Thyroglobulin immunoassay and kit therefor

Abstract

Provided is a novel method that can accurately measure the amount of thyroglobulin in a sample even for samples that are anti-thyroglobulin antibody positive. The immunoassay is a method that carries out an immunoassay-based measurement of the thyroglobulin in a sample isolated from an organism, wherein this method comprises a pretreatment step in which the sample isolated from the organism is treated with a reducing agent. The immunoassay uses a monoclonal antibody, or an antigen-binding fragment thereof, for which the corresponding antigen is reducing agent-treated thyroglobulin.

Description

[Technical Field]

[0001] This invention relates to an immunoassay of thyroglobulin and a kit therefor. [Background technology]

[0002] Thyroglobulin (Tg) is a glycoprotein with a molecular weight of 660,000 that is produced only by thyroid follicular cells. Biosynthesized Tg is released into the follicular lumen. During this process, iodine molecules bind to the tyrosine group of the Tg molecule through the action of peroxidase, leading to the synthesis of thyroid hormones. The Tg in the follicular lumen is taken up again by follicular cells, broken down within the follicular cells, and released into the thyroid. This process is also activated by the action of thyroid-stimulating hormone (TSH). Therefore, under normal conditions, only a very small amount of Tg itself is released into the bloodstream, and the release of Tg into the bloodstream indicates some kind of thyroid abnormality. Thus, Tg is a highly organ-specific and extremely useful marker for thyroid diseases. In particular, serum Tg is used as a marker for surgical evaluation of differentiated thyroid cancer and to determine the presence or absence of postoperative recurrence or metastasis. In addition, it is useful as an indicator of the effectiveness of treatment and remission in Graves' disease, for determining and differentiating the type of congenital hypothyroidism, and for monitoring treatment. Furthermore, combining this with imaging studies has been suggested to potentially enable preoperative diagnosis of nodular goiter and differentiation between benign thyroid diseases and malignant tumors.

[0003] However, if a subject is positive for anti-thyroglobulin antibodies (TgAb), actual Tg levels may be underestimated due to measurement problems. For example, in thyroid cancer, 20-30% of patients are TgAb positive, so it was necessary to measure TgAb simultaneously when measuring Tg. Also, accurately measuring the amount of Tg is difficult in Hashimoto's disease where TgAb is positive, and similarly, there was a risk that the amount of Tg was not being accurately measured in other autoimmune diseases (such as Graves' disease) where TgAb positivity is observed.

[0004] A method for measuring thyroglibulin is known in which the sample is pretreated with a surfactant or acidifying agent in order to accurately measure the amount of Tg (Patent Document 1). Patent Document 1 states that a reducing agent may be included as an optional component in the pretreatment solution, but it is not used in the examples. A method for measuring thyroglibulin is also known in which the sample is pretreated with an alkaline substance (Patent Document 2). Furthermore, a method for measuring thyroglibulin is also known in which an anti-Tg monoclonal antibody prepared using a synthetic peptide as an immunogen is used (Patent Document 3). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO 2018 / 047792 [Patent Document 2] WO 2020 / 0241443 [Patent Document 3] Japanese Patent Application Publication No. 7-46966 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a novel method that can accurately measure the amount of thyroglobulin in a sample, even in samples that are positive for anti-thyroglobulin antibodies. [Means for solving the problem]

[0007] As a result of diligent research, the inventors of this invention discovered that thyroglobulin / anti-thyroglobulin antibody immune complexes can be dissociated by reduction treatment. They succeeded in producing a monoclonal antibody using the reduced thyroglobulin after dissociation as the corresponding antigen, and found that thyroglobulin can be accurately measured even in samples positive for anti-thyroglobulin antibodies by immunoassay using this monoclonal antibody, thus completing the present invention.

[0008] In other words, the present invention provides the following: (1) A method for measuring thyroglobulin in a sample isolated from a living organism by immunoassay, comprising a pretreatment step of treating the sample isolated from a living organism with a reducing agent, wherein the immunoassay is an immunoassay using a monoclonal antibody or an antigen-binding fragment thereof as the corresponding antigen, with thyroglobulin treated with a reducing agent. (2) The method according to (1), wherein the corresponding epitope of the monoclonal antibody is included in the amino acid sequence represented by any of SEQ ID NOs. 1 to 5. (3) The method according to (1) or (2), wherein the immunoassay is a sandwich method, and the monoclonal antibody or its antigen-binding fragment is used in at least one of the first antibody and the second antibody of the sandwich method. (4) The method according to (3), wherein the first antibody comprises at least one selected from the group consisting of an antibody whose amino acid sequence of SEQ ID NO: 1 contains the corresponding epitope and an antibody whose amino acid sequence of SEQ ID NO: 5 contains the corresponding epitope, and the second antibody comprises at least one selected from the group consisting of an antibody whose amino acid sequence of SEQ ID NO: 2 contains the corresponding epitope, an antibody whose amino acid sequence of SEQ ID NO: 3 contains the corresponding epitope and an antibody whose amino acid sequence of SEQ ID NO: 4 contains the corresponding epitope. (5) The method according to (4), wherein the first antibody is a capture antibody and the second antibody is a labeled antibody. (6) An immunoassay kit for performing the method described in (1), comprising a reducing agent and a monoclonal antibody or an antigen-binding fragment thereof, wherein the thyroglobulin treated with the reducing agent is the corresponding antigen. [Effects of the Invention]

[0009] According to the present invention, thyroglobulin can be accurately measured even in samples that are positive for anti-thyroglobulin antibodies. [Brief explanation of the drawing]

[0010] [Figure 1A]It is a figure showing the results of epitope analysis obtained in the Examples below. [Figure 1B] Same as above [Figure 1C] Same as above [Figure 2] It is a figure showing the effect of pretreatment with various reducing agents obtained in the Examples below. [Figure 3] It is a figure showing the correlation between the Tg-SH measurement system and the Tg measurement system obtained in the Examples below. [Figure 4] It is a figure showing the correlation between the Tg-SH measurement system and the Tg measurement system in the low-concentration region obtained in the Examples below. MODE FOR CARRYING OUT THE INVENTION

[0011] Unless otherwise specified, the concentration indicated by "%" described in the present specification is a weight / volume (w / v) concentration indication. In addition, the temperature is room temperature unless otherwise specifically stated.

