Compositions, kits and methods for detecting autoantibodies

Abstract

Kits, compositions, and methods useful in the diagnosis of autoantibody-mediated thyroid disease are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 817,458, filed March 12, 2019, which is incorporated by reference in its entirety into this specification.

[0002] Technical Field FIELD OF THE DISCLOSURE

[0002] The subject matter described herein relates to kits, compositions, and methods useful in the diagnosis of autoantibody-related thyroid disease. [Background technology]

[0003] background

[0003] Thyroid dysfunction affects an estimated 1-10 percent of adults in the general population. Many thyroid disorders, such as Graves' disease, Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism (including neonatal hypothyroidism), non-goitrous hypothyroidism, euthyroid or hypothyroid autoimmune thyroiditis, and primary and idiopathic myxedema, involve the action of autoantibodies (thyroid-stimulating immunoglobulin (TSI) and / or thyroid-blocking immunoglobulin (TBI)) that recognize and bind to receptors present on the thyroid gland, resulting in undesirable alterations in thyroid hormone production.

[0004]

[0004] While diagnostic techniques are available to detect these autoantibodies, many of these techniques are cumbersome and labor-intensive, cannot distinguish between TSI and TBI, and / or lack sufficient sensitivity and / or specificity. Summary of the Invention [Means for solving the problem]

[0005] Quick Overview

[0005] The following aspects and embodiments thereof described and illustrated below are intended to be exemplary and illustrative and not limiting in scope.

[0006]

[0006] The present disclosure provides kits, compositions, and methods for detecting and / or distinguishing between thyroid stimulating antibodies (TSI) and thyroid blocking antibodies (TBI).

[0007] In one aspect, kits are provided that include transgenic cells stably transfected with a first expression vector and a second expression vector, and a reaction buffer without a cell lysis agent, optionally including a substrate for a reporter. In these provided kits, the first expression vector includes a nucleotide sequence encoding a chimeric or wild-type TSH receptor, while the second expression vector includes a synthetic nucleotide sequence encoding a reporter, the synthetic nucleotide sequence (1) operably linked to an inducible cAMP-inducible promoter, and / or (2) further encoding a heterologous cAMP binding protein, the cAMP binding protein being fused to the reporter. In these provided kits, expression of the reporter is associated with an intracellular signal to be detected.

[0008] In some embodiments, the intracellular signal is detected without lysing the transgenic cells. In other embodiments, the intracellular signal is produced intracellularly and detected extracellularly. In other embodiments, the reporter is a protein that is continuously expressed to produce a reporter that produces a signal intracellularly upon catalytic reaction with a substrate.

[0009] In another aspect, methods are provided for detecting thyroid stimulating and / or thyroid blocking autoantibodies in a sample, the methods comprising: (a) contacting a transgenic cell with a sample suspected of containing thyroid stimulating and / or thyroid blocking autoantibodies, the transgenic cell being stably transfected with a first expression vector and a second expression vector; and (b) detecting an intracellular signal associated with expression of a reporter. In these provided methods, the first expression vector comprises a nucleotide sequence encoding a chimeric or wild-type TSH receptor, while the second expression vector comprises a synthetic nucleotide sequence encoding a reporter, the synthetic nucleotide sequence (1) operably linked to a cAMP-inducible promoter, and / or (2) further encoding a heterologous cAMP binding protein, the cAMP binding protein fused to the reporter. [Brief description of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows the signal-to-background ratio (S / B) for neat versus diluted samples (normal, low TSI, and high TSI) obtained from the disclosed in vivo assay for TSI / TBI (see Example 1). [Diagram 2]

[0011] Figure 2 shows results from an experiment comparing a protocol that includes a wash step with a protocol that excludes the wash step. Figure 2 shows the signal to background ratio for samples (normal, low TSI, and high TSI) obtained from the disclosed in vivo assay for TSI / TBI (see Example 1). [Figure 3A]

[0012] Figure 3A shows results from an experiment comparing a protocol including an overnight cell seeding step with a protocol including a 2 hour cell seeding step, and shows titration curves and calculated EC50 values ​​using an antibody standard (thyroid stimulating antibody M22). [Figure 3B]

[0012] Figure 3B shows results from an experiment comparing a protocol including an overnight cell seeding step with a protocol including a 2 hour cell seeding step, and shows the signal to reference ratios for four negative (NS1, NS2, NS3, and NS4) and four positive (PS1, PS2, PS3, and PS4) samples. [Figure 3C]

[0012] Figure 3C shows results from an experiment comparing a protocol including an overnight cell seeding step with a protocol including a 2 hour cell seeding step, and shows the response for each positive sample calculated as the fold response relative to the average value of the negative samples (see Example 1). [Figure 4A]

[0013] Figure 4A shows the results from an experiment comparing a protocol including a 2 hour cell seeding step with a protocol in which cells are used immediately after thawing or shortly thereafter. Figure 4A shows the titration curves and calculated EC50 values ​​obtained from the M22 standard. [Figure 4B]

[0013] Figure 4B shows results from an experiment comparing a protocol including a 2 hour cell seeding step with a protocol in which cells are used immediately after thawing or shortly thereafter. Figure 4B shows the signal to reference ratios for four negatives (NS1, NS2, NS3, and NS4) and four positives (PS1, PS2, PS3, and PS4). [Figure 4C]

[0013] Figure 4C shows results from an experiment comparing a protocol including a 2 hour cell seeding step with a protocol in which cells are used immediately after thawing or shortly thereafter, and shows the response for each positive sample calculated as the fold response relative to the average value of the negative samples (see Example 1). [Figure 5A]

[0014] FIG. 5A shows the titration curves and calculated EC50 values ​​obtained from the M22 standard (see Example 1). [Figure 5B]

[0015] FIG. 5B shows the signal to reference ratios for the four negative and four positive samples. [Figure 5C]

[0016] FIG. 5C shows the response for each positive sample calculated as the fold response relative to the average of the negative samples (see Example 1). [Figure 6]

[0017] FIG. 6 shows results from an experiment to assess the specificity of the TSI assay of the present disclosure, depicting the signal response ratios (SRRs) observed using the TSI assay of the present disclosure performed on serial dilutions of a thyroid blocking antibody (K170) or a thyroid stimulating antibody (M22) (see Example 1). [Figure 7]

[0018] Figure 7 shows real-time measurements of results from the rapid TSI assay. Normal serum and three TSI positive samples were tested with the rapid assay of the present disclosure. Results (%SRR) were calculated using RLU data measured every 10 minutes up to 90 minutes (see Example 1). [Figure 8]

[0019] Figure 8 shows the results from an experiment to assess whether TSHR blocking antibodies also react with the ChR4 chimeric TSHR receptor. Figure 8 shows the percent inhibition of thyroid blocking antibodies K170, 3H10, and 4C1 observed in the TSI assay of the present disclosure (see Example 2). [Figure 9]

[0020] 9 shows results from an experiment comparing protocols with different incubation times (10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, or 90 min). The first three bars for each time point (samples DLS004, DLS052, and DLS034) correspond to signals from normal samples; the last four bars for each time point (samples DLS079, DLS060, DLS016, and DLS122) correspond to signals from TSI-positive samples (see Example 2). [Figure 10]

[0021] 1 shows titration curves and calculated IC50 values ​​obtained from TSHR blocking antibody K170 against different densities of CHO-ChR4 / 22F transgenic cells (see Example 3). [Figure 11A]

[0022] Figure 11A shows the results from an experiment designed to determine the sensitivity of the disclosed method. Figure 11A shows the titration curves and calculated EC50 values ​​for the disclosed assay and for the THYRETAIN® TSI assay. The signal obtained from the M22 standard using the disclosed method was about 10-fold higher than that obtained using the THYRETAIN® TSI assay. The analytical sensitivity of the two assays was similar (see Example 4). [Figure 11B]

[0022] Figure 11B shows results from an experiment designed to determine the sensitivity of the disclosed method. Figure 11B shows the titration curves and calculated EC50 values ​​for the disclosed assay and for the THYRETAIN® TSI assay. The signal obtained from the M22 standard using the disclosed method was about 10-fold higher than that obtained using the THYRETAIN® TSI assay. The analytical sensitivity of the two assays was similar (see Example 4). [Figure 12]

[0023] Figure 12 shows results on clinical (human serum) samples using different incubation conditions: (1) room temperature for 90 minutes ("RT90") or (2) 1 hour at 37°C, followed by 30 minutes at room temperature ("37°C-60 / RT30"). For comparison, Figure 12 also shows results for the same samples using the THYRETAIN® TSI assay (see Example 5). [Figure 13]

