Direct immunoassay method for detecting autoantibodies

Abstract

Disclosed herein are immunoassays for detecting antibodies in biological samples from subjects and / or for diagnosing autoimmune diseases of subjects. [Solution] The disclosed immunoassay evaluates the level of antibodies in a biological sample from a subject by simultaneously binding an antibody to a capture antigen (e.g., an unlabeled antigen bound to a solid support) and a labeled antigen not bound to a solid support, using a one-step direct process.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Application No. 62 / 693,439, filed on Jul. 2, 2018, the entire content of which is incorporated herein by reference.

[0002] Disclosed herein are methods for detecting antibodies in a biological sample from a subject and methods for diagnosing an autoimmune disease of the subject.

Background Art

[0003] The evaluation of antibodies such as autoantibodies in a biological sample from a subject has been hampered by the lack of sensitivity and specificity of reagents and assays. Currently, competitive assays are used to detect autoantibodies in a biological sample from a subject. In these assays, a labeled control antibody is bound to an antigen, and then these are bound to a solid support, and this labeled control antibody / antigen complex is incubated with a suspected biological sample containing autoantibodies. If autoantibodies are present in the sample, the labeled control antibody dissociates from the labeled control antibody / antigen complex, causing a decrease in the signal generated from the complex. The amount of autoantibodies present in the sample is inversely proportional to the decrease in the signal. Such competitive assays are based on the premise that autoantibodies and control antibodies compete around the same epitope of the antigen, but this is not always the case. Therefore, the usefulness of the competitive assay format is limited.

[0004] Indirect binding assays have also been developed in which an antigen is bound to a solid phase, incubated with a biological sample, washed / isolated, and incubated with a labeled anti - human secondary antibody. However, such assays are time - consuming and require additional reagents.

Summary of the Invention

Means for Solving the Problems

[0005] Disclosed herein is a method for detecting antibodies in a biological sample from a subject, the method comprising: a) incubating the biological sample from the subject with a solid support bound to an unlabeled antigen, wherein the unlabeled antigen is specifically recognized by an antibody; and a labeled antigen, wherein the labeled antigen does not bind to the solid support but is specifically recognized by an antibody, thereby forming a solid support / labeled antigen complex in the presence of the antibody; and b) detecting the solid support / labeled antigen complex, wherein the presence of the solid support / labeled antigen complex indicates the presence of antibodies in the biological sample. This includes.

[0006] A method for diagnosing the target autoimmune disease is also provided. This method includes: a) incubating a biological sample from a subject with a solid support bound to an unlabeled antigen, wherein the unlabeled antigen is specifically recognized by autoantibodies from the subject; and a labeled antigen, wherein the labeled antigen does not bind to the solid support and is specifically recognized by autoantibodies, wherein a solid support / labeled antigen complex is formed in the presence of autoantibodies; and b) diagnosing the subject with an autoimmune disease if the solid support / labeled antigen complex is detected.

[0007] 1) a solid support, an unlabeled antigen, and a labeled antigen; or 2) a kit comprising a solid support to which an unlabeled antigen is conjugated and a labeled antigen is further disclosed herein.

[0008] The abstract and the following detailed description will be better understood when read in conjunction with the attached drawings. For illustrative purposes, examples of embodiments of the disclosed method and kit are shown in the drawings, but the method and kit are not limited to the specific embodiments disclosed. The drawings are described below. [Brief explanation of the drawing]

[0009] [Figure 1]This figure shows an example of a reaction scheme for a disclosed immunoassay called an antigen crosslinking immunoassay. [Figure 2] This figure shows an example timeline for performing the disclosed immunoassay. [Figure 3] This figure shows an example of a dose-response curve from a disclosed immunoassay (concentration of anti-thyroglobulin antigen as a function of the observed signal (RLU)). [Figure 4] This figure shows an example of dose-response curves comparing undiluted (diamond) and diluted (square) antibody samples using the disclosed immunoassay. [Figure 5] This figure shows example dose-response curves analyzing the effect of increasing the amount of acridinium ester. Triangle = 20 × acridinium ester; square = 10 × acridinium ester; and rhombus = 5 × acridinium ester. [Modes for carrying out the invention]

[0010] The disclosed methods and kits can be more readily understood by referring to the following detailed description provided in conjunction with the accompanying drawings that form part of this disclosure. It should be understood that the disclosed methods and kits are not limited to the specific methods and kits described and / or presented herein, and that the terms used herein are for illustrative purposes only to describe specific embodiments and do not limit the methods and kits described in the claims.