[0012] <Method for Measuring Thyroglobulin> The thyroglobulin (Tg) measured in the present invention is Tg derived from any animal, but is preferably Tg derived from a mammal (e.g., primates such as humans, monkeys and chimpanzees; rodents such as mice, rats and rabbits; companion animals such as dogs and cats; livestock such as pigs and cattle; working animals such as horses and sheep), more preferably Tg derived from a primate, and particularly preferably Tg derived from a human.

[0013] 1. Pretreatment step The method of the present invention is a method for measuring thyroglobulin (Tg) present in a specimen by an immunoreaction in which the specimen is reacted with an antibody, and comprises a pretreatment step of mixing the specimen and a pretreatment solution before the immunoreaction (reaction step). Through the pretreatment step, Tg can be released from autoantibodies (TgAb) and the like. The pretreatment solution contains a reducing agent. As the reducing agent, any existing reducing agents can be used, including 2-(diethylamino)ethanethiol hydrochloride (2-DEAET), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 2-mercaptoethanol, mercaptoethylamine, and cysteine, among which 2-DEAET, TCEP, and DTT are particularly preferably usable. The concentration of the reducing agent is preferably a concentration capable of dissociating the Tg / anti-Tg antibody immune complex and eliminating the influence of TgAb. This concentration varies depending on the reducing agent used, and can be determined by the method specifically described in the examples below. Briefly, for specimens pretreated with various concentrations of reducing agent, immunoassay is performed under the same other conditions in the presence of TgAb and in the absence of TgAb, and the concentration is one at which the measured values of the immunoassay do not differ between the presence of TgAb and the absence of TgAb. As specifically described in the examples below, this concentration (the final concentration after mixing with the specimen during pretreatment) is about 150 mM or more for 2-DEAET, about 10 mM or more for DTT, and about 5 mM or more for TCEP. The upper limit of the concentration of the reducing agent is not particularly limited, but there is no point in increasing the concentration of the reducing agent as long as it is a concentration that is not affected by TgAb. Therefore, it is usually 4 times or less, preferably 2 times or less, of each of the above concentrations. That is, the upper limit of the concentration of 2-DEAET is, for example, 600 mM, preferably 300 mM; the upper limit of the concentration of DTT is, for example, 40 mM, preferably 20 mM; and the upper limit of the concentration of TCEP is, for example, 20 mM, preferably 10 mM.

[0014] The specimen used in the present invention is not particularly limited as long as it can contain Tg, and examples include serum, plasma, whole blood, urine, stool, oral mucosa, pharyngeal mucosa, intestinal mucosa, and biopsy specimens (e.g., fine needle aspiration (FNA) specimens, intestinal specimens, liver specimens). Preferably, the specimen is a blood specimen (whole blood, serum, or plasma). More preferably, the specimen is serum or plasma.

[0015] The pretreatment solution may contain other protein denaturants such as urea and thiourea, as needed. The concentration of the denaturant is preferably 0.1 M or higher at the time of treatment, and more preferably 0.5 M or higher and less than 4 M. In addition, monosaccharides, disaccharides, citric acid, and citrates, or a combination thereof, may be added to the pretreatment solution to enhance the treatment effect. Furthermore, the pretreatment solution may contain chelating agents such as EDTA. The pretreatment solution may also contain surfactants. Any of anionic surfactants, cationic surfactants, amphoteric surfactants, or nonionic surfactants can be used. Examples of amphoteric surfactants include C10APS (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), C12APS (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), C14APS (N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), and C16APS (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). Examples of anionic surfactants include sodium dodecyl sulfate (SDS), N-lauroyl sarcosine (NLS), lithium dodecyl sulfate, sodium dodecylbenzenesulfonate, and deoxycholic acid. Examples of cationic surfactants include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride (C16TAC), decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide (CTAB), laurylpyridinium chloride, tetradecylpyridinium chloride, and cetylpyridinium chloride. Examples of nonionic surfactants include polyoxyethylene isooctylphenyl ethers such as Triton X100 and Triton X114, polyoxyethylene nonylphenyl ethers such as NP-40, and polyoxyethylene sorbitan alkyl esters such as Tween 80.The surfactant content is, for example, 1 to 10%, preferably 3 to 8%, based on the weight of the pretreatment solution.

[0016] The pretreatment step is not particularly limited as long as it does not adversely affect the immunoassay; for example, it can be performed at 0°C to 37°C, but it can be carried out without any problems at room temperature, so it is simpler and preferable to do it at room temperature.

[0017] 2. Reaction process The sample mixture obtained in the above pretreatment step of the method of the present invention is then subjected to the reaction step of an immunoassay. In the reaction step, the sample mixture is optionally mixed with a buffer solution, and the antigen in the mixture is reacted with an antibody against Tg or its antigen-binding fragment. Various methods for Tg immunoassays are well known, and any immunoassay capable of quantifying Tg can be used.

[0018] Examples of the aforementioned buffers include those based on MES buffer, phosphate buffer, Tris buffer, and carbonate buffer, with phosphate buffer-based buffers being particularly preferable.

