plasma.

16. The method of claim 11 or 12, wherein the lower reference value for TSH is 0.1 to 1.0 μIU / mL and / or the upper reference value for TSH is 4.0 to 9.0 μIU / mL.

17. The method of claim 11 or 12, wherein the lower reference limit for FT4 is 0.9 ng / dL, the upper reference limit for FT4 is 1.7 ng / dL, and / or the lower reference limit for FT3 is 2.3 pg / mL, and the upper reference limit for FT3 is 4.0 pg / mL.

18. The method according to claim 11 or 12, wherein the subject is a pregnant woman or a woman wishing to become pregnant.

19. A TSH activity measurement kit according to claim 6 or 7 for use in the method according to claim 11 or 12.

Images