[0011] Unless otherwise stated, any description of possible mechanisms, mechanisms of action, or reasons for improvement is for illustrative purposes only, and the disclosed methods and kits should not be limited by the accuracy of such suggested mechanisms, mechanisms of action, or reasons for improvement.

[0012] Throughout this document, the description refers to methods for detecting antibodies and methods for diagnosing autoimmune diseases. Where this disclosure describes or claims a configuration or embodiment relating to a method for detecting antibodies, such configuration or embodiment is similarly applicable to methods for diagnosing autoimmune diseases. Similarly, where this disclosure describes or claims a configuration or embodiment relating to a method for diagnosing autoimmune diseases, such configuration or embodiment is similarly applicable to methods for detecting antibodies.

[0013] Where a range of numbers is described or specified herein, that range includes its endpoints and all individual integers and fractions within that range, and also includes each of the narrower ranges formed by all possible combinations of their endpoints and the integers and fractions within them, to the same extent as each of these narrower ranges being explicitly described, in order to form subgroups within the larger group of values ​​within the described range. Even where a range of numbers is described herein as being greater than a given value, the range is finite and its upper limit is limited by values ​​that can be manipulated within the context of the invention as described herein. Even where a range of numbers is described herein as being less than a given value, the range is limited by non-zero values. The ranges of the invention are not intended to be limited to the specific values ​​described when defining the range. All ranges are inclusive and combinable.

[0014] When a value is expressed as an approximation, the use of the antecedent "approximately" should be understood as indicating that the specific value forms another embodiment. When referring to a specific numerical value, it should include at least that specific value unless explicitly indicated otherwise in the context.

[0015] For clarity, it should be understood that certain configurations of the disclosed methods and kits described in this specification in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various configurations of the disclosed methods and kits described in the context of a single embodiment for the sake of brevity may also be provided separately or in any partial combination.

[0016] As used herein, the singular forms "a," "an," and "the" include plural forms.

[0017] Various terms related to the aspects described are used throughout the specification and claims. Unless otherwise indicated, such terms should be given their common meanings in the art. Other terms that are defined in detail should be interpreted in a manner consistent with the definitions provided herein.

[0018] The term "comprising" means "consisting essentially of" and "consisting The term "consisting essentially of" is intended to include examples that fall under the umbrella term "consisting of"; similarly, the term "consisting essentially of" is intended to include examples that fall under the umbrella term "consisting of".

[0019] Disclosed herein are immunoassays for detecting antibodies in biological samples from subjects and / or for diagnosing autoimmune diseases of subjects. The disclosed immunoassays assess the level of antibodies in a sample by simultaneously binding antibodies to a capture antigen (e.g., an unlabeled antigen bound to a solid support) and a labeled antigen not bound to a solid support using a single direct step. Due to the bivalent nature of antibodies, antibodies present in a biological sample simultaneously bind to both the unlabeled antigen and the labeled antigen bound to a solid support, forming a complex containing the solid support and the labeled antigen. The reading from the assay (i.e., the signal of the labeled antigen bound to the solid support by the antibody) is directly proportional to the level of antibodies present in the sample.