[0019] The antibody used in the method of the present invention is a monoclonal antibody whose corresponding antigen is Tg (sometimes referred to as "Tg-SH") treated with a reducing agent. Here, "with Tg-SH as the corresponding antigen" means a monoclonal antibody that is induced using Tg-SH as an immunogen and reacts with Tg-SH as an antigen-antibody. A monoclonal antibody with Tg-SH as the corresponding antigen can be obtained by the conventional hybridoma method (see examples below) using Tg-SH as an immunogen. The isotype of the monoclonal antibody is not limited and may be any of the isotypes of IgG, IgM, IgA, IgD, IgE, or IgY. It is also possible to use antigen-binding fragments of the above monoclonal antibody, such as F(ab')2, Fab', Fab, or Fv (in the following description (up to the examples), the term "antibody" means "antibody or its antigen-binding fragment" unless it is clear from the context). Furthermore, when two types of antibodies are used, such as in the sandwich method, at least one of them may be a monoclonal antibody that corresponds to Tg-SH as the antigen, and the other may be a polyclonal antibody. However, it is preferable if both are monoclonal antibodies that correspond to Tg-SH as the antigen, as this improves the reproducibility of the measured values.

[0020] In the following example, five different monoclonal antibodies were obtained by hybridoma using Tg-SH as an immunogen. The epitopes of each monoclonal antibody are included in SEQ ID NOs. 1-5. Since five different monoclonal antibodies were obtained by hybridoma using Tg-SH as an immunogen, it was demonstrated that monoclonal antibodies corresponding to Tg-SH can be reproducibly obtained by a conventional hybridoma method using Tg-SH as an immunogen.

[0021] Antibodies against Tg-SH may be immobilized on a solid phase. In this specification, antibodies immobilized on a solid phase or antibodies that are immobilized on a solid phase may simply be referred to as immobilized antibodies. Examples of solid phases include solid phases capable of containing or mounting a liquid phase (e.g., supports such as plates, membranes, and test tubes, and containers such as well plates, microfluidics, glass capillaries, nanopillars, and monolithic columns), and solid phases that can be suspended or dispersed in a liquid phase (e.g., solid phase supports such as particles). Examples of solid phase materials include glass, plastic, metal, and carbon. Non-magnetic or magnetic materials can also be used as solid phase materials, but magnetic materials are preferred from the viewpoint of ease of operation. The solid phase is preferably a solid phase support, more preferably a magnetic solid phase support, and even more preferably magnetic particles. Conventional known methods can be used for immobilizing antibodies. Examples of such methods include physical adsorption, covalent bonding, methods utilizing affinity substances (e.g., biotin, streptavidin), and ionic bonding. In certain embodiments, the antibody against Tg-SH is an antibody immobilized on a solid phase, preferably an antibody immobilized on a magnetic solid phase, and more preferably an antibody immobilized on magnetic particles.

[0022] The reaction step may involve mixing the pretreatment mixture with a buffer and then contacting the immobilized antibody, or it may involve pre-adding the immobilized antibody, for example on particles, to the buffer to create a particle solution, and then mixing the mixture with the particle solution. The reaction step may consist of only a primary reaction step, such as in immunoaggregation or competitive methods, or it may include a secondary reaction step, such as in the sandwich method. If a secondary reaction step is included, a washing step to remove unreacted components may be included between the primary and secondary reaction steps.

[0023] Antibodies against Tg-SH may be labeled with a labeling substance. In this specification, antibodies labeled with a labeling substance may simply be referred to as labeled antibodies. Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin), fluorescent substances or proteins (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), light-emitting or light-absorbing substances (e.g., luciferin, aequorin, acridinium, ruthenium), and radioactive substances (e.g., 3 H, 14 C, 32 P, 35 S, 125 I) is one example. Furthermore, if a secondary reaction is provided in the method of the present invention, the antibody used in the secondary reaction may be labeled with such a labeling substance.

[0024] In certain embodiments, the method of the present invention includes, as the antibody used in the secondary reaction, another antibody against Tg-SH that recognizes a different epitope than the antibody against Tg-SH. The combination of the epitope recognized by the antibody against Tg-SH and the epitope recognized by the other antibody against Tg-SH is not particularly limited. The use of such another antibody is preferred, for example, when a sandwich method is used. As mentioned above, one of the antibodies may be a polyclonal antibody that undergoes an antigen-antibody reaction with Tg-SH.

[0025] 3. Detection process When a primary or secondary antibody is labeled, detection is performed using a method suitable for the label used, for example, by adding the enzyme substrate if an enzyme label is used. For example, if alkaline phosphatase (ALP) is used as the labeled antibody, a chemiluminescent enzyme immunoassay (CLEIA) system using 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane·2sodium salt (AMPPD) as the enzyme substrate can be used.

[0026] The method of the present invention is an immunoassay using an antibody against Tg-SH. Examples of such immunoassays include direct competitive methods, indirect competitive methods, and sandwich methods. Other examples of such immunoassays include chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), immunoturbidimetry (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioactive immunoassay (RIA), latex agglutination reaction, fluorescence immunoassay (FIA), and immunochromatography. These immunoassays themselves are well known and do not need to be described in detail here, but each will be briefly explained.

[0027] The direct competitive method involves immobilizing an antibody against the target antigen to be measured (Tg-SH in this invention) onto a solid phase (as described above for the solid phase and immobilization), performing a blocking treatment to prevent non-specific adsorption (treating the solid phase with a protein solution such as serum albumin), reacting this antibody with a test sample containing the target antigen (a sample that has undergone the pretreatment steps described above in this invention) and a fixed amount of labeled antigen (as described above for labeling), washing, and then quantifying the label bound to the solid phase. Since the antigen in the test sample and the labeled antigen bind competitively to the antibody, the more antigen in the test sample there is, the less label will bind to the solid phase. Various antigen standard solutions of known concentrations are prepared, and the amount of label immobilized on the solid phase (absorbance, luminescence intensity, fluorescence intensity, etc., depending on the properties of the label, the same applies hereinafter) is measured for each, and a calibration curve is created with antigen concentration on the x-axis and label amount on the y-axis. For an unknown test sample, the amount of labeling can be measured, and the amount of antigen in the unknown test sample can be determined by applying the measured labeling amount to a calibration curve. The direct competition method itself is well known in this field and is described, for example, in US 20150166678 A1.