[0024] FIG. 13 shows endpoint measurements from assays using either CHO-ChR4 / 22F transgenic cells or pre-balanced CHO-ChR4 / 22F transgenic cells, and for comparison, results from a THYRETAIN® TSI assay (see Example 5). [Figure 14A]

[0025] FIG. 14A shows kinetic measurements from assays using either CHO-ChR4 / 22F transgenic cells or pre-equilibrated CHO-ChR4 / 22F transgenic cells, and for comparison, results from a THYRETAIN® TSI assay, where kinetic measurements were taken at various time points between 5 and 90 minutes of incubation time (see Example 5). [Figure 14B]

[0025] Figure 14B shows kinetic measurements from assays using either CHO-ChR4 / 22F transgenic cells or pre-equilibrated CHO-ChR4 / 22F transgenic cells, and for comparison, results from a THYRETAIN (registered trademark) TSI assay, where kinetic measurements were taken at various time points between 5 and 90 minutes of incubation time (see Example 5). [Figure 14C]

[0025] Figure 14C shows kinetic measurements from assays using either CHO-ChR4 / 22F transgenic cells or pre-equilibrated CHO-ChR4 / 22F transgenic cells, and for comparison, results from a THYRETAIN (registered trademark) TSI assay, where kinetic measurements were taken at various time points between 5 and 90 minutes of incubation time (see Example 5). [Figure 14D]

[0025] Figure 14D shows kinetic measurements from assays using either CHO-ChR4 / 22F transgenic cells or pre-equilibrated CHO-ChR4 / 22F transgenic cells, and for comparison, results from a THYRETAIN (registered trademark) TSI assay, where kinetic measurements were taken at various time points between 5 and 90 minutes of incubation time (see Example 5). [Figure 15A]

[0026] Figure 15A shows the %SRR in 130 anti-thyroid peroxidase (TPO) antibody positive serum samples. In Figure 15A, which shows results from the THYRETAIN® TSI assay, the black dotted line indicates the assay cutoff of 140% SRR. [Figure 15B]

[0026] Figure 15B shows the %SRR in 130 anti-thyroid peroxidase (TPO) antibody positive serum samples. In Figure 15B, which shows results from the TSI assay of the present disclosure, the purple dotted line indicates a tentative assay cutoff of 31% SRR (see Example 6). [Figure 16]

[0027] FIG. 16 shows a standard curve generated using the World Health Organization (WHO) international standard for TSI for use with the TSI assay of the present disclosure (see Example 7). [Figure 17]

[0028] FIG. 17 shows Table 4, which provides a summary of the results of the THYRETAIN® TSI assay (see Example 5). [Figure 18]

[0029] FIG. 18 shows Table 5, which provides a summary of the results of the THYRETAIN® TSI assay using CHO-ChR4 / 22F cells (see Example 5). [Figure 19]

[0030] FIG. 19 shows Table 6, which provides a summary of the results of the THYRETAIN® TSI assay using pre-equilibrated CHO-ChR4 / 22F cells (see Example 5). [Figure 20]

[0031] FIG. 20 provides the nucleotide sequence of a synthetic artificial nucleic acid encoding a ChR4 chimeric thyroid stimulating hormone receptor (TSHR) as provided in SEQ ID NO:1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description

[0032] The present disclosure provides kits, compositions, and methods for detecting thyroid blocking immunoglobulin (TBI) and / or thyroid stimulating immunoglobulin (TSI). Compared to methods known in the art for detecting TSI and / or thyroid blocking immunoglobulin (TBI), the methods of the present disclosure involve fewer steps and shorter test times while retaining sensitivity and specificity for TSI and / or TBI. For example, the kits, compositions, and methods of the present disclosure allow for detection of TSI and / or TBI without cell lysis, which not only reduces processing time but also allows for kinetic measurements over time in addition to end-point measurements.

[0012]

[0033] These features, among others, allow for large-scale use of the disclosed kits, compositions, and methods, making them more accessible to a wider range of potential users in a variety of settings.

[0013] I. Definition

[0034] The terms "about" and "approximately" as used herein in reference to numerical values ​​are used herein to include numbers that fall within a range of 20%, 10%, 5%, or 1% in either direction (greater than or less than) that numerical value unless otherwise stated or otherwise clear from the context (except where such number exceeds 100% of the possible value).

[0014]

[0035] The term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three amino acids. However, the term also refers to specific functional classes of polypeptides, such as luciferase polypeptides. For each such class, the present specification may refer to a known reference polypeptide having a defined sequence. However, those skilled in the art will recognize that the term "polypeptide" is sufficiently general to include not only polypeptides having the complete sequence of the reference polypeptide, but also polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Furthermore, those skilled in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Furthermore, circular substitution of protein sequences may also retain activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity (e.g., circular permutations), often more than about 50%, 60%, 70%, or 80%, and more usually contains at least one region of very high identity, often more than 90%, or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, and usually encompasses at least 3-4, and often up to 20 or more amino acids with another polypeptide of the same class, is encompassed by the related term "polypeptide" as used herein. One skilled in the art can identify other regions of similarity and / or identity by analyzing the sequences of the various polypeptides referenced herein.

[0015] II. Kit

[0036] In one aspect, a kit for detecting thyroid stimulating immunoglobulin (TSI) and / or thyroid blocking immunoglobulin (TBI) is provided. The kit generally includes (a) a transgenic cell stably transfected with a first expression vector and a second expression vector, and / or (b) a reaction buffer without a cell lysis agent. Generally, the first expression vector includes a nucleotide sequence encoding a chimeric thyroid stimulating hormone (TSH) receptor, and the second expression vector includes a reporter, (i) operably linked to a cAMP-inducible promoter, and / or (ii) includes a synthetic nucleotide sequence further encoding a heterologous cAMP binding protein, the cAMP binding protein being fused to the reporter. In some embodiments, the kit provided further includes one standard or control reagent. In some embodiments, the kit provided does not include a cell lysis agent, and is not used with a cell lysis agent.

[0016]

[0037] When the kit is used according to the methods further described herein, the transgenic cell expresses the chimeric TSHR on its cell surface. Upon binding to a TSI and / or TBI, cAMP levels in the transgenic cell increase, which results in (1) expression of a reporter and / or (2) binding of cAMP to a fusion protein comprising the reporter. In some embodiments, binding of cAMP to the fusion protein results in a conformational change in the reporter, which allows detection of the reporter or increases the detectability of the reporter.

[0017] A. Transgenic Cells 1. First Expression Vector

[0038] The first expression vector comprises a nucleotide sequence encoding a chimeric thyroid stimulating hormone (TSH) receptor (chimeric TSHR). Generally, such chimeric TSHR binds to TSI, TBI, or both. In some embodiments, the chimeric TSHR comprises a portion of human TSHR (hTSHR) and a portion of another receptor. For example, the chimeric TSHR can be a chimera of human TSHR (hTSHR) and luteinizing hormone chorionic gonadotropin receptor (LH-CGR). Non-limiting examples of suitable hTSHR / LH-CGR chimeric receptors include: (1) a chimeric receptor having amino acid residues 8-165 replaced by the equivalent residues from rat LH-CGR (hereinafter "ChR1"); (2) a chimeric receptor having amino acid residues 90-165 replaced by the equivalent residues from rat LH-CGR (hereinafter "ChR2"), (3) a chimeric receptor having amino acid residues 262-335 of hTSHR replaced by the equivalent residues from rat LH / CGR (hereinafter "ChR3"); and (4) a chimeric receptor having amino acid residues 262-368 of hTSHR replaced by residues 262-334 from rat luteinizing hormone / chorionic gonadotropin receptor (LH / CGR), hereinafter "ChR4" (see, e.g., U.S. Pat. No. 9,739,775, the entire contents of which are incorporated herein by reference). Non-limiting examples of suitable TSHRs are those whose nucleotide sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or 100% identical to the nucleotide sequence of SEQ ID NO:1 and also provided in FIG.

[0018]

[0039] Generally, the chimeric TSHR is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter.

[0019] 2. Second Expression Vector

[0040] The second expression vector comprises a synthetic nucleotide sequence encoding a reporter, as further described herein.

[0020]

[0041] Generally, the synthetic nucleotide sequence is operably linked to a promoter. In some embodiments, the synthetic nucleotide sequence is operably linked to a constitutive promoter.

[0021]

[0042] In some embodiments, the synthetic nucleotide sequence is operably linked to a cAMP-inducible promoter; thus, in some embodiments, the presence of cAMP induces expression of the reporter.

[0022]

[0043] In some embodiments, the synthetic nucleotide sequence further encodes a heterologous cAMP binding protein, the cAMP binding protein being fused to a reporter, in some such embodiments, the presence of cAMP induces a conformational change in the reporter upon binding to the cAMP binding protein, the conformational change corresponding to increased activity of the reporter.

[0023] 3. Cell line

[0044] A number of cell lines are suitable for generating stably transfected cells, including cell lines used as components of protein expression systems. In some embodiments, the transgenic cells comprise mammalian cells. Non-limiting examples of suitable mammalian cell lines include adenocarcinoma human alveolar basal epithelial cells (e.g., A549 cells), African monkey (African monkey kidney cells (e.g., COS and Vero cells), baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, mouse myeloma cells (e.g., J558L, NS0, and Sp2 / 0 cells), human osteosarcoma cells (e.g., U2OS), human breast cancer cells (e.g., MCF-7), human cervical cancer cells (e.g., HeLa), human embryonic kidney cells (e.g., HEK293), human fibrosarcoma cells (e.g., HT1080), human hepatoma cells (e.g., HepG2), human rhabdomyosarcoma RD cells ("human RD cells"), human retinoblastoma cells (e.g., SO-Rb5 and Y79), mouse embryonic carcinoma cells (e.g., P19), mouse fibroblast cells (e.g., L929 and NIH3T3), mouse neuroblastoma cells (e.g., N2a), and derivatives of any of the above cell lines.

[0024]

[0045] In some embodiments, the mammalian cell comprises a Chinese Hamster Ovary (CHO) cell (including any derivative thereof) or a human RD cell (including any derivative thereof). Non-limiting examples of CHO cell line derivatives include CHO-K1, CHO pro-3, and DHFR-deficient cell lines, such as DUKX-X11 and DG44.

[0025]

[0046] In some embodiments, the transgenic cell further comprises a substrate for the reporter.

[0026] B. Reporters and Substrates

[0047] In some embodiments, the presence of the reporter can be detected without lysing the transgenic cell. For example, the reporter itself can include a detectable moiety, such as a fluorescent label. Alternatively, or in addition, the reporter can bind to or act on a substrate, and the binding or action of the reporter on the substrate can be detected within the cell. For example, the reporter can include an enzyme whose action on the substrate can be detected within the cell.

[0027]

[0048] In some embodiments, the reporter is a light-emitting reporter, such as a chemiluminescent or bioluminescent reporter. For example, the reporter may include an enzyme whose action on a substrate results in the emission of light.

[0028]

[0049] For example, the reporter can include a luciferase polypeptide, including, but not limited to, firefly luciferase (e.g., Photinus pyralis luciferase), Renilla luciferase (e.g., Renilla reformis luciferase), Gaussia luciferase (e.g., Gaussia princeps luciferase), Oplophorus luciferase (e.g., Oplophorus gracilirostris luciferase), or variants or combinations thereof. In some embodiments, the reporter is a modified luciferase. Examples of modified luciferases include, but are not limited to, those described in U.S. Patent Nos. 5,670,356; 7,729,118; and 8,008,006, the entire contents of each of which are incorporated by reference herein. In some embodiments, the modified luciferase is a circularly permuted luciferase.

[0029]

[0050] In some embodiments, the conformational change in the luciferase polypeptide is associated with increased luciferase activity.

[0030]

[0051] In some embodiments, the kits provided further include a substrate for the reporter. The substrate can be provided as a separate reagent. Alternatively, or in addition, the substrate can be provided as part of another reagent. For example, the transgenic cells provided herein can include the substrate.

[0031]

[0052] Any substrate suitable for the reporter can be used. In some embodiments, the substrate can diffuse through the cell membrane into the cytoplasm. In some embodiments, the substrate is actively transported through the cell membrane into the cytoplasm.

[0032]

[0053] For example, where the reporter comprises a luciferase polypeptide, the substrate can be any corresponding luciferin. A luciferin is a "corresponding luciferin" if the luciferase polypeptide can oxidize the luciferin to generate an excited molecule (e.g., oxyluciferin) that then emits light when relaxed to the ground state.

[0033]

[0054] For example, D-luciferin or its derivatives can be substrates for various luciferase polypeptides, such as firefly luciferase. As another example, coelenterazine or its derivatives can be substrates for various luciferase polypeptides, such as Renilla luciferase, Gaussia luciferase, and Oplophorus luciferase.

[0034] C. Reaction Buffer

[0055] The reaction buffer lacks a cell lysis agent and generally contains a mixture of salts. For example, the reaction buffer may contain CaCl 2 , KCl, KH 2 PO 4 , MgSO 4 , Na 2 HPO 4 , NaHCO 3 , NaCl, HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), or any combination thereof. In some embodiments, the reaction buffer may contain a salt selected from the group consisting of CaCl 2 , KCl, KH 2 PO 4 , MgSO 4 , Na 2 HPO 4 In some embodiments, the reaction buffer contains CaCl 2 , KCl, KH 2 PO 4 , MgSO 4 , Na 2 HPO 4 , NaHCO 3 , and NaCl.

[0035]

[0056] The reaction buffer may include one or more components in addition to the salt mixture. For example, in some embodiments, the reaction buffer further includes sucrose. In some embodiments, the reaction buffer includes at least 5 g / L, at least 10 g / L, at least 15 g / L, or at least 20 g / L of sucrose. In some embodiments, the reaction buffer includes at most 100 g / L, at most 90 g / L, at most 80 g / L, at most 70 g / L, at most 60 g / L, at most 50 g / L, at most 40 g / L of sucrose, at most 30 g / L, or at most 20 g / L of sucrose. In some embodiments, the reaction buffer comprises between 5 g / L and 100 g / L, between 10 g / L and 90 g / L, between 10 g / L and 80 g / L, between 10 g / L and 70 g / L, between 10 g / L and 60 g / L, between 10 g / L and 50 g / L, between 10 g / L and 40 g / L, between 10 g / L, or between 10 g / L and 30 g / L of sucrose. In some embodiments, the reaction buffer comprises about 5 g / L, about 7.5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 22.5 g / L, about 25 g / L, about 27.5 g / L, about 30 g / L, about 32.5 g / L, about 35 g / L, about 37.5 g / L, about 40 g / L, about 42.5 g / L, about 45 g / L, about 47.5 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, or about 75 g / L of sucrose.

[0036]

[0057] In some embodiments, the reaction buffer comprises at least 10 mM, at least 20 mM, at least 30 mM sucrose, at least 40 mM, at least 50 mM, at least 75 mM, or at least 100 mM sucrose. In some embodiments, the reaction buffer comprises at most 300 mM, at most 275 mM, at most 250 mM, at most 225 mM, at most 200 mM, at most 175 mM, at most 150 mM, at least 125 mM, at most 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM, or at most 50 mM sucrose. In some embodiments, the reaction buffer comprises 10-300 mM, 20-200, 30-150 mM, 30-125 mM, or 30-100 mM sucrose. In some embodiments, the reaction buffer comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, about 200 mM, or about 220 mM sucrose.

[0037]

[0058] In some embodiments, the reaction buffer further comprises polyethylene glycol (PEG), e.g., PEG having a molecular weight of 100 to 20,000 (e.g., PEG-100, PEG-200, PEG-300, Peg-400, PEG-600, PEG-1000, PEG-1500, PEG-2000, PEG-2050, PEG-3000, PEG-3350, PEG-4000, PEG-4600, PEG-6000, PEG-8000, PEG-10,000, PEG-12,000, PEG-20,000, or mixtures thereof). In some embodiments, the reaction buffer comprises at least 0.5% PEG, at least 1% PEG, at least 2% PEG, at least 3% PEG, at least 4% PEG, at least 5% PEG, at least 6% PEG, at least 7% PEG, or at least 8% PEG. In some embodiments, the reaction buffer comprises at most 12% PEG, at most 11% PEG, at most 10% PEG, at most 9% PEG, at most 8% PEG, at most 7% PEG, at most 6% PEG, or at most 5% PEG. In some embodiments, the reaction buffer comprises 0.5%-12% PEG, 1%-10% PEG, or 2%-6% PEG. In some embodiments, the reaction buffer comprises about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, or about 8% PEG.

[0038]

[0059] In some embodiments, the reaction buffer comprises albumin, such as bovine serum albumin. In some embodiments, the reaction buffer comprises at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% albumin. In some embodiments, the reaction buffer comprises at most 12%, at most 10%, at most 8%, at most 6%, at most 4%, or at most 2% albumin.

[0039]

[0060] In some embodiments, the reaction buffer comprises salt, PEG, sucrose, and albumin. In some embodiments, the reaction buffer comprises salt, PEG, and sucrose, but no albumin. In some embodiments, the reaction buffer comprises salt and PEG, but no sucrose or albumin.

[0040]

[0061] Non-limiting examples of reaction buffer formulations include those disclosed in U.S. Pat. No. 9,739,775 (referred to therein as "Stimulation Medium"), the entire contents of which are incorporated herein by reference.

[0041]

[0062] In some embodiments, the reaction buffer contains a reagent that inhibits binding of TSH to the wild-type or chimeric TSHR receptor, thereby preventing false signals due to, for example, the presence of normal physiological or high levels of TSH in the test blood sample. The TSH inhibitor can be an antibody that specifically binds to TSH, or another agent that blocks the binding of TSH to the wild-type or chimeric TSHR without binding to the TSHR, thus allowing the assay to proceed without interference from TSH or from a TSH blocking reagent.

[0042] D. Controls and Standards

[0063] In some embodiments, the kit provided further comprises one or more control thyroid stimulating agents and / or one or more control thyroid blocking agents. The term control thyroid stimulating agent as used herein refers to an agent known to stimulate TSHR expressed on mammalian cells. Binding of the control thyroid stimulating agent to TSHR induces intracellular signaling events including cAMP upregulation. Examples of suitable control thyroid stimulating agents include, but are not limited to, thyroid stimulating antibodies (e.g., M22 anti-TSHR mAb and NIBSC08 / 204 (WHO International Standard for Thyroid Stimulating Antibodies) and thyroid stimulating hormone (TSH) (e.g., bovine TSH). The term control thyroid blocking agent as used herein refers to an agent known to block the action of TSHR expressed on mammalian cells, for example, by preventing TSH binding to TSHR or by binding to TSHR and thereby inhibiting the binding of a stimulating agent, for example, a TSI-specific antibody. Examples of suitable control thyroid stimulating agents include, but are not limited to, thyroid blocking antibodies, for example, 3H10 and K170.

[0043]

[0064] In some embodiments, the kits provided include one or more control samples, such as a control negative sample lacking TSI or TBI, a control negative sample containing a control thyroid blocking agent, and / or a control positive sample containing TSI, TBI, or a control thyroid stimulating agent. Non-limiting examples of suitable control negative samples include serum samples known to be negative for TSI and TBI, clinical samples (e.g., human serum samples), artificial compositions lacking TSI and TBI, clinical samples known to contain a control thyroid blocking agent, or artificial samples containing a control thyroid blocking agent. Non-limiting examples of suitable control positive samples include serum samples spiked with TSI, TBI, or a control thyroid stimulating agent; clinical samples (e.g., human serum samples) known to be positive for TSI and / or TBI, or artificial compositions spiked with TSI, TBI, or a control thyroid stimulating agent.

[0044]

[0065] In some embodiments, the kits provided include a quantification standard or set of standards, e.g., for quantifying the amount of TSI and / or TBI in a sample. The standards can be characterized with a known amount or concentration of an agent, e.g., a control thyroid stimulating agent and / or a control thyroid blocking agent. The kit can include instructions for diluting the standard to create the set of standards, or the kit can include a set of standards, each with a different known amount or concentration of an agent.

[0045]

[0066] When used in accordance with the methods provided, in some embodiments, at least one control sample or standard is processed in parallel with other samples.

[0046] III. Assay Methods

[0067] In one aspect, methods are provided for detecting thyroid stimulating immunoglobulin (TSI) and / or thyroid blocking immunoglobulin (TBI) that generally include the steps of (a) contacting a transgenic cell (as described herein) with a sample suspected of containing thyroid stimulating and / or thyroid blocking autoantibodies, and (b) detecting an intracellular signal associated with expression of a reporter.

[0047]

[0068] In some embodiments, the contacting step comprises contacting in a buffer containing a substrate for the reporter. The buffer generally excludes cell lysis agents and can be, for example, any of the reaction buffers described above.

[0048]

[0069] In some embodiments, the methods provided further comprise the step of exposing the transgenic cell to a substrate for the reporter prior to the contacting step.

[0049]

[0070] The sample may be obtained from any subject as further described herein. In some embodiments, the sample comprises serum. In some embodiments, the sample is a non-diluted sample.

[0050]

[0071] In some embodiments, the detection step is performed 240 minutes or less (about 240 minutes or less), 180 minutes or less, 90 minutes or less, 60 minutes or less, 30 minutes or less, 15 minutes or less, or 5 minutes or less after the contacting step. For example, in some embodiments, the detection step is performed less than 240 minutes, 180 minutes, 90 minutes, or 60 minutes after the contacting step. In some embodiments, the detection step is performed about 5 minutes to about 60 minutes after the contacting step, for example, about 10 minutes, about 15 minutes, 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0051]

[0072] In some embodiments, the detection step is performed without adding or removing transgenic cells or a sample after the contacting step. Thus, in some embodiments, the intracellular signal is detected from a composition comprising the transgenic cells and a sample. In some embodiments, at least a portion (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%) of the transgenic cells in the composition are intact (i.e., not lysed) at the time of detection.

[0052]

[0073] In some embodiments, the detecting step is performed without adding or removing a substrate after the contacting step. In some embodiments, the detecting step is performed without adding or removing a substrate, a transgenic cell, or a sample after the contacting step.

[0053]

[0074] In some embodiments, the contacting step is performed at room temperature (e.g., entirely at room temperature). In some embodiments, the method is performed entirely at room temperature, i.e., all steps in the method are performed at room temperature.

[0054]

[0075] In some embodiments, the methods provided further comprise thawing the transgenic cells prior to the contacting step, in some embodiments, the contacting step is performed less than 120 minutes, less than 90 minutes, less than 60 minutes, or less than 30 minutes after said thawing.

[0055]

[0076] In some embodiments, the methods provided are performed using multi-well plates, such as 96-well plates, 384-well plates, etc., such as black, white, white plates with clear bottoms, or clear plates. In some embodiments, the methods provided are performed using white plates. In some embodiments, the methods provided are performed on uncoated or untreated plates.

[0056] A. Detection of TSI

[0077] In some embodiments, the methods provided are used to detect a TSI. In these embodiments, the chimeric TSHR binds to a TSI and may or may not also bind to a TBI, and the sample is suspected of containing a TSI and may or may not also be suspected of containing a TBI.

[0057]

[0078] When detecting a TSI using the methods provided, the signal from a sample suspected of containing a TSI is generally compared to a control baseline value. A signal greater than the control baseline value may indicate the presence of a TSI.

[0058]

[0079] A control baseline value can be provided (e.g., in instructions included in a provided kit) or it can be obtained from control wells that contain transgenic cells and substrate, but do not contain a TSI. For example, the control wells contain (1) a control sample that does not contain a TSI and is intended to be processed in parallel with a sample suspected of containing a TSI; and / or (2) a composition that does not need to be processed in parallel with a sample suspected of containing a TSI. Alternatively or additionally, the control wells can lack a sample, but are otherwise processed in parallel with a sample suspected of containing a TSI.

[0059] B. Detection of TBI

[0080] In some embodiments, the methods provided are used to detect TBI. In these embodiments, the chimeric TSHR binds to TBI and may or may not bind to TSI, and the sample is suspected of containing TBI and may or may not be suspected of containing TSI.

[0060]

[0081] Generally, when using the methods provided to detect TBI, the contacting step further comprises contacting the transgenic cell with a control thyroid stimulating agent (described further herein) that binds to the chimeric TSHR encoded by the first expression vector. In some embodiments, the transgenic cell is contacted with the control thyroid stimulating agent prior to contacting the transgenic cell with the sample.

[0061]

[0082] Generally, when performing an assay to detect TBI, the signal from a sample suspected of containing TBI is compared to a control baseline value. If the signal associated with the sample is reduced compared to the control baseline value, the sample may contain TBI.

[0062]

[0083] A control baseline value can be provided (e.g., in instructions included in a provided kit) or it can be obtained from a control well that contains thyroid stimulating agent, transgenic cells, and substrate, but does not contain TBI. For example, the control well contains (1) a control sample that does not contain TBI and is intended to be processed in parallel with a sample suspected of containing TBI; and / or (2) a composition that does not need to be processed in parallel with a sample suspected of containing TBI. Alternatively or additionally, the control well can lack a sample, but can otherwise be processed in parallel with a sample suspected of containing TBI.

[0063] IV.Applications

[0084] The kits, compositions, and methods provided herein can be used to detect thyroid stimulating immunoglobulin (TSI) and / or thyroid blocking antibodies (TBI), which may be useful in the diagnosis of one or more diseases associated with the presence of autoantibodies to the TSHR, such as TSI and / or TBI.

[0064]

[0085] In some embodiments, the sample is obtained from a subject suspected of having or at risk of developing an autoimmune thyroid disease. In some embodiments, the autoimmune thyroid disease is associated with the presence of TSI. In some embodiments, the autoimmune thyroid disease is associated with the presence of TBI. In some embodiments, the autoimmune thyroid disease is associated with the presence of both TSI and TBI. Some subjects may have more than one disorder, or their disorders may change over time, such that the profile of any autoantibodies in their system changes over time, for example switching from predominating one type of autoantibody to another (e.g., from TSI to TBI or vice versa).

[0065]

[0086] For example, hyperthyroidism is often associated with the production of TSI. A non-limiting example of a disease characterized by hyperthyroidism is Graves' disease.

[0066]

[0087] Some hypothyroid disorders are associated with TBI, including, but not limited to, Hashimoto's disease, neonatal hypothyroidism, non-goitreotic hypothyroidism, primary myxedema, and idiopathic myxedema.

[0067]

[0088] In some embodiments, the subject from which the sample is obtained is a mammal, hi some embodiments, the subject is a human.

[0068]

[0089] In some embodiments, the sample is a blood or serum sample.

[0069] V. Related Kits and Methods

[0090] Those skilled in the art will recognize that the present disclosure provides sufficient guidance for creating further products for the detection and screening of other biological molecules in addition to TSHR autoantibodies that modify TSH signaling activity.For example, in light of the teachings herein, those skilled in the art can construct cell-based reporter assay systems to detect stimulating or blocking antibodies or other stimulating or blocking agents that have an effect on intracellular signaling that exerts its effect via G protein-coupled cell surface receptors (GPCRs), resulting in changes in intracellular cAMP levels.

[0070]

[0091] GPCRs are a large family of integral membrane proteins that respond to a variety of extracellular stimuli. Each GPCR binds to and is activated by a specific ligand, which ranges in size from small molecule catecholamines, lipids, or neurotransmitters to large protein hormones. When a GPCR, such as the TSH receptor, is activated by its extracellular ligand TSH, a conformational change is induced in the receptor that is transmitted to the attached intracellular heterotrimeric G protein complex. In a cAMP-dependent pathway, the activated protein G s The alpha subunit binds to and activates an enzyme called adenylyl cyclase, which then catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP).

[0071]

[0092] Increases or decreases in the concentration of the second messenger cAMP can be detected, for example, by a cAMP-inducible promoter reporter construct in a host transgenic cell that also expresses the relevant GPCR. Alternatively, increases or decreases in the concentration of cAMP can be detected by the use of modified / heterologous cAMP binding proteins that are fused to a reporter moiety that can be detected and / or quantified.

[0072]

[0093] It is known that a wide variety of peptide and polypeptide moieties can be added to either the N- or C-terminus of a GPCR molecule without substantially destroying the signaling activity of the receptor, an observation that permits the construction of other detection systems in addition to the TSHR system described in this disclosure.

[0073]

[0094] In one embodiment, when a GPCR is modified to contain a fusion protein with one or more Mycobacterium tuberculosis (TB) antigens, it is possible to use the cell-based detection system described herein that uses the modified / chimeric GPCR to trigger a signaling cascade (e.g., a cAMP signal). The system can then detect antibodies that may be present, for example, in the blood of a subject, and anti-TB antibodies can bind to the TB antigen portion of the chimeric GPCR fusion receptor on the surface of a reporter cell, with anti-TB antibody binding resulting in activation or inhibition of the GPCR signaling portion of the fusion protein, inducing an increase or decrease in cAMP production.

[0074]

[0095] Assessment of an increase or decrease in cAMP production in response to anti-TB antibodies binding to a TB antigen on the chimeric GPCR molecule can be detected, for example, by using a cAMP-sensitive promoter reporter construct, or a cyclic AMP binding protein fused to a suitable reporter moiety.

[0075]

[0096] In yet another embodiment, this same principle can be used to employ a modified version of the system described herein that allows transgenic detector cells to detect specific T cells activated by TB antigens that induce a T cell specific response. This approach works for any antigen that initiates a cell mediated immune response.

[0076]

[0097] Interferon-γ (IFN-γ or gamma) release assays (IGRAs), such as IGRAs for the diagnosis of both latent (LTBI) and active tuberculosis (TB), may also be used when used with the methods and kits described herein. IGRAs rely on the fact that T lymphocytes release IFN-γ when exposed to a specific antigen, which is quantified by an ELISA-type assay. A variety of commercially available IGRAs are available for the diagnosis of TB infection. For example, the QIAGEN® QuantiFERON®-TB Gold assay is a whole blood test to quantify the amount of IFN-γ produced in response to a mixture of two synthetic peptides corresponding to Mycobacterium tuberculosis antigens. In addition, the Oxford Immunotec Ltd. T-SPOT.TB™ IGRA is also available, which counts the number of anti-mycobacterial effector T cells that produce interferon-gamma in a blood sample in response to exposure to Mycobacterium tuberculosis specific antigens.

[0077] VI. Identification of inhibitory anti-TSHR autoantibodies (TBI)

[0098] Thyroid-blocking immunoglobulin (TBI) is an autoantibody that binds to the thyroid-stimulating hormone receptor (TSHR) and blocks the action of thyroid-stimulating hormone (TSH). Although the presence of TBI leads to Hashimoto's disease, TBI is not the only cause of Hashimoto's disease.

[0078]

[0099] The ability to distinguish between thyroid stimulating immunoglobulin (TSI) and thyroid blocking immunoglobulin (TBI) requires a biological test system rather than a simple immunoassay, because both TSI and TBI autoantibodies bind to the TSHR, thus invalidating this type of serological detection system as distinguishing between inhibitory and stimulatory biological effects. A biological test system is required to test for the biological effects of autoantibodies to the TSHR.

[0079]

[0100] The present disclosure provides modifications of the cell-based biological assay protocols described herein, which specifically distinguish TBI autoantibodies as opposed to TSI autoantibodies. These protocols include the following modifications:

[0080]

[0101] (1) In a cell-based assay, a control number of a TSI-specific monoclonal antibody (MAb) is added to test wells containing a small aliquot of patient serum.

[0081]

[0102] (2) In the absence of TBI autoantibodies in patient test serum, there is stimulation / activation of the cell-based biological test system and detection of the reporter, resulting in a given reporter signal range, designated as the negative signal.

[0082]

[0103] (3) In the presence of TBI autoantibodies in the patient test serum, the assay shows a loss of reporter signal (signal inhibition) of 30% or more. This reduction in reporter activity indicates the presence of TBI autoantibodies in the subject's serum.

[0083]

[0104] A 30% reduction in reporter signal compared to a given "TBI negative signal" is only one embodiment, since any statistically significant reduction in reporter activity is also contemplated as a positive test for the presence of TBI autoantibodies in patient serum. For example, other quantitative thresholds indicating the presence of TBI autoantibodies can include any reduction in reporter activity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. EXAMPLES

[0084] Working Example Example 1 Assays for detecting thyroid-stimulating immunoglobulin (TSI)

[0105] This example describes the development of an improved method for detecting thyroid stimulating immunoglobulin (TSI) in a sample. In the improved method disclosed herein, the sample is incubated with double transgenic cells expressing (1) a chimeric thyroid stimulating hormone receptor (TSHR) receptor on the cell surface and (2) a modified luciferase fused to a cAMP binding protein. For example, binding of the chimeric TSHR receptor to thyroid stimulating immunoglobulin in the sample results in cAMP signaling in the transgenic cells. cAMP binds to the cAMP binding protein, which causes a conformational change in the modified luciferase, which improves the activity of the modified luciferase. When the modified luciferase cleaves its substrate, a light signal is generated. The signal is then read from the incubation mixture without lysing the cells.

[0085]

[0106] Thus, this method allows, among other things, kinetic measurements over time (e.g., homogeneous real-time assays). Moreover, this method is compatible with automation and has a much shorter test turnaround time than other TSI and / or thyroid blocking immunoglobulin (TBI) detection assays. For example, an assay performed according to this method may allow results to be read in 90 minutes or less from the start of the protocol.

[0086] A. Materials and Methods

[0107] Chinese hamster ovary (CHO) cells were transiently transfected with two plasmids: a first plasmid encoding GLOSENSOR™ (available from Promega) luciferase and cAMP receptor ("p22F"), and a second plasmid encoding the thyroid stimulating hormone receptor (TSHR). One set of CHO cells was transfected with a plasmid encoding the wild-type TSHR (pTSHR-WT); another set of CHO cells was transfected with a plasmid encoding ChR4, a chimeric TSHR containing human TSHR sequences and rat luteinizing hormone receptor (LHR) sequences ("pChR4"). ChR4 is described in U.S. Pat. Nos. 8,986,937 and 9,739,775, the contents of each of which are incorporated herein by reference.

[0087]

[0108] Parallel assays were performed on both types of transfected cells (cells transfected with wt TSHR and those transfected with ChR4). The following samples were tested: a sample containing 2 mIU / mL bTSH, three TSI-positive serum samples, and a normal serum sample as a negative control. The three TSI-positive samples were selected based on the results from the THYRETAIN® TSI assay, which showed %SRR values ​​of 285%, 376% and 501% (THYRETAIN® TSI cutoff is 140% SRR). Reaction buffer with 6% cAMP reagent was used as assay blank, and results were reported as signal-to-blank (S / B) ratio. Cells transfected with p22F / pTSHR-wt showed a significantly higher biological response to bTSH stimulation than cells transfected with p22F / pChR4, although both receptors showed similar activity when tested with low TSI-positive samples (data not shown).

[0088]

[0109] Using medium or high TSI positive samples, higher S / B ratios were observed for TSHR-ChR4 transfected cells than for TSHR-wt transfected cells. Similar results were obtained when this experiment was repeated (data not shown). Based on the transient transfection results, the TSHR-ChR4 receptor was selected for further development in this assay.

[0089]

[0110] To generate a stable transfected cell line expressing both the luciferase reporter and the ChR4 receptor, the p22F and pChR4 plasmids were linearized by restriction enzyme digestion and co-transfected into CHO K1 cells. After selection, the cells were screened using TsAb Mab M22 in a TSHR stimulation assay. Six clones were selected for further cloning by limiting dilution based on the level of M22-induced luminescence. A single clone (2C1E3) had the highest signal-to-background ratio and was selected for assay development. The cells were frozen either in vials or in microtiter plate wells (e.g., black or white 96-well plates) for subsequent use in the assay.

[0090]

[0111] Genomic integration of the ChR4 receptor was confirmed by polymerase chain reaction (PCR) amplification using primers designed based on the full-length TSHR gene and sequencing of the amplified products. The PCR product was obtained only from the template of the TSHR-ChR4 stable cell line with a predicted size of 2.1 kb. To confirm the receptor sequence, the PCR product was sequenced and analyzed using Clone Manager software. The deduced amino acid sequence of the PCR product was aligned against the predicted TSHR ChR4 protein sequence and the TSHR wild-type (wt) protein sequence. The sequence alignment results confirmed the integration of the TSHR-ChR4 gene in the genome of the cell line. The cells were thawed, mixed with reaction buffer containing substrate, transferred to or kept in 96-well microtiter plates, and handled as further described below depending on the experiment. For many of the experiments described in parts B-E, white 96-well plates were used.

[0091]

[0112] TSI-negative ("normal"), low TSI, and high TSI serum samples were tested undiluted ("neat"), diluted 1:2, or diluted 1:4. TSI levels were determined by the THYRETAIN® TSI Reporter Bioassay Kit (Quidel Corporation, Athens, Ohio) ("THYRETAIN®" TSI assay) and assigned as "normal", "low TSI", "moderate TSI", or "high TSI" according to Table 1.

[0092] [Table 1]

[0093] B. Sample Dilution

[0113] To test whether a sample dilution step was required, CHO-ChR4 / 22F cells were seeded onto plates overnight (approximately 17-18 hours) and incubated in CHO growth medium, then the medium was removed. One hundred microliters of 6% GLOSENSOR™ substrate in reaction buffer was added to each well. Ten microliters of sample was added in duplicate to each well. The samples tested in this example were normal serum, low TSI, or high TSI, each tested undiluted ("neat"), diluted 1:2, and diluted 1:4.

[0094]

[0114] Plates were incubated at 37° C. for up to 1 hour and then transferred to room temperature. Luminometer readings were then taken from each well.

[0095]

[0115] Table 2 and Figure 1 show the results from all samples. While signal-to-background ratios of over 100 were obtained from all high TSI samples (including "neat" samples), the ratios were significantly lower for low TSI and normal serum samples. Furthermore, the assay distinguished samples (e.g., high TSI vs. low TSI or low TSI vs. normal) in undiluted ("neat") samples at least as well as the assay could distinguish in diluted samples.

[0096] [Table 2]

[0097]

[0116] Therefore, this assay does not require a sample dilution step.

[0098] C. Washing after cell seeding

[0117] CHO-ChR4 / 22F cells were seeded as described above in Example 1, part B. Ten microliters of undiluted sample was added to each well in duplicate. For some wells, the seeded cell monolayer was washed with reaction buffer before adding the sample. For other wells, no washing step was used before adding the sample. The plate was incubated at 37° C. for up to 1 hour and then transferred to room temperature. After 30 minutes at room temperature, luminometer readings were taken from each well.

[0099]

[0118] Figure 2 shows the results from this experiment. As Figure 2 shows, elimination of the wash step did not affect the ability of the assay to distinguish between normal, low and high TSI samples. Therefore, this assay does not require a wash step.

[0100] D. Cell seeding

[0119] To assess whether reducing the seeding time affects assay performance, CHO-ChR4 / 22F cells were seeded either overnight or for 2 hours as described in Example 1, part B. Ten microliters of undiluted normal or TSI positive samples were added to each well in duplicate. Additionally, M22 standards were added to one set of wells to generate a titration curve. Plates were incubated at 37° C. for up to 1 hour and then transferred to room temperature. After 30 minutes at room temperature, luminometer readings were then taken from each well.

[0101]

[0120] Figure 3A shows the titration curves and calculated EC50 values ​​obtained from the M22 standard. Figure 3B shows the signal to reference ratios for the four negative and four positive samples. Figure 3C shows the response for each positive sample calculated as the fold response relative to the mean value of the negative samples. Both assays (overnight or 2 hour cell seeding) were able to distinguish normal samples from TSI positive samples (Figure 3B). Furthermore, the fold increase in signal for TSI positive samples was comparable between both assays (Figure 3C).

[0102]

[0121] Thus, the assay worked properly even with a reduced cell seeding time of 2 hours.

[0103]

[0122] To evaluate whether optional cell seeding was necessary, a similar set of experiments was performed, except that some CHO-ChR4 / 22F cells were seeded 2 h prior to incubation with substrate and reaction buffer, whereas some CHO-ChR4 / 22F cells were suspended directly in substrate and reaction buffer immediately after thawing.

[0104]

[0123] Figure 4A shows the titration curves and calculated EC50 values ​​obtained from the M22 standard. Figure 4B shows the signal to reference ratios for the four negative and four positive samples. Figure 4C shows the response for each positive sample calculated as the fold response relative to the mean value of the negative samples. Both assays performed comparably in terms of their ability to distinguish between normal and TSI positive samples (Figure 5B) and in terms of fold change relative to values ​​from normal samples (Figure 5C).

[0105]

[0124] These results suggest that the methods of the present disclosure do not require a cell seeding step.

[0106] E. Incubation Conditions

[0125] To optimize the incubation conditions, a set of experiments was performed comparing two conditions for incubating CHO-ChR4 / 22F cells with substrate and reaction buffer. Immediately after thawing, CHO-ChR4 / 22F cells were resuspended in 6% GLOSENSOR™ in reaction buffer and added to the microwells. Ten microliters of undiluted normal or TSI positive sample was added to each microwell, and the resulting mixture was incubated either (1) at 37°C for 1 hour, then at room temperature for 30 minutes, or (2) at room temperature for 90 minutes. Luminometer readings were taken at the end of the incubation period. As with the experiments described in parts C and D of Example 1, M22 standards were also assayed to establish a titration curve.

[0107]

[0126] Figure 5A shows the titration curves and calculated EC50 values ​​obtained from the M22 standard. Figure 5B shows the signal to reference ratios for the four negative and four positive samples. Figure 5C shows the response for each positive sample calculated as the fold response relative to the mean value of the negative samples. Under both incubation conditions, the assay was able to distinguish between normal and TSI positive samples (Figure 5B), and the fold change relative to values ​​from normal samples was comparable between the two assay conditions (Figure 5C).

[0108]

[0127] To assess whether shortening the incubation period would affect assay performance, a series of experiments used various incubation times at room temperature (10, 20, 30, 40, 50, 60, 70, 80, or 90 min). Figure 6 shows the results from these experiments. The first three bars for each time point (samples DLS004, DLS052, DLS034) correspond to the signal from normal samples; the last four bars for each time point (samples DLS079, DLS060, DLS016, and DLS122) correspond to the signal from TSI-positive samples. As Figure 6 shows, the signal from the TSI-positive samples can be distinguished from the signal from the TSI-negative samples with incubation times as short as 30 min at room temperature. As expected, the separation between normal and TSI-positive samples generally increased with increasing incubation times across the time points tested.

[0109] F. Development of a Rapid TSI Assay

[0128] Both M22 and TSI positive serum samples were used to determine optimal cell density, sample volume, assay temperature, and incubation time. An assay reference control was prepared using bTSH at 2mIU / mL, and assay results were reported as signal to reference ratio (%SRR). The assay was performed in a homogenous format with only three major steps. First, 10 μl of each sample is added to duplicate wells of a white 96-well plate. Then, one vial of CHO-ChR4 / 22F cells is thawed at 37°C and transferred to 10 mL of reaction buffer containing 6% luciferase substrate. Finally, 100 μl of cell suspension (6.5 × 10 5 100 cells / ml) are dispensed into each well. The plate is kept at room temperature and luminescence is measured every 10 minutes up to 90 minutes. The data in Figure 7 show that the %SRR values ​​for the TSAb positive samples increase with time while the %SRR values ​​for the negative samples remain constant.

[0110] G. Summary

[0129] The disclosed method eliminates several steps characteristic of other TSI or TBI detection assays and detects TSI and / or TBI with a short test turnaround time and a simple protocol. The total test turnaround time of the disclosed method is significantly reduced to about 60 minutes or less compared to the 21-22 hour test turnaround time characteristic of other TSI or TBI detection assays.

[0111] Example 2 Specificity of assays for detecting thyroid-stimulating antibodies

[0130] To assess the specificity of the TSI assay of the present disclosure described in Example 1, serial dilutions of a thyroid blocking antibody (K170) and a thyroid stimulating antibody (M22) were tested simultaneously. As shown in Figure 8, the TSI assay of the present disclosure was highly sensitive to M22 stimulation, reaching a plateau at 50 ng / mL. In contrast, K170 did not induce luciferase activity in the TSI assay of the present disclosure, even at the highest tested concentration of 1000 ng / mL (Figure 8).

[0112]

[0131] The ChR4 receptor has been shown to interact with both the stimulatory antibody M22 and the blocking antibody K170 in the THYRETAIN® TSI assay. To determine whether thyroid blocking antibodies interact with the ChR4 receptor in the TSI assay of the present disclosure, 10 ng / mL of M22 was mixed with serial dilutions (1000 ng / mL to 2 ng / mL) of K170 and two other mouse TSHR blocking antibodies 3H10 (DSMZ, Braunschweig, Germany) and 4C1 (Santa Cruz BioTech, Dallas, Taxes). All three blocking antibodies had an inhibitory effect on M22-induced activity of the ChR4 receptor, but the blocking activity of K170 was significantly more potent than the other two mouse blocking antibodies (Figure 9).

[0113]

[0132] These results support the conclusions presented by Furmaniak et al. (Auto Immun Highlights, 2013, 4(1):11-26) and Nunez et al. (J Mol. Endocrinol. 2012, 49(2):137-151) that TSHR antibodies with distinct functional activities have overlapping binding sites on the concave surface of the TSHR. Because TSHR blocking antibodies do not cause changes in the intracellular concentration of cAMP, the TSI assay of the present disclosure can only detect TSHR inhibiting antibodies, or the net inhibitory effect of TBI, when both types of antibodies coexist in a sample.

[0114]

[0133] However, these results demonstrate that the assay of the present disclosure can be used or developed as a rapid homogeneous TBI assay.

[0115] Example 3 Assays for detecting thyroid-blocking autoantibodies

[0134] This example demonstrates a method for detecting thyroid blocking immunoglobulin (TBI) in a sample.

[0116]

[0135] Samples and three assay controls (reference, positive and negative) are added in duplicate to a multi-well microtiter plate (10 μL per well). Reaction buffer (RB) containing an optimal concentration of bovine TSH is added to each well of the plate (50 μL per well).

[0117]

[0136] CHO-ChR4 / 22F cells produced as described in Example 1 are thawed and resuspended in RB containing 12% GLOSENSOR™ substrate. Fifty microliters of the cell suspension is added to each well of the plate; the final concentration of GLOSENSOR™ substrate is 6%.

[0118]

[0137] Luminometer readings are taken from each well as described in Example 1, with the following modifications.

[0119]

[0138] After the samples are added, bovine TSH (thyroid stimulating hormone) is added to each well (except for one or more control wells). The following controls are also used: (1) "Positive control": at least one well containing no sample but containing a TSHR blocking antibody (e.g., 3H10 or K170), CHO cells, GLOSENSOR™ substrate, and reaction buffer. (2) "Negative Control": at least one well containing neither a TSHR blocking antibody nor any sample, but containing CHO cells, GLOSENSOR™ substrate, normal serum, and reaction buffer. (3) "Reference Control": at least one well containing neither a TSHR blocking antibody nor any sample, but containing bovine TSH, CHO cells, GLOSENSOR™ substrate, and reaction buffer.

[0120]

[0139] The signal from the wells containing the sample is compared to the signal from the reference control wells using the following Equation 1. A decrease in signal relative to the negative control indicates the presence of TBI in the sample.

number

[0121]

[0140] FIG. 10 shows the titration curves and calculated IC obtained from the assay for samples containing K170 TSHR blocking antibody. 50 Values ​​are shown. Each curve represents a titration curve for different densities of CHO-ChR4 / 22F transgenic cells.

[0122] Example 4 Assay reproducibility and sensitivity

[0141] To determine the reproducibility and sensitivity of the disclosed method, the assay was repeated on replicates of the following samples: 1) reaction buffer only (blank); 2) bovine TSH in normal human serum at 2 mIU / mL ("reference sample"); 3) normal (TSI negative) sample; 4) TSI positive sample; and 5) a sample containing 3 ng / mL M22 in normal serum.

[0123]

[0142] The assays were performed as described in Example 1, Part E (hereafter "modified TSI assay"), with incubation periods ranging from 10 minutes to 90 minutes, all at room temperature.

[0124]

[0143] Table 3 presents a summary of the results including %CV. As shown in Table 3, the %CV for both RLU and %SRR was below 8.1% for each sample.

[0125] [Table 3]

[0126]

[0144] To assess the sensitivity of the assays of the present disclosure, 1) using an assay performed as described in Example 1, Part E, with an incubation period of 60 minutes at room temperature; and 2) the M22 standard was assessed using the THYRETAIN® TSI assay according to the manufacturer's protocol.

[0127]

[0145] M22 antibody in an amount of 100 ng / mL was serially diluted to seven concentrations and tested simultaneously in both assays.

[0128]

[0146] 11A-11B show titration curves and calculated EC 50 Values ​​are shown. The signal obtained from the M22 antibody standard using the method of the present disclosure was approximately 10-fold higher than that obtained using the THYRETAIN® TSI assay, but the dose-response curves from the two assays were very similar. EC for the modified TSI assay 50 The value was 4.7 ng / mL and approximately 5.5 ng / mL for the THYRETAIN® TSI assay, indicating that the analytical sensitivity of the two assays was similar.

[0129]

[0147] These results demonstrate that the method of the present disclosure is reproducible and as sensitive as the THYRETAIN® TSI assay.

[0130] Example 5 TSI detection in clinical samples

[0148] To evaluate the performance of the disclosed method on clinical samples and to optimize the assay conditions, the assay was performed on clinical serum samples as described in Example 1, Part E, with minor modifications as explained below. For comparison, each sample was also tested using a THYRETAIN® TSI Reporter Bioassay Kit (Quidel, Athens, Ohio) ("THYRETAIN® TSI Assay").

[0131] sample

[0149] The experiments described in this Example used nine "normal" (negative for TSI), nine "low TSI", five "medium TSI", and five "high TSI" serum samples from human patients as determined by the THYRETAIN® TSI assay (see Table 1).

[0132] Incubation conditions

[0150] In one set of experiments, CHO-ChR4 / 22F transgenic cells were incubated with samples (as well as GLOSENSOR™ substrate and reaction buffer) for (1) 90 minutes at room temperature ("RT90") or (2) 1 hour at 37°C, followed by 30 minutes at room temperature ("37°C-60 / RT30"). Other conditions were as described in Example 1, Part E.

[0133]

[0151] Figure 12 shows the results for both incubation conditions and for the THYRETAIN® TSI assay. As Figure 12 shows, separation between normal and "low TSI" samples was more apparent for the RT90 assay than for the 37°C-60 / RT30 assay. Furthermore, both the RT90 and 37°C-60 / RT30 assays generally distinguished "high TSI" samples better than the THYRETAIN® TSI assay.

[0134] Pre-equilibration

[0152] In another set of experiments, assays were performed on clinical serum samples using (1) CHO-ChR4 / 22F cells (described in Example 1) and (2) CHO-ChR4 / 22F cells that were pre-equilibrated with 6% GLOSENSOR™ substrate for 2 hours, then frozen and subsequently thawed for use in the assay. In this set of experiments, the cells were incubated at room temperature for 90 minutes.

[0135]

[0153] Figures 13 and 14A-14D show measurements from the assay described in Example 1 using CHO-ChR4 / 22F cells or pre-equilibrated CHO-ChR4 / 22F cells. For comparison, Figures 13 and 14A-D also show results from a THYRETAIN® TSI assay. Figure 10 shows the endpoint measurements, and Figures 14A-14D show kinetic measurements taken at various time points during the 5-90 minute incubation period. Tables 4-6 (Figures 17-19) show summary data on signal to response ratios obtained using the THYRETAIN® TSI Reporter BioAssay kit (Table 4; Figure 17) and for assays of the present disclosure using CHO-ChR4 / 22F cells and pre-equilibrated CHO-ChR4 / 22F cells; see Table 5 (Figure 18) and Table 6 (Figure 19), respectively.

[0136]

[0154] As shown in FIG. 13 and Tables 4 (FIG. 17) and 5 (FIG. 18), pre-equilibration of cells with substrate increased the sensitivity of the assay. In FIG. 14, signals from negative samples tended to decrease over time, while signals from TSI-positive samples tended to increase over time. Furthermore, both assays (using CHO-ChR4 / 22F cells and pre-equilibrated CHO-ChR4 / 22F cells) performed at least as well as the THYRETAIN® TSI assay. For "high TSI" samples, the assay of the present disclosure produced a greater signal than the THYRETAIN® TSI assay; see FIG. 13, and compare Tables 4 (FIG. 17) and 5 (FIG. 18) to Table 3.

[0137] conclusion

[0155] Thus, the disclosed method can distinguish between normal, low TSI, medium TSI, and high TSI clinical samples with results qualitatively similar to or better than those of the THYRETAIN® TSI assay. These results also demonstrate that (1) the method can distinguish between normal and TSI-positive clinical samples even when cells are incubated at room temperature; and (2) pre-equilibration of cells with substrate resulted in improved assay sensitivity.

[0138] Example 6 Correlation Between Results from the TSI Assay of the Present Disclosure and the THYRETAIN® TSI Assay on Clinical Samples

[0156] To further evaluate the performance of the TSI assay of the present disclosure on clinical samples, a provisional cut-off value for the TSI assay of the present disclosure was determined by measuring 145 human serum samples that tested negative in the THYRETAIN® TSI assay. This provisional assay cut-off for the TSI of the present disclosure was calculated to be 31% SRR (mean % SRR value of 145 normal serum samples + 2 x standard deviation), and this value was used as the cut-off to identify TSI-positive samples.

[0139]

[0157] To evaluate the performance of the presently disclosed assay on samples from unselected patients with autoimmune thyroid disease (AITD), 130 human serum samples positive for thyroid peroxidase (TPO) antibodies were tested by both the presently disclosed TSI and THYRETAIN® TSI assays. Samples with %SRR values ​​greater than 31% were identified as positive for TSI in the presently disclosed TSI assay. Following the THYRETAIN® TSI assay instructions, samples with %SRR values ​​greater than 140% were identified as positive for TSI. The results from the two assays were comparable for those 130 samples, as evidenced by the fact that the positive and negative agreement rates (PPA and NPA) between the two bioassays were 96% (95% CI: 0.79-0.99) and 95% (95% CI: 0.90-0.98), respectively (Table 7 and Figures 15A and 15B). The results from the two assays showed a strong correlation, with a correlation coefficient R value of 0.71, despite the fact that the %SRR values ​​generated by the TSI assay of the present disclosure were significantly higher than those generated by the THYRETAIN® assay for the same highly TSI positive samples (data not shown).

[0140] [Table 4]

[0141] Example 7 Quantitative detection of thyroid stimulating antibodies by the disclosed TSI assay

[0158] To determine whether the TSI assay of the present disclosure can be used to quantitatively detect TSI, a three-point standard panel was developed using three concentrations of the WHO international standard for TSI. A standard panel was prepared using normal serum as a matrix and tested on four days with TSI-positive serum samples. The TSI concentration of each TSI-positive sample was calculated based on a formula derived from the standard curve generated on the same plate, and the %CV was determined for data obtained from four experiments performed on four different days. All four standard curves generated on four days were reproducible, with coefficient of determination values ​​greater than 0.99 (Figure 16). The calculated TSI concentrations for low, medium, and high (TSI) positive samples were also consistent with %CV values ​​of less than 10% (Table 8). These data indicate that the TSI assay of the present disclosure has the potential to be used for quantitative measurement of TSI in patient samples.

[0142] [Table 5]

Claims

1. (a) (i) a first expression vector comprising a nucleotide sequence encoding a chimeric thyroid-stimulating hormone (TSH) receptor; and (ii) a second expression vector comprising a synthetic nucleotide sequence encoding a reporter operably linked to a constitutive promoter, wherein a cAMP binding protein is fused to said reporter; and frozen transgenic cells stably transfected with (b) a reaction buffer without a cytolytic agent, the reaction buffer containing a substrate for the reporter; A kit comprising: The kit, wherein expression of the reporter is associated with an intracellular signal.

2. 2. The kit of claim 1, wherein the chimeric TSH receptor comprises a human TSH receptor sequence.

3. 3. The kit of claim 2, wherein the chimeric TSH receptor is ChR4.

4. The kit of any one of claims 1 to 3, wherein the reporter comprises a luciferase polypeptide and the buffer comprises a substrate for the luciferase polypeptide.

5. The kit of claim 4 , wherein the luciferase polypeptide is a modified luciferase.

6. The kit of claim 5 , wherein the modified luciferase is a circularly permuted luciferase.

7. The kit according to any one of claims 4 to 6, wherein the substrate comprises luciferin.

8. The kit according to any one of claims 1 to 7, wherein the intracellular signal comprises luminescence.

9. The kit of any one of claims 1 to 8, wherein the transgenic cells comprise mammalian cells.

10. 10. The kit of claim 9, wherein the mammalian cells comprise Chinese hamster ovary (CHO) cells or human RD cells.

11. The kit according to any one of claims 1 to 10, wherein the transgenic cells further comprise a substrate for the reporter.

12. The kit according to any one of claims 1 to 11, wherein the reaction buffer comprises polyethylene glycol and sucrose.

13. The kit of claim 12 , wherein the reaction buffer further comprises albumin.

14. 14. The kit of claim 13, wherein the albumin comprises bovine serum albumin.

15. The kit according to any one of claims 1 to 14, which does not contain a cell lysis agent.

16. 1. A method for detecting thyroid-stimulating and / or thyroid-blocking autoantibodies in a sample, comprising: (a) (i) a first expression vector comprising a nucleotide sequence encoding a chimeric thyroid-stimulating hormone (TSH) receptor; and (ii) a second expression vector comprising a synthetic nucleotide sequence encoding a reporter operably linked to a constitutive promoter, wherein a cAMP binding protein is fused to said reporter; thawing transgenic cells that have been stably transfected with (b) contacting the thawed transgenic cells with a sample suspected of containing thyroid-stimulating and / or thyroid-blocking autoantibodies; and (c) detecting an intracellular signal associated with expression of the reporter; A method comprising:

17. The method of claim 16 , wherein the transgenic cell further comprises a substrate for the reporter.

18. 18. The method of claim 16 or 17, wherein the contacting step comprises contacting in a buffer containing a substrate for the reporter.

19. 20. The method of claim 18, wherein the buffer does not contain a cell lysing agent.

20. 20. The method of claim 18 or 19, further comprising exposing the transgenic cell to the substrate for the reporter prior to the contacting step.

21. The method of any one of claims 16 to 20, wherein the contacting step comprises contacting with an undiluted sample.

22. 22. The method of any one of claims 16 to 21, wherein the detecting step is carried out less than 240 minutes, less than 180 minutes, less than 90 minutes, less than 60 minutes, or less than 30 minutes after said contacting.

23. The method according to any one of claims 16 to 22, wherein the intracellular signal is detected from a composition comprising the transgenic cell and the sample.

24. 24. The method of claim 23, wherein at least 75% of the transgenic cells in the composition are intact at the time of detection.

25. The method of any one of claims 16 to 24, wherein the contacting step is carried out at room temperature.

26. The method of any one of claims 16 to 24, further comprising thawing the transgenic cells prior to the contacting step.

27. 27. The method of claim 26, wherein the contacting step is performed less than 120 minutes, less than 90 minutes, less than 60 minutes, less than 30 minutes, or less than 5 minutes after the thawing.

28. 28. The method of any one of claims 16-27, wherein the contacting step further comprises contacting the transgenic cells with a control thyroid stimulating agent.

29. 29. The method of claim 28, wherein the transgenic cells are contacted with the control thyroid stimulating agent prior to contacting the transgenic cells with the sample.

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