[0020] In some embodiments, the immunoassay may include an "antigen crosslinking" immunoassay, and an example of such a reaction scheme is shown in Figure 1. A biological sample known to contain or suspected to contain the antibody 10 of interest is incubated with a solid support 30 to which labeled antigen 20 and unlabeled antigen are bound. If the antibody is not present, the labeled antigen will not bind to or interact with the solid support. Therefore, if the antibody is not present, the labeled antigen will remain in the solution, and isolating the solid support will not isolate the labeled antigen. If the antibody 10 is present in the biological sample, the antibody 10 will simultaneously bind to the unlabeled antigen and labeled antigen 20 bound to the solid support 30, thereby linking the labeled antigen 20 and the solid support 30 and causing the formation of a solid support / labeled antigen complex 40. It should be understood that the order in which incubation takes place may differ from the order illustrated in Figure 1. For example, a biological sample known to contain or suspected to contain the antibody 10 of interest may first be incubated with a solid support 30 to which unlabeled antigen is bound, and then subsequently incubated with the labeled antigen 20. Alternatively, a biological sample known to contain or suspected to contain the target antibody 10 is incubated simultaneously with a solid support 30 bound to an unlabeled antigen and a labeled antigen 20. It can be done.

[0021] An example timeline for performing the disclosed immunoassay is illustrated in FIG. 2. In FIG. 2, a biological sample known or suspected to have the antibody of interest 10 is incubated with the labeled antigen 20 for less than about 3 minutes. A solid support 30 to which an unlabeled antigen is bound is added and incubated with the antibody / labeled antigen mixture for about 6.5 minutes. Thereafter, the solid support / labeled antigen complex 40 can be detected (not shown).

[0022] The disclosed immunoassay includes a method for detecting an antibody in a biological sample from a subject. The method for detecting an antibody in a biological sample from a subject is: a) incubating a biological sample from a subject with: a solid support to which an unlabeled antigen is bound, wherein the unlabeled antigen is specifically recognized by the antibody; and a labeled antigen, wherein the labeled antigen is not bound to the solid support and is specifically recognized by the antibody such that, in the presence of the antibody, a solid support / labeled antigen complex is formed; and b) detecting the solid support / labeled antigen complex, wherein the presence of the solid support / labeled antigen complex indicates the presence of the antibody in the biological sample, and includes.

[0023] In some embodiments, the antibody is an autoantibody. Thus, the disclosed method can be used to detect autoantibodies in a biological sample from a subject. Examples of autoantibodies that can be detected by the disclosed method are autoantibodies that specifically bind to thyroglobulin (i.e., anti-thyroglobulin antibodies). In embodiments where the autoantibody specifically binds to thyroglobulin, the unlabeled antigen is thyroglobulin and the labeled antigen is labeled thyroglobulin.

[0024] The disclosed immunoassays include methods for diagnosing the target autoimmune disease. The methods for diagnosing the target autoimmune disease are: a) Biological samples from the subject: A solid support to which an unlabeled antigen is bound, wherein the unlabeled antigen is specifically recognized by the target autoantibody; and A labeled antigen, wherein the labeled antigen does not bind to a solid support and is specifically recognized by an autoantibody. This involves incubation together, Here, in the presence of autoantibodies, a solid support / labeled antigen complex is formed; b) If a solid support / labeled antigen complex is detected, the subject should be diagnosed with an autoimmune disease. Includes.

[0025] The disclosed method can be used to diagnose any autoimmune disease in which an antigen recognized by an autoantibody is known. In some embodiments, the autoimmune disease is an autoimmune thyroid disease, including Graves' disease (GD) and Hashimoto's thyroiditis (HT). In embodiments where the autoimmune disease is an autoimmune thyroid disease, the antigen (unlabeled and labeled) is thyroglobulin.

[0026] The following disclosures also apply to methods for detecting antibodies and methods for diagnosing autoimmune diseases.

[0027] Appropriate biological samples include, but are not limited to, serum, plasma, whole blood, saliva, urine, semen, sweat, tears, and body tissues, any biological sample from a subject that contains or is suspected of containing the antigen of interest.

[0028] In some embodiments, the biological sample from the subject can be diluted to reduce the sample concentration before the incubation process. Suitable dilutions include, for example, 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:1000, etc.

[0029] The solid support may be any material to which unlabeled antigens can be directly or indirectly bound or linked. Examples of solid supports include, but are not limited to, column matrix materials, culture plates, tubes, dishes, flasks, microtiter plates, beads / particles, prokaryotic or eukaryotic cells fixed with thermally sterilized formalin (or by other chemical means), microscope slides, ACLAR® films, or any other optically transparent polymer or combination thereof. The solid support may be composed entirely or partially of plastics, cellulose, cellulose derivatives, nitrocellulose, glass, glass fibers, latex, or combinations thereof. In some embodiments, the solid support may include magnetic particles. Suitable magnetic particles include paramagnetic particles (PMPs) and latex magnetic particles (LMPs).

[0030] Unlabeled antigens can be directly bound to or indirectly linked to a solid support, thereby forming a solid support to which the unlabeled antigen is bound. Unlabeled antigens can be directly bound to a solid support. Suitable techniques for directly binding unlabeled antigens to a solid support include, for example, covalent bonding, adsorption, non-covalent interactions, or a combination thereof. Alternatively, unlabeled antigens can be indirectly linked to a solid support. Suitable means for indirectly linking unlabeled antigens to a solid support include, for example, linkage via peptides, proteins, antibodies, linkers, or a combination thereof. In some embodiments, unlabeled antigens can be indirectly linked to a solid support via streptavidin and biotin. For example, unlabeled antigens can be biotinylated, and the solid support may contain streptavidin.

[0031] A solid support bound to an unlabeled antigen can be present in a buffer containing one or more salts, one or more stabilizers, and one or more surfactants. In some embodiments, the buffer may contain HEPES salt, sodium chloride, bovine albumin, bovine globulin, and Tween20. In some embodiments, the buffer may contain HEPES salt (16.9 g / L HEPES acid and 7.6 g / L HEPES sodium salt), 300 mM sodium chloride, 1% bovine albumin, 0.1% bovine globulin, and 0.2% Tween20.

[0032] The labeled antigen does not bind to or otherwise interact with the solid support in the absence of an antibody. Therefore, in embodiments where the solid support contains streptavidin, the labeled antigen is not biotinylated.

[0033] The labeled antigen includes a detectable label. Suitable detectable labels include, but are not limited to, enzyme conjugates (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase), fluorescent probes, radioisotopes, chemiluminescent and bioluminescent labels, or combinations thereof. In some embodiments, the detectable label includes acridinium esters or their analogues. Acridinium ester analogues include dimethylacridinium ester (DMAE), N-sulfopropyldimethylacridinium ester (NSP-DMAE), high quantum yield acridinium ester (HQYAE), and zwitterionic acridinium. The detectable labels include, but are not limited to, esters (ZAE), hexa(ethylene) glycol acridinium esters (HEGAE), N-sulfopropyl-2-isopropoxydimethylacridinium esters (iso-Di-ZAE), trisulfopropyl acridinium esters (TSP-AE), or N-sulfopropyldimethylacridinium esters containing hexa(ethylene) glycol linkers (HEG-GLU-AE). In some embodiments, the detectable labels include ruthenium esters or analogs thereof. The detectable labels may be present in a 1:1 molar ratio with the antigen, or in a molar excess. For example, the detectable labels may be present in molar excesses of 5, 10, 20, or 50.

[0034] The labeled antigen may be present in a buffer containing one or more salts, one or more stabilizers, and one or more surfactants. In some embodiments, the buffer may contain HEPES salt, sodium chloride, bovine albumin, bovine globulin, and Tween20. In some embodiments, the buffer may contain HEPES salt (16.9 g / L HEPES acid and 7.6 g / L HEPES sodium salt), 300 mM sodium chloride, 1% bovine albumin, 0.1% bovine globulin, and 0.2% Tween20.

[0035] The disclosed method can be performed faster than currently used indirect two-step assays, taking approximately one hour. For example, in the disclosed method, a biological sample known or suspected to contain antibodies can be incubated in the reaction mixture for approximately 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or less than 10 minutes. A labeled antigen can be added and incubated with the biological sample for approximately 1 minute, 2 minutes, 3 minutes, 4 minutes, or less than 5 minutes. A solid support conjugated with an unlabeled antigen can be added to the mixture of the biological sample and the labeled antigen and incubated for approximately 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, or less than 10 minutes. In some embodiments, the incubation step is performed in a total of approximately 10 to 20 minutes. The subsequent detection or diagnostic step can be performed in less than 5 minutes. It should be understood that the length of time required for the assay can vary based on several factors, including the level of antibodies in the biological sample and the affinity of the antibodies to the antigen. Therefore, the disclosed method can be performed for any appropriate length of time.

[0036] This method may further include determining the level of antibodies in a biological sample from a subject. The level of antibodies in a biological sample from a subject is directly proportional to the level of the solid support / labeled antigen complex. Therefore, the level of antibodies can be determined by determining the level of the solid support / labeled antigen complex, which can be done, for example, by measuring the signal from the complex. Similarly, in embodiments where this method is used to diagnose an autoimmune disease in a subject, the solid support / labeled antigen complex is detected by measuring the signal from the labeled antigen linked to the solid support.

[0037] The disclosed method can be performed manually or automated. For example, the disclosed method can be performed using the ADVIA CENTAUR® immunoassay system or the ATELLICA® system.

[0038] Kits for carrying out the disclosed methods are also provided. The kits may include a solid support, an unlabeled antigen, and a labeled antigen. Suitable solid supports include those disclosed above. The solid support binds to the unlabeled antigen but not to the labeled antigen. Therefore, in some embodiments, the kit includes a solid support to which the unlabeled antigen is bound and a labeled antigen. In some embodiments, the antigen is thyroglobulin and the labeled antigen is labeled thyroglobulin. Suitable labels include those disclosed above in the methods. [Examples]

[0039] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are illustrative of the disclosed embodiments and are not limiting.

[0040] Reagent manufacturing The solid-phase reagent of the thyroglobulin assay contained streptavidin-coated magnetic particles (Thermo Fisher Scientific, Dynabeads® M270 REF 34353) conjugated to biotinylated human thyroglobulin at a target particle concentration of 0.6 g / L in buffered saline containing HEPES salts (HEPES acid 16.9 g / L and HEPES sodium salt 7.6 g / L), 300 mM sodium chloride to provide ionic strength, 1% bovine albumin and 0.1% bovine globulin as stabilizers, and 0.2% Tween 20.

[0041] Labeled Thyroglobulin Reagent - The assay labeling reagent contained human thyroglobulin labeled with ZAE-type acridinium ester at a thyroglobulin target concentration of 1.2 μg / mL in buffered saline containing HEPES salt (HEPES acid 16.9 g / L and HEPES sodium salt 7.6 g / L), 300 mM sodium chloride to provide ionic strength, 1% bovine albumin and 0.1% bovine globulin as stabilizers, and 0.2% Tween 20.

[0042] Sample preparation Samples (10 levels) of the WHO reference material (anti-thyroglobulin serum, human NIBSC code 65 / 093) were prepared according to the recommended dilution procedure. The predicted concentrations of anti-thyroglobulin in each sample are shown in Table 1. Similarly, patient serum standards were prepared using high concentrations of human anti-thyroglobulin antibody (e.g., 10,000–50,000 IU / mL) and diluted to 10 levels over a wide concentration range (Table 2). All samples were subjected to the disclosed immunoassay (referred to as “antigen-crosslinking immunoassay”) and ADVIA. Analysis was performed using competitive assays currently in use with the CENTAUR® system.

[0043] Reaction procedure for antigen crosslinking immunoassay As illustrated in Figure 2, the following steps were performed using the ADVIA CENTAUR® system: Dispense 25 μL of the sample containing anti-thyroglobulin antibody into a cuvette; • Incubate the sample for 4.75 minutes; Dispense 100 μL of reagent probe 1 of the labeled thyroglobulin reagent and incubate the mixture for 2.75 minutes; Dispense 200 μL of reagent probe 2 of the thyroglobulin-conjugated solid-phase reagent and incubate the mixture at 37°C for 6.5 minutes; The formed complex is separated using a magnet, the complex is aspirated, and washed with washing buffer; Dispense 300 μL each of the acid reagent (HCl) and the base reagent (NaOH) to initiate the chemiluminescence reaction; • The results for relative luminescence units (RLU) were reported.

[0044] Reaction procedure for competitive immunoassay The following steps were performed manually or using the ADVIA CENTAUR® system. Dispense 40 μL of the sample containing anti-thyroglobulin antibody into a cuvette; • 100 μL of the labeling reagent was added to the cuvette and incubated at 37°C for 2.5 minutes. The labeling reagent was prepared by mixing human thyroglobulin (approximately 0.38 μg / mL) with acridinium ester in a buffer saline containing BSA, a protein stabilizer, and a preservative; • 200 μL of solid phase was mixed in a cuvette and incubated at 37°C for 5.0 minutes. The solid phase reagent was prepared by mixing polyclonal human anti-thyroglobulin antibody (approximately 1.98 μg / mL) conjugated to polyclonal goat anti-human antibody (approximately 49.5 μg / mL) covalently bound to paramagnetic particles in a buffer containing BSA, a protein stabilizer, and a preservative; Next, apply a magnet to separate and attract the contents, then wash the cuvette with reagent water; Dispense 300 μL each of the acid reagent (HCl) and the base reagent (NaOH) to initiate the chemiluminescence reaction; • Reported RLU results.

[0045] Data Analysis The RLUs resulting from the reaction were determined as a function of the predicted concentration of antibody activity in international units per mL (IU / mL) (Table 1 for WHO reference samples, Table 2 for patient serum samples). In antigen-crosslinked immunoassays, the signal (RLU) was directly proportional to the concentration of autoantibodies present in the sample, whereas in competitive assays, the signal was inversely proportional to the concentration of autoantibodies present in the sample.

[0046] [Table 1]

[0047] [Table 2]

[0048] The crosslinking assay showed a significantly higher relative increase in signal compared to the relative decrease in signal observed in the competing assay at low concentrations. A relative increase or decrease in signal at low concentrations directly affects the detection capability of the assay. For example, the limit of detection (LoD), which is the lowest concentration at which the antibody can be detected in 95% of cases, depends on the detection capability of the assay. In the experiments described above, the LoD was calculated to be 1 IU / mL and 45 IU / mL for the crosslinking assay and the competing assay, respectively. These results demonstrate that the disclosed antigen crosslinking immunoassay is more sensitive than the currently used competing assay.

[0049] Optimization of antigen crosslinking immunoassays One potential drawback of one-step immunoassays is the "hook effect," where the detectable signal decreases as the analyte concentration increases, potentially leading to falsely low results. As shown in Figure 3, for example, at lower anti-thyroglobulin antibody concentrations, the antigen crosslinking assay showed a direct relationship between antibody concentration and relative signal (RLU). However, as the antibody concentration increased, the relative signal began to saturate in Figure 3 (approximately 500 UI / mL). At low anti-thyroglobulin antibody concentrations, a limited number of antibodies encounter a large number of solid support / unlabeled thyroglobulin complexes. As the antibody concentration increases, the solid support / unlabeled thyroglobulin complexes become scarce, and therefore only a small fraction of the total antibody binds to the solid support / unlabeled thyroglobulin complexes, making it impossible to fully detect the portion of antibody bound to the solid support / unlabeled thyroglobulin complexes. In the disclosed immunoassay, this "hook effect" could be overcome by optimizing the molar ratio of antibody to labeled thyroglobulin and bound unlabeled thyroglobulin. As shown in Figure 4, when the anti-thyroglobulin antibody sample was diluted (square), the antibody measurement range expanded, and a linear relationship was obtained between the signal and antibody concentration compared to the undiluted sample (diamond shape).

[0050] To analyze the effect of labeling on the relative signals obtained by immunoassay, the amount of acridinium ester added to thyroglobulin was varied to achieve ratios of 5, 10, and 20 molar excess of acridinium ester labeling. As shown in Figure 5, higher amounts of acridinium ester resulted in higher relative signals.

[0051] Similarly, optimizing the ratio of anti-thyroglobulin antibody to solid support / unlabeled thyroglobulin complex can achieve the desired dose-response and expand analytical sensitivity and measurement range.

[0052] Those skilled in the art will understand that numerous modifications and alterations can be made to preferred embodiments of the present invention, and that such modifications and alterations can be made without departing from the spirit of the invention. Accordingly, the appended claims are intended to encompass all such equivalent modifications that fall within the true spirit and scope of the invention.

Claims

1. A method for diagnosing autoimmune thyroid disease in a subject: a) Dilute the biological sample from the subject to form a diluted biological sample, and take the diluted biological sample from the subject: A solid support to which unlabeled thyroglobulin is bound, wherein the unlabeled thyroglobulin is specifically recognized by an anti-thyroglobulin autoantibody from the subject, and the solid support comprises magnetic beads or magnetic particles; and A chemiluminescently labeled thyroglobulin comprising a chemiluminescent label bound to thyroglobulin in a molar excess of at least five times the amount of thyroglobulin, wherein the chemiluminescently labeled thyroglobulin does not bind to a solid support and is specifically recognized by the anti-thyroglobulin autoantibody. It involves incubation together, Here, a solid support / chemiluminescent labeled thyroglobulin complex is formed in the presence of an anti-thyroglobulin autoantibody; b) Using a magnet to separate the solid support / chemiluminescent labeled thyroglobulin complex from chemiluminescent labeled thyroglobulin that is not bound to the anti-thyroglobulin autoantibody; c) detecting the luminescence level of the solid support / chemiluminescent labeled thyroglobulin complex; and d) If a chemiluminescence level of the solid support / chemiluminescent labeled thyroglobulin complex is detected, the subject shall be diagnosed with an autoimmune thyroid disease. The method comprising the above.

2. The method according to claim 1, wherein the magnetic beads or magnetic particles include paramagnetic particles or latex magnetic particles.

3. The method according to claim 1, wherein the unlabeled thyroglobulin is indirectly bound to magnetic beads or magnetic particles.

4. The method according to claim 3, wherein the unlabeled thyroglobulin contains biotin and the magnetic beads or magnetic particles contain streptavidin.

5. The method according to claim 1, wherein the unlabeled thyroglobulin is directly bound to a solid support.

6. The method according to claim 1, wherein the chemiluminescent labeling of the chemiluminescently labeled thyroglobulin comprises an acridinium ester or an analogue thereof.

7. The method according to claim 1, wherein the level of anti-thyroglobulin autoantibody in the biological sample of interest is directly proportional to the level of the solid support / chemiluminescent labeled thyroglobulin complex.

8. The method according to claim 1, wherein the autoimmune thyroid disease is Graves' disease or Hashimoto's thyroiditis.

9. The method according to claim 1, wherein the incubation is carried out without washing or separating an unlabeled solid support / anti-thyroglobulin autoantibody complex or a chemiluminescent labeled thyroglobulin / anti-thyroglobulin autoantibody complex prior to the formation of a solid support / chemiluminescent labeled thyroglobulin complex.

10. The method according to claim 1, wherein the chemiluminescent label is bound to thyroglobulin in a molar excess of at least 10 times the amount of thyroglobulin.

11. The method according to claim 1, wherein the chemiluminescent label is bound to thyroglobulin in a molar excess of at least 20 times relative to thyroglobulin.

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