[0028] In the indirect competitive method, the target antigen (Tg-SH in this invention) is immobilized on a solid phase (as described above for the solid phase and immobilization). Next, after blocking the solid phase, a test sample containing the target antigen (a sample that has undergone the pretreatment step as described above in this invention) is mixed with a certain amount of anti-target antigen antibody and reacted with the immobilized antigen. After washing, the anti-target antigen antibody bound to the solid phase is quantified. This can be done by reacting the anti-target antigen antibody with a labeled secondary antibody (labeling as described above), washing, and then measuring the amount of labeling. Various antigen standard solutions of known concentrations are prepared, and for each, the amount of labeling is measured after immobilization on the solid phase to create a calibration curve. For an unknown test sample, the amount of labeling is measured, and the amount of antigen in the unknown test sample can be measured by applying the measured amount of labeling to the calibration curve. It is also possible to use a labeled primary antibody instead of a labeled secondary antibody. The indirect competitive method itself is well known in this field and is described, for example, in US 20150166678 A1 mentioned above.

[0029] Among sandwich methods, for example, the forward sandwich method involves immobilizing an anti-target antigen antibody on a solid phase (as described above for solid phase and immobilization), optionally treating with blocking, reacting with a test sample containing the target antigen (in this invention, a sample that has undergone the pretreatment steps described above), washing, reacting with a labeled secondary antibody against the target antigen (labeling as described above), washing, and then quantifying the label bound to the solid phase. Among sandwich methods, for example, the reverse sandwich method involves first reacting a labeled antibody against the target antigen (label) with the test sample, reacting the antigen-antibody complex formed by the binding of the labeled antibody and the target antigen with the immobilized antibody, washing, and then quantifying the label bound to the solid phase. There is also a sandwich method in which the immobilized antibody, the test sample, and the labeled antibody are reacted simultaneously. In the sandwich method, an antibody that is immobilized on the solid phase after reacting with the test sample may be used as the immobilized antibody. When using antibodies immobilized on a solid phase, for example, the immobilized antibody may be reacted with the test sample and labeled antibody, and then the immobilized antibody may be immobilized on the solid phase; or the immobilized antibody may be reacted with the test sample, and then the immobilized antibody may be immobilized on the solid phase, and then reacted with the labeled antibody. Various antigen standard solutions of known concentrations are prepared, and the amount of labeling immobilized on the solid phase is measured for each to create a calibration curve. For an unknown test sample, the amount of labeling is measured, and the measured amount of labeling is applied to the calibration curve to determine the amount of antigen in the unknown test sample. The sandwich method itself is well known in this field and is described, for example, in US 20150309016 A1.

[0030] For example, in the sandwich method, a first antibody and a second antibody are used, and it is preferable that at least one of the first and second antibodies is an antibody in which the amino acid sequence represented by any of SEQ ID NOs: 1 to 5 contains an epitope. More preferably, the first antibody is at least one, preferably both, selected from the group consisting of the antibody in which the amino acid sequence of SEQ ID NO: 1 contains an epitope and the antibody in which the amino acid sequence of SEQ ID NO: 5 contains an epitope, and the second antibody is at least one, preferably at least two, more preferably all three, selected from the group consisting of the antibody in which the amino acid sequence of SEQ ID NO: 2 contains an epitope, the antibody in which the amino acid sequence of SEQ ID NO: 3 contains an epitope, and the antibody in which the amino acid sequence of SEQ ID NO: 4 contains an epitope.

[0031] Preferably, the first antibody is used as a capture antibody (immobilized antibody), and the second antibody is used as a labeled antibody. Alternatively, the second antibody may be used as a capture antibody, and the first antibody may be used as a labeled antibody.

[0032] Among the various immunoassays described above, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA) are immunoassays classified based on the type of label used when carrying out the aforementioned direct competitive method, indirect competitive method, sandwich method, etc. Chemiluminescent enzyme immunoassay (CLEIA) is an immunoassay that uses an enzyme as a label (e.g., the alkaline phosphatase described above) and a substrate that generates a chemiluminescent compound as a substrate (e.g., the AMPPD described above). Enzyme immunoassay (EIA) is an immunoassay that uses an enzyme as a label (e.g., the aforementioned peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc.). As the substrate for each enzyme, a compound that can be quantified by absorbance measurement or the like is used. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), etc.; in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP), etc.; in the case of β-galactosidase, MG: 4-methylumbelliferyl galactoside, NG: nitrophenyl galactoside, etc.; and in the case of luciferase, luciferin, etc. are used. Radioimmunoassay (RIA) is a method that uses a radioactive substance as a label, and as the radioactive substance, as described above 3 H, 14 C, 32 P, 35 S, 125 I, and other radioactive elements can be mentioned. Fluorescent immunoassay (FIA) is a method that uses a fluorescent substance or fluorescent protein as a label, and examples of the fluorescent substance or fluorescent protein include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein, etc., as described above. Immunoassays themselves using these labels are well known in the art, and are described, for example, in US 8039223 B and US 20150309016 A1.

[0033] Immunoturbidimetric assay (TIA) is an immunoassay that utilizes the phenomenon of increased turbidity due to the antigen-antibody complex generated by the antigen-antibody reaction between a target antigen to be measured (Tg-SH in this invention) and an antibody against that antigen. Various known concentrations of antigen are added to an anti-target antigen-antibody solution, and the turbidity is measured for each to create a calibration curve. Similarly, the turbidity of an unknown test sample is measured, and by applying the measured turbidity to the calibration curve, the amount of antigen in the unknown test sample can be determined. Immunoturbidimetric assay itself is well known and is described, for example, in US 20140186238 A1. Latex agglutination is similar to immunoturbidimetric assay, but instead of the antibody solution used in immunoturbidimetric assay, a suspension of latex particles with anti-target antigen-antibody immobilized on its surface is used. Immunoturbidimetric assay and latex agglutination are well known in this field and are described, for example, in US 7,820,398B.

[0034] Immunochromatography is a method in which the above-described sandwich method or competitive method is carried out on a substrate (also referred to as a matrix or strip) formed of a porous material such as filter paper, cellulose membrane, glass fiber, or nonwoven fabric. For example, in the case of immunochromatography based on the sandwich method, a detection zone on which an anti-target antigen antibody is immobilized is provided on the substrate, a test sample containing a target antigen (in the present invention, a sample that has been subjected to the pretreatment step as described above) is added to the substrate, and a developing solution is allowed to flow from the upstream side to move the target antigen to the detection zone and immobilize the target antigen on the detection zone. The immobilized target antigen is sandwiched with a labeled secondary antibody, and the label immobilized on the detection zone is detected, thereby detecting the target antigen in the test sample. By forming a labeling zone containing a labeled secondary antibody on the upstream side of the detection zone in advance, a conjugate of the target antigen and the labeled secondary antibody is immobilized on the detection zone. When the label is an enzyme, a substrate zone containing a substrate for the enzyme is also provided on the upstream side of the detection zone. In the case of the competitive method, for example, a target antigen is immobilized on the detection zone in advance, allowing competition between the target antigen in the test sample and the target antigen immobilized on the detection zone. A labeled antibody zone is provided upstream of the detection zone, the target antigen in the test sample is reacted with the labeled antibody, and unreacted labeled antibody is immobilized on the detection zone to detect or quantify the label, thereby detecting or quantifying the target antigen in the test sample. Immunochromatography itself is well known in the art and is described, for example, in US 6210898 B.

[0035] <Kit for measuring Tg-SH> The kit for measuring Tg-SH of the present invention is a measurement reagent capable of implementing the above-described method for measuring Tg-SH. The measurement reagent of the present invention comprises at least a reducing agent and a monoclonal antibody that uses Tg-SH as a corresponding antigen.

[0036] The kit of the present invention may contain each component in a form isolated from the others or in the form of a composition. Specifically, each component may be provided in a form contained in a different container (e.g., a tube, a plate), or some components may be provided in the form of a composition (e.g., in the same solution). Alternatively, the kit of the present invention may be provided in the form of a device. Specifically, all components may be provided in a form contained within a device. Alternatively, some components may be provided in a form contained within a device, and the remainder may be provided not contained within a device (e.g., in a form contained in different containers). In this case, the components not contained within the device may be used by being injected into the device when measuring the target substance.

[0037] In preferred embodiments, the kit of the present invention may have a configuration depending on the type of immunoassay to be employed. For example, when a sandwich method is employed, the kit of the present invention may include, as essential components, i) a pretreatment solution, ii) an antibody against Tg-SH, iii) a buffer, and as optional components, iv) another antibody against Tg-SH, v) a labeling agent, vi) a diluent, and, if necessary, vii) a substrate that reacts with the labeling agent. Components ii) and iii) may be contained in the same solution. Component iv) may be labeled with v) the labeling agent. Preferably, the antibody against Tg-SH may be immobilized on magnetic particles.

[0038] The present invention will be described in detail below based on examples. However, the present invention is not limited to the following examples. [Examples]

[0039] 1. Obtaining anti-Tg-SH monoclonal antibodies (A) Preparation of immunoantigens Natural Human Thyroglobulin protein (abcam, ab96518) was prepared to a concentration of 10 mg / mL using phosphate buffer (pH 7.2) containing 150 mM NaCl. The reducing agent TCEP (tris(2-carboxyethyl)phosphine) was added to a final concentration of 10 mM, and the mixture was reacted at 37°C for 30 minutes. Reduced thyroglobulin (Tg-SH) was prepared by buffer exchange using a desalting column with phosphate buffer (pH 6.0) containing 1 mM EDTA and 150 mM NaCl.

[0040] (B) Immunotherapy and cell fusion The antigen protein Tg-SH prepared by the method described above was diluted to a final concentration of 1.0 mg / ml using 10 mM phosphate-buffered saline (pH 7.3) (PBS) containing 150 mM NaCl, mixed with an equal volume of Freund's adjuvant, and administered subcutaneously to 4-6 week old BALB / c mice at a dose of 50-100 μg. Similar booster immunizations were performed every two weeks, and 100 μg of antigen protein dissolved in PBS was administered intraperitoneally as a final immunization. Three days after the final immunization, the spleen was aseptically removed from these mice, hybridomas were prepared using a conventional hybridoma method, and the culture supernatant was collected and used for the screening described below.

[0041] (C) Screening of anti-Tg-SH antibody-producing hybridomas Hybridomas producing the target antibody were searched for using the aforementioned antigen protein Tg-SH via ELISA. Tg-SH antigen, diluted to 1 μg / mL with TBS containing 1 mM TCEP and 6 M urea, was added to 50 μL per well in a Nunk multimodule plate and left to stand overnight at 4-8°C. After removing the antigen diluent, 100 μL of blocking solution (0.1% casein, 1 mM EDTA, PBS) was added per well, and the plate was left to stand at room temperature for 1 hour to prepare an antigen-solid-phase plate.

[0042] 50 μL of the culture supernatant of hybridomas diluted with reaction solution (0.1% casein, 1 mM EDTA, PBS) was added to each well of an antigen-solid-phase plate and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20 (trade name), 50 μL of horseradish peroxidase (HRP)-labeled anti-mouse IgG Fc-specific antibody was added and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20 (trade name), 50 μL of TMB solution was added to each well to induce color development. Subsequently, 2 M H2SO4 was added to each well to stop the reaction, and the absorbance was measured at a wavelength of 450 nm to obtain antibody-producing hybridomas with the desired reaction specificity.

[0043] (D) Cloning of hybridomas The obtained hybridomas were cloned using the limiting dilution method to establish antibody-producing hybridomas. The obtained hybridomas were named 9E12, A1010, A1024, A2029, and A2068.

[0044] (E) Preparation of anti-Tg-SH monoclonal antibody The established antibody-producing hybridomas were acclimatized in serum-free medium (Hybridoma-SFM, GIBCO). They were then cultured in 50 mL T75 flasks until the cell density reached approximately 5 × 10⁶. 5 When the cell count reached 500 mL, the cells were transferred to a culture bag (Nipro Corporation) filled with 500 mL of serum-free medium. After culturing for 2–4 weeks, the culture supernatant was collected. The culture medium was applied to a column packed with Protein G Sepharose (GE Healthcare), and the bound antibody was eluted with a pH 3 buffer. The eluate was rapidly neutralized with 2 M Tris (pH 8), and the buffer was exchanged to PBS using a desalting column. 5–20 mg of anti-Tg-SH monoclonal antibody could be obtained from 500 mL of culture medium.

[0045] 2. Identification of the reaction region of anti-Tg-SH antibodies using thyroglobulin partial-length antigens. Six transglottic antigens (TGN, 20-377 aa; TG2F, 359-726 aa; TG4F, 1074-1469 aa; TG5F, 1470-1891 aa; TG6F, 1892-2187; TGC, 2336-2768 aa; Table 1 shows the amino acid sequences of each antigen) expressed in E. coli were used to identify the reaction sites of each antibody. Each antigen, including the Tg-SH used as an immunogen, was diluted to 1 μg / mL with 1 mM TCEP and 6 M urea-containing TBS, and 50 μL was added per well to a Nunk multimodule plate, which was left to stand overnight at 4-8°C. After removing the antigen diluent, 100 μL of blocking solution (0.1% casein, 1 mM EDTA, PBS) was added to each well, and the plate was left to stand at room temperature for 1 hour to prepare an antigen-solid-phase plate.

[0046] 50 μL of anti-Tg-SH antibody diluted in reaction solution (0.1% casein, 1 mM EDTA, PBS) was added to each well of an antigen-solid-phase plate and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20 (trade name), 50 μL of horseradish peroxidase (HRP)-labeled anti-mouse IgG Fc-specific antibody was added and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20 (trade name), 50 μL of TMB solution was added to each well to induce color development. Subsequently, 2 M H2SO4 was added to each well to stop the reaction, and the absorbance was measured at a wavelength of 450 nm. The results showed that 9E12 recognized TGN, A1010 recognized TGC, and A1024, A2029, and A2068 recognized TG5F (Table 2).

[0047] [Table 1] Each subsequence in Table 1 is shown as sequence numbers 6-11 in the sequence listing, respectively.

[0048] [Table 2]

[0049] 3. Epitope analysis of anti-Tg-SH antibodies To analyze the epitopes of the obtained anti-Tg-SH antibodies, the aforementioned partial-length thyroglobulin antigens TGN, TG5F, and TGC deletion mutants were expressed in E. coli, and the results obtained by dot blotting using the E. coli lysates, as shown in Figures 1A-C, indicate that the 9E12 antibody has an epitope within the CELQRETAFLKQADYVP sequence (SEQ ID NO: 1) at 34-50 aa, A1024 has an epitope within VIFDANAPVAVRSK (SEQ ID NO: 2) at 1579-1592 aa, A2029 has an epitope within VPDSEFPVMQCLTDCT (SEQ ID NO: 3) at 1593-1608a.a., A2068 has an epitope within the LGDQEFIKSLTPLEGTQ sequence (SEQ ID NO: 4) at 1793-1809 aa, and A1010 has an epitope within 2683-2698 The presence of an epitope within the TPWPDFVPRAGGENYK sequence (SEQ ID NO: 5) of aa was demonstrated. The numbers shown in Figures 1A-C (for example, 20-377 in TGN20-377) represent the amino acid positions of the full-length thyroglobulin protein (SEQ ID NO: 12). Anti-TrpE confirms that the target protein is immobilized on the nitrocellulose membrane used in this analysis.

[0050] 4. Reactivity evaluation of anti-Tg-SH monoclonal antibodies The reactivity of the obtained Tg-SH monoclonal antibody against non-reduced Tg and reduced Tg (Tg-SH) was compared. Natural Human Thyroglobulin protein (abcam, ab96518) was diluted with PBS to 1 μg / mL to obtain non-reduced Tg, and diluted with PBS containing 5 mM TCEP to obtain Tg-SH. 50 μL of these antigens were added per well to a Nunk multimodule plate and left to stand overnight at 4-8°C. After removing the antigen diluent, 100 μL of blocking solution (0.1% casein, 1 mM EDTA, PBS) was added per well, and the plate was left to stand at room temperature for 1 hour to prepare an antigen-solid-phase plate. 50 μL of anti-Tg antibody and anti-Tg-SH antibody diluted with reaction solution (0.1% casein, 1 mM EDTA, PBS) were added to each well of the antigen-solid-phase plate and left to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20 (trade name), 50 μL of alkaline phosphatase (ALP)-labeled anti-mouse IgG Fc-specific antibody was added and the mixture was allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20 (trade name), 50 μL of substrate solution containing AMPPD (Lumipulse® substrate solution, Fujirebio Corporation) was added to each well, and the chemiluminescence due to the enzymatic reaction was measured using a plate reader.

[0051] The results are shown in Table 3. While typical anti-Tg antibodies A and B show less than 10% reactivity towards Tg-SH compared to reactivity towards unreduced Tg, the obtained anti-Tg-SH antibody showed higher reactivity towards Tg-SH than towards unreduced Tg.

[0052] [Table 3]

[0053] 5. Construction of a highly sensitive automated analytical measurement system The following steps were taken to construct a highly sensitive automated analytical measurement system: (A) preparation of antibody-conjugated ferrite particles and (B) preparation of alkaline phosphatase (ALP)-labeled antibodies.

[0054] (A) Preparation of antibody-conjugated magnetic particles Magnetic particles were reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride) at room temperature for 30 minutes. After washing the ferrite particles, anti-Tg-SH antibody or anti-Tg antibody was added and the mixture was stirred in an end-over mixer at room temperature for 60 minutes. To stop the reaction, 1 M Tris (pH 7.0) was added and the mixture was stirred in an end-over mixer at room temperature for 30 minutes. These particles were dispersed in storage buffer (50 mM Tris, 2.0% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% NaN3, pH 7.2) to prepare antibody-conjugated magnetic particles.

[0055] (B) Preparation of alkaline phosphatase (ALP) labeled antibody Desalted monoclonal antibodies were digested with pepsin and purified by gel filtration using coupling buffer (0.1 M phosphate buffer, 1 mM EDTA, pH 6.0) to obtain antibodies with the Fc region removed. Next, 2-mercaptoethylamine (final concentration 10 mM) was added and the mixture was allowed to stand at 37°C for 3 hours to perform thiolation. Further desalting with coupling buffer was performed to obtain Fab' antibody. Meanwhile, desalted alkaline phosphatase and N-(4-maleimidobutyryloxy)-succinimide (GMBS) were mixed in 0.1 M phosphate buffer (pH 7.0) and allowed to stand at 30°C for 1 hour to perform maleimidation. After desalting with coupling buffer, Fab' and maleimidized alkaline phosphatase were mixed in a molar ratio of 1:1 and allowed to stand at 25°C for 30 minutes to perform coupling. 2-mercaptoethylamine (final concentration 2 mM) was added to the coupling solution and allowed to stand at room temperature for 30 minutes to stop the reaction. Furthermore, iodoacetamide (final concentration 10 mM) was added and the mixture was allowed to stand at room temperature for 30 minutes to block the free thiol groups. The mixture was concentrated and purified by gel filtration to obtain ALP-labeled antibody.

[0056] (C) Antibody combinations For the non-reducing Tg measurement system (hereinafter referred to as the Tg measurement system), anti-Tg antibody A-conjugated ferrite particles and ALP-labeled anti-Tg antibody B were combined to construct the respective measurement systems. For the Tg-SH measurement system, co-conjugated ferrite particles of 9E12 and A1010 as capture antibodies were mixed with A1024, A2029, and A2068 as ALP-labeled substances (labeled antibodies) to construct the respective measurement systems.

[0057] 6. Test to confirm the effectiveness of pre-reduction treatment (A) Preparation of pre-reduction treatment solution A reduction pretreatment solution was prepared by adding the following reducing agents: 2M arginine hydrochloride, 50 mM Tris, pH 7.5, and the reducing agents 2-DEAET (2-diethylaminoethanethiol hydrochloride) in a concentration range of 0-500 mM, DTT (dithiothreitol) in a concentration range of 0-50 mM, and TCEP (tris(2-carboxyethyl)phosphine hydrochloride) in a concentration range of 0-50 mM.

[0058] (B) Preparation of TgAb-positive model specimens A TgAb-positive model sample [TgAb(+)] was prepared by diluting Natural Human Thyroglobulin protein (abcam, ab96518) with horse serum and adding Lumipulse Presto TgAb (Fujirebio) TgAb calibrator (3000 IU / mL) to a concentration of 500 IU / mL. On the other hand, a TgAb-negative model sample [TgAb(-)] was prepared by adding TgAb calibrator (0 IU / mL) using the same method.

[0059] (C)Measurement method A pre-treated sample was prepared by mixing 30 μL of the sample with 90 μL of pre-treatment solution and allowing it to stand at room temperature for 30 minutes. Of this, 50 μL was mixed with 50 μL of a magnetic particle solution containing anti-Tg-SH antibody or anti-Tg antibody-conjugated magnetic particles (50 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA 3Na, 5% Gelatin from cold water fish skin, 1 M arginine hydrochloride, 100 mM iodoacetamide, pH 7.5) and reacted at 37°C for 8 minutes. Magnetic particles were collected and washed to remove unbound components, and 50 μL of a labeled diluent (50 mM MES, 100 mM NaCl, 0.3 mM ZnCl2, 1 mM MgCl2, 2% BSA, 5 μg / mL Inactive ALP, 0.1% ProClin300, pH 6.8) containing ALP-labeled anti-Tg-SH antibody or anti-Tg antibody diluted to 0.2 μg / mL was added. The mixture was reacted at 37°C for 8 minutes. The magnetic particles were collected and washed to remove unbound components, and 200 μL of a substrate solution containing AMPPD (Lumipulse® substrate solution, manufactured by Fujirebio Corporation) was added to count the amount of luminescence due to the enzymatic reaction. All steps from sample pretreatment onward in this example were performed using the automated analyzer Lumipulse L2400 (registered trademark, manufactured by Fujirebio Corporation).

[0060] (A) Results Figure 2 shows the results of pretreatment with various reducing agents. In the Tg measurement system, when no reducing agent is added, the signal of the TgAb(+) sample is lower than that of the TgAb(-) sample, indicating that Tg measurement is being interfered with by TgAb. This effect decreases with increasing reducing agent concentration, but the signal also decreases significantly, making it difficult to measure at a reducing agent concentration where the TgAb(+) and TgAb(-) values ​​match (TgAb is completely liberated). On the other hand, the Tg-SH measurement system shows a significant increase in reactivity with the addition of a reducing agent, demonstrating that measurement is possible at reducing agent concentrations where TgAb is completely liberated. At pretreatment concentrations of 150 mM for 2-DEAET, 10 mM for DTT, and 5 mM or higher for TCEP, Tg can be measured without interference from TgAb.

[0061] 7. Efficacy confirmation test of reduction pretreatment using human samples (A) Preparation of pre-reduction treatment solution To prevent protein aggregation and increased sample viscosity during reduction treatment, improve operability, and enhance measurement sensitivity, the pretreatment solution composition was investigated. The resulting conditions were 2.4 M urea, 6.4% C16APS (N-hexadodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 0.16 M NaCl, 50 mM Tris, 20 mM TCEP, and pH 7.5. For comparative Tg measurement systems, a pretreatment solution with the TCEP removed from this composition was used.

[0062] (B) Preparation of TgAb-positive model specimens Model samples were prepared by adding a sample diluent (Fujirebio) or a sample with high Tg levels to serum from thyroid-related diseases or healthy individuals, to which TgAb levels were measured using TgAb from Lumipulse Presto TgAb (Fujirebio). In addition, samples #1-26 shown in Table 4 were prepared by diluting Natural Human Thyroglobulin protein (nhTg, abcam, ab96518) with Lumipulse sample diluent (Fujirebio) and by adding an nhTg dilution series to which Lumipulse Presto TgAb (Fujirebio) TgAb calibrator was added to a concentration of 750 IU / mL.

[0063] (C)Measurement method A pre-treated sample was prepared by mixing 30 μL of the sample with 90 μL of the pre-treatment solution and allowing it to stand at room temperature for 30 minutes. Of this, 50 μL was mixed with 50 μL of a magnetic particle solution containing anti-Tg-SH antibody or anti-Tg antibody-conjugated magnetic particles (100 mM Tris-HCl, 200 mM NaCl, 20 mM EDTA 3Na, 0.1% ProClin 300, 2% BSA, pH 7.5) and reacted for 8 minutes. The magnetic particles were collected and washed to remove unbound components, and 50 μL of ALP-labeled anti-Tg-SH antibody or anti-Tg antibody, diluted to 0.2 μg / mL with a labeled diluent (50 mM MES, 0.1 mM ZnCl2, 1 mM MgCl2, 3% BSA, 5 μg / mL Inactive ALP, 0.10% NaN3, pH 6.8), was added and reacted for 8 minutes. Magnetic particles were collected and washed to remove unbound components, and 200 μL of a substrate solution containing AMPPD (Lumipulse® substrate solution, manufactured by Fujirebio Corporation) was added. The amount of luminescence due to the enzymatic reaction was counted, and the Tg value in the sample was calculated from the count using a calibration curve. The calibration curve was created based on the amount of luminescence obtained for each standard solution of Natural Human Thyroglobulin protein (nhTg, abcam, ab96518) corresponding to Tg levels of 0, 10, 50, 200, and 1000 ng / mL, measured in the same manner as the sample. All steps from sample pretreatment onward in this example were performed using the automated analyzer Lumipulse L2400 (registered trademark, manufactured by Fujirebio Corporation).

[0064] (A) Results Table 4 shows the results of Tg-SH measurement and Tg measurement. Figure 3 shows the correlation between Tg-SH measurement and Tg measurement, and Figure 4 is an enlarged view of the low-concentration range in Figure 3. Compared to the Tg measurement system, the Tg-SH measurement system often yields Tg quantitative values ​​that are more than twice as high. In particular, in samples #7 and #13, Tg that could not be detected by the Tg measurement system was detected by the Tg-SH measurement system. From the above, it has been shown that the Tg-SH measurement of the present invention can avoid interference with measurement by TgAb and improve false low values ​​in Tg measurement diagnosis.

[0065] [Table 4]

Claims

1. A method for measuring thyroglobulin in a sample isolated from a living organism by immunoassay, comprising a pretreatment step of treating the sample isolated from a living organism with a reducing agent, wherein the immunoassay uses a monoclonal antibody or an antigen-binding fragment thereof as the corresponding antigen, and the corresponding epitope of the monoclonal antibody is contained in an amino acid sequence represented by any of SEQ ID NOs: 1 to 5.

2. The method according to claim 1, wherein the immunoassay is a sandwich method, and the monoclonal antibody or its antigen-binding fragment is used in at least one of the first antibody and the second antibody of the sandwich method.

3. The first antibody comprises at least one selected from the group consisting of an antibody whose amino acid sequence of SEQ ID NO: 1 contains the corresponding epitope and an antibody whose amino acid sequence of SEQ ID NO: 5 contains the corresponding epitope. The method according to claim 2, wherein the second antibody comprises at least one selected from the group consisting of an antibody whose amino acid sequence of SEQ ID NO: 2 contains the corresponding epitope, an antibody whose amino acid sequence of SEQ ID NO: 3 contains the corresponding epitope, and an antibody whose amino acid sequence of SEQ ID NO: 4 contains the corresponding epitope.

4. The method according to claim 3, wherein the first antibody is a capture antibody and the second antibody is a labeled antibody.

5. An immunoassay kit for performing the method according to claim 1, comprising a reducing agent and a monoclonal antibody or an antigen-binding fragment thereof, wherein the corresponding epitope of the monoclonal antibody is included in an amino acid sequence represented by any of SEQ ID NOs: 1 to 5.

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