Detection method and detection kit for thyroglobulin

Abstract

The invention relates to the technical field of biomedicine, in particular to a thyroglobulin detection method and a thyroglobulin detection kit. The detection method comprises the following steps: providing a to-be-detected sample, and pretreating the to-be-detected sample to obtain a pretreated sample; detecting the thyroglobulin in the pretreated sample; wherein the pretreatment comprises the following steps: mixing the sample to be detected with an acidic reagent, and incubating; the acidic reagent comprises organic acid or salt thereof. The invention also provides a corresponding kit. By applying the detection method or the kit provided by the invention, interference of the thyroglobulin antibody TgAb in a sample on a detection result can be avoided, and accurate quantification of thyroglobulin is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method and a kit for detecting thyroglobulin. Background Art

[0002] Thyroglobulin (Tg) is a glycoprotein secreted by thyroid follicular epithelial cells with a molecular weight of 660kD. It is the precursor of thyroid hormone (T3, T4). Almost all Tg exists in the thyroid gland, but a small amount of Tg enters the blood during its synthesis and transportation. Therefore, a certain amount of Tg can be detected in normal human blood.

[0003] In clinical practice, Tg testing is mainly used for long-term monitoring of differentiated thyroid cancer (DTC) patients for disease recurrence and metastasis after total or subtotal thyroidectomy. Since the thyroid gland is the only source of Tg, serum Tg levels will drop to very low or even undetectable levels after complete thyroidectomy. If Tg can still be detected or the Tg level is elevated, it indicates residual or recurrent DTC. In addition, when thyroid diseases occur, serum Tg will also be elevated in many patients, such as goiter, benign thyroid nodules, thyroid adenoma, multinodular goiter, thyrotoxicosis, and thyroiditis in the toxic phase. Serum Tg testing can also be used to evaluate the cause of congenital hypothyroidism in infants. Tg testing can also help identify artificial hyperthyroidism caused by excessive intake of T4.

[0004] Currently, most commercial Tg test kits use a two-site antibody sandwich-based immunoassay, which has the following limitations: 10% of healthy people and 20% of DTC patients have thyroglobulin autoantibodies (TgAb), which can affect the detection of Tg and lead to erroneous low test results. Therefore, it is usually recommended to test TgAb while measuring serum Tg. If TgAb is positive, it indicates that Tg detection may be interfered with. In this case, the test results interfered with by TgAb are difficult to use as a basis for clinical diagnosis.

[0005] Therefore, in order to better meet clinical needs, the art needs to develop Tg detection methods and kits that are not interfered by TgAb. Summary of the invention

[0006] The purpose of the present invention is to provide a detection method and a detection kit for thyroglobulin, which can avoid the interference of TgAb in the sample on the detection result and realize the accurate quantification of thyroglobulin.

[0007] To this end, in a first aspect, the present invention provides a method for detecting thyroglobulin, comprising the following steps:

[0008] Providing a sample to be tested, pre-treating the sample to be tested to obtain a pre-treated sample; detecting thyroglobulin in the pre-treated sample; wherein,

[0009] The pretreatment includes mixing the sample to be tested with an acidic reagent and incubating the mixture; the acidic reagent includes an organic acid or a salt thereof.

[0010] In some embodiments, the organic acid comprises at least one of the following group: sulfonic acid, halogenated sulfonic acid.

[0011] In some embodiments, the organic acid includes at least one of the following group: C1-C6 alkyl sulfonic acid, C1-C6 halogenated alkyl sulfonic acid, C6-C12 aryl sulfonic acid, C6-C12 halogenated aryl sulfonic acid.

[0012] In some embodiments, the halogen substitution is selected from at least one of fluorine substitution, chlorine substitution, and bromine substitution.

[0013] In some embodiments, the organic acid comprises at least one of the following group: methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, trifluoromethanesulfonic acid, trichloromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid.

[0014] In some embodiments, the organic acid comprises a halocarboxylic acid.

[0015] In some embodiments, the halogenated carboxylic acid comprises a C1-C6 halogenated alkyl carboxylic acid.

[0016] In some embodiments, the halogen substitution is selected from at least one of fluorine substitution, chlorine substitution, and bromine substitution.

[0017] In some embodiments, the organic acid comprises at least one of the following group: tribromoacetic acid, trifluoroacetic acid, trichloroacetic acid, trifluoropropionic acid, trifluorobutyric acid, monochloroacetic acid, dichloroacetic acid.

[0018] In some embodiments, the organic acid includes at least one of the following group: C1-C6 monoalkyl phosphate, C1-C6 dialkyl phosphate.

[0019] In some embodiments, the organic acid includes at least one of the following group: monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, diethyl phosphate, mono-n-butyl phosphate, and di-n-butyl phosphate.

[0020] In some embodiments, the pH of the acidic reagent is ≤ 2; and / or,

[0021] In the acidic reagent, the concentration of the organic acid or its salt is 0.1-1M.

[0022] In some embodiments, the acidic reagent further includes a surfactant.

[0023] In some embodiments, the surfactant includes at least one of the following: a nonionic surfactant, a cationic surfactant, an anionic surfactant, and a zwitterionic surfactant.

[0024] In some embodiments, the surfactant comprises at least one of the following: hexadecyltrimethylammonium bromide, sodium lauryl sulfate, polysorbate, CHAPS.

[0025] In some embodiments, a neutralization step is further included after the incubation to make the pH value of the pre-treated sample between 6.0 and 8.5.

[0026] In some embodiments, the neutralization step is performed before the detection, or the neutralization step is performed simultaneously with the detection.

[0027] In some embodiments, thyroglobulin is detected in the pretreatment sample by immunoassay.

[0028] In some embodiments, the immunoassay comprises any one method selected from the group consisting of enzyme-linked immunosorbent assay, western blotting, immunoprecipitation assay, immunodiffusion assay, and fluorescent immunoassay.

[0029] In some embodiments, the ELISA comprises any one method selected from the group consisting of an indirect ELISA, a sandwich ELISA, and a competitive ELISA.

[0030] A second aspect of the present invention provides a method for detecting thyroglobulin, comprising the following steps:

[0031] Providing a sample to be tested, pre-treating the sample to be tested to obtain a pre-treated sample; detecting thyroglobulin in the pre-treated sample; wherein,

[0032] The pretreatment includes mixing the sample to be tested with an acidic reagent and incubating the mixture; the acidic reagent includes an inorganic acid or a salt thereof; the inorganic acid includes at least one of the following groups: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid.

[0033] In some embodiments, the pH of the acidic reagent is ≤ 2; and / or,

[0034] In the acidic reagent, the concentration of the inorganic acid or its salt is 0.1-1M.

[0035] In some embodiments, the acidic reagent further includes a surfactant.

[0036] In some embodiments, the surfactant includes at least one of the following: a nonionic surfactant, a cationic surfactant, an anionic surfactant, and a zwitterionic surfactant.

[0037] In some embodiments, the surfactant comprises at least one of the following: hexadecyltrimethylammonium bromide, sodium lauryl sulfate, polysorbate, CHAPS.

[0038] In some embodiments, a neutralization step is further included after the incubation to make the pH value of the pre-treated sample between 6.0 and 8.5.

[0039] In some embodiments, the neutralization step is performed before the detection, or the neutralization step is performed simultaneously with the detection.

[0040] In some embodiments, thyroglobulin is detected in the pretreatment sample by immunoassay.

[0041] In some embodiments, the immunoassay comprises any one method selected from the group consisting of enzyme-linked immunosorbent assay, western blotting, immunoprecipitation assay, immunodiffusion assay, and fluorescent immunoassay.

[0042] In some embodiments, the ELISA comprises any one method selected from the group consisting of an indirect ELISA, a sandwich ELISA, and a competitive ELISA.

[0043] The third aspect of the present invention provides the use of an acidic reagent in the preparation of a thyroglobulin detection product, wherein the acidic reagent includes at least one of the following groups: an organic acid or a salt thereof, an inorganic acid or a salt thereof; the inorganic acid includes at least one of the following group: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid.

[0044] In a fourth aspect of the present invention, a thyroglobulin detection kit is provided, which comprises a pretreatment reagent and a detection reagent; wherein:

[0045] The pretreatment reagent includes an acidic reagent; the acidic reagent includes at least one of the following groups: an organic acid or a salt thereof, an inorganic acid or a salt thereof; the inorganic acid includes at least one of the following groups: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid;

[0046] The detection reagent is used for detecting thyroglobulin.

[0047] In some embodiments, the organic acid comprises at least one of the following:

[0048] (i) sulfonic acid, halogenated sulfonic acid; the halogenated acid is preferably at least one of fluorine substitution, chlorine substitution and bromine substitution; preferably, C1-C6 alkyl sulfonic acid, C1-C6 halogenated alkyl sulfonic acid, C6-C12 aryl sulfonic acid, C6-C12 halogenated aryl sulfonic acid; more preferably, methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, trifluoromethanesulfonic acid, trichloromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid;

[0049] (ii) halogenated carboxylic acid; the halogenated carboxylic acid is preferably at least one of fluorine substitution, chlorine substitution and bromine substitution; preferably, a C1-C6 halogenated alkyl carboxylic acid; more preferably, tribromoacetic acid, trifluoroacetic acid, trichloroacetic acid, trifluoropropionic acid, trifluorobutyric acid, monochloroacetic acid, dichloroacetic acid;

[0050] (iii) C1-C6 monoalkyl phosphates, C1-C6 dialkyl phosphates; preferably, monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, diethyl phosphate, mono-n-butyl phosphate, di-n-butyl phosphate.

[0051] In some embodiments, the pH of the acidic reagent is ≤ 2; and / or,

[0052] In the acidic reagent, the concentration of the organic acid or its salt and the inorganic acid or its salt is 0.1-1M.

[0053] In some embodiments, the kit further comprises a neutralizing reagent; the neutralizing reagent comprises an alkaline substance or a buffer salt.

[0054] In some embodiments, the neutralizing agent includes the alkaline substance, and the concentration of the alkaline substance is 0.5% to 4% (w / v).

[0055] In some embodiments, the neutralizing agent includes the buffer salt, the concentration of the buffer salt is 300-1000 mM; and / or the pH value of the neutralizing agent is 6.0-9.0.

[0056] In some embodiments, the detection reagent includes a first antibody; the first antibody is an antibody or antigen-binding fragment that specifically binds to thyroglobulin.

[0057] In some embodiments, the detection reagent further comprises a second antibody, which is suitable for forming a sandwich immune complex of the first antibody-thyroglobulin-second antibody.

[0058] In some embodiments, the first antibody and / or the second antibody is coupled to a detection label; the detection label includes at least one selected from the following group: a radioactive label, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, a quantum dot or an enzyme.

[0059] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0061] Figure 1 : Detecting the Tg content in the serum sample, and analyzing the correlation between the detection results of the kit provided in Example 1-1 and the TGMS detection results;

[0062] Figure 2 :The Tg content in serum samples was detected, and the correlation analysis between the test results of the commercially available Elecsys Tg II kit and the TGMS test results was performed;

[0063] Figure 3 :The Tg content in serum samples was detected, and the correlation analysis between the test results of the kit without methanesulfonic acid and the TGMS test results was performed;

[0064] Figure 4 : The TgAb negative samples were pretreated with the R3 reagent in Example 1-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0065] Figure 5 : The TgAb positive samples were pretreated with the R3 reagent in Example 1-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0066] Figure 6 : Detecting the Tg content in the serum sample, and analyzing the correlation between the test results of the kit provided in Example 2-1 and the TGMS test results;

[0067] Figure 7 : The TgAb negative samples were pretreated with the R3 reagent in Example 2-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0068] Figure 8 : The TgAb positive samples were pretreated with the R3 reagent in Example 2-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0069] Fig. 9: Detecting the Tg content in the serum sample, and analyzing the correlation between the test results of the kit provided in Example 3-1 and the TGMS test results;

[0070] Fig.10 : The TgAb negative samples were pretreated with the R3 reagent in Example 3-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0071] Fig.11 : The TgAb positive samples were pretreated with the R3 reagent in Example 3-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0072] Fig.12 : Detecting the Tg content in the serum sample, and analyzing the correlation between the test results of the kit provided in Example 4-1 and the TGMS test results;

[0073] Fig.13 : The TgAb negative samples were pretreated with the R3 reagent in Example 4-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0074] Fig.14 : The TgAb positive samples were pretreated with the R3 reagent in Example 4-1, and the pretreated and untreated samples were subjected to size exclusion chromatography separation results;

[0075] Fig.15 : Detecting the Tg content in the serum sample, and analyzing the correlation between the test results of the kit provided in Example 5-1 and the TGMS test results;

[0076] Fig.16 : Detecting the Tg content in the serum sample, and analyzing the correlation between the test results of the kit provided in Example 6-1 and the TGMS test results;

[0077] Fig.17 : Detecting the Tg content in the serum sample, and analyzing the correlation between the test results of the kit provided in Example 7-1 and the TGMS test results;

[0078] Fig.18 : The Tg content in the serum sample was detected, and the correlation analysis between the detection results of the kit provided in Example 8-1 and the TGMS detection results was performed. DETAILED DESCRIPTION

[0079] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0080] Clinically, thyroglobulin (Tg) can be used for the diagnosis or auxiliary diagnosis of a variety of diseases. However, thyroglobulin autoantibodies (TgAb) may exist in the subjects. When TgAb exists, it will bind to Tg and interfere with the Tg test results, making the test results low. Therefore, it is usually recommended to test TgAb while measuring serum Tg. If TgAb is positive, it indicates that Tg detection may be interfered with. In this case, the test results interfered with by TgAb are difficult to serve as the basis for clinical diagnosis.

[0081] The present invention mainly improves the pretreatment step of Tg detection so that TgAb is dissociated from Tg, thereby avoiding the interference of TgAb and realizing accurate detection of Tg.

[0082] In a specific embodiment of the present invention, a method for detecting thyroglobulin using an organic acid or a salt thereof is provided, which comprises the following steps:

[0083] Providing a sample to be tested, pre-treating the sample to be tested to obtain a pre-treated sample; detecting thyroglobulin in the pre-treated sample; wherein,

[0084] The pretreatment includes mixing the sample to be tested with an acidic reagent and incubating the mixture; the acidic reagent includes an organic acid or a salt thereof.

[0085] In some embodiments, the present invention provides another method for detecting thyroglobulin using inorganic acid, comprising the following steps:

[0086] Providing a sample to be tested, pre-treating the sample to be tested to obtain a pre-treated sample; detecting thyroglobulin in the pre-treated sample; wherein,

[0087] The pretreatment includes mixing the sample to be tested with an acidic reagent and incubating the mixture; the acidic reagent includes an inorganic acid or a salt thereof; the inorganic acid includes at least one of the following groups: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid.

[0088] In some embodiments, for the above-mentioned detection method using an organic acid, and for the above-mentioned detection method using an inorganic acid, the volume ratio of the sample to be tested to the acidic reagent is (1-5):(1-5); for example, it can be about 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:1, 3:2, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, 5:4, etc.

[0089] In some embodiments, for the above detection method using organic acid and for the above detection method using inorganic acid, the sample to be tested includes a body fluid sample, a cell sample or a tissue sample.

[0090] In some embodiments, the sample to be tested includes at least one of the following: blood, serum, plasma, urine, saliva, cerebrospinal fluid, lymph, spinal fluid, amniotic fluid, synovial fluid, tears, saliva, milk, mucosal secretions, exudate, sweat, vitreous fluid, peritoneal fluid, nasopharyngeal swab, oral swab.

[0091] In some embodiments, for the above-mentioned detection method using an organic acid and for the above-mentioned detection method using an inorganic acid, the incubation temperature is 20-37°C, for example, it can be about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, etc.

[0092] In some embodiments, for the above-mentioned detection method using organic acid and for the above-mentioned detection method using inorganic acid, the incubation time is 1 to 20 minutes, for example, it can be about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 15 minutes, 17 minutes, 20 minutes, etc.

[0093] In some embodiments, for the above detection method using organic acid and for the above detection method using inorganic acid, the pH of the acidic reagent is ≤ 2, for example, it can be 1, 1.2, 1.5, 1.8, 2, etc.

[0094] In some embodiments, the present invention provides a thyroglobulin detection kit, comprising a pretreatment reagent and a detection reagent; wherein,

[0095] The pretreatment reagent includes an acidic reagent; the acidic reagent includes at least one of the following groups: an organic acid or a salt thereof, an inorganic acid or a salt thereof; the inorganic acid includes at least one of the following groups: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid;

[0096] The detection reagent is used for detecting thyroglobulin.

[0097] In some embodiments, in the thyroglobulin detection kit, the pH of the acidic reagent is ≤2, for example, it can be 1, 1.2, 1.5, 1.8, 2, etc.

[0098] Organic Acid

[0099] In some embodiments, in the detection of the thyroglobulin, an acidic reagent comprising an organic acid or a salt thereof is used. In some embodiments, an organic acid or a salt thereof is used as an acidic reagent in the thyroglobulin detection kit. The organic acid may be selected from, for example, at least one of sulfonic acid, halogenated sulfonic acid, halogenated carboxylic acid, and alkyl phosphate.

[0100] In some embodiments, the organic acid comprises at least one of the following group: sulfonic acid, halogenated sulfonic acid.

[0101] In some embodiments, the organic acid includes at least one of the following group: C1-C6 alkyl sulfonic acid, C1-C6 halogenated alkyl sulfonic acid, C6-C12 aryl sulfonic acid, C6-C12 halogenated aryl sulfonic acid.

[0102] In some embodiments, the halogen substitution is selected from at least one of fluorine substitution, chlorine substitution, and bromine substitution.

[0103] In some embodiments, the organic acid comprises at least one of the following group: methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, trifluoromethanesulfonic acid, trichloromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid.

[0104] In some embodiments, the organic acid comprises a halocarboxylic acid.

[0105] In some embodiments, the halogenated carboxylic acid comprises a C1-C6 halogenated alkyl carboxylic acid.

[0106] In some embodiments, the halogen substitution is selected from at least one of fluorine substitution, chlorine substitution, and bromine substitution.

[0107] In some embodiments, the organic acid comprises at least one of the following group: tribromoacetic acid, trifluoroacetic acid, trichloroacetic acid, trifluoropropionic acid, trifluorobutyric acid, monochloroacetic acid, dichloroacetic acid.

[0108] In some embodiments, the organic acid includes at least one of the following group: C1-C6 monoalkyl phosphate, C1-C6 dialkyl phosphate.

[0109] In some embodiments, the organic acid includes at least one of the following group: monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, diethyl phosphate, mono-n-butyl phosphate, and di-n-butyl phosphate.

[0110] In some embodiments, in the acidic reagent, the concentration of the organic acid or its salt is 0.1 to 1 M; for example, it can be about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, etc.

[0111] Inorganic acid

[0112] In some embodiments, for the thyroglobulin detection method or the thyroglobulin detection kit, an acidic reagent including an inorganic acid or its salt is used; the inorganic acid can be selected from at least one of phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid.

[0113] In some embodiments, in the acidic reagent, the concentration of the inorganic acid or its salt is 0.1 to 1 M; for example, it can be about 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, etc.

[0114] Surfactants

[0115] In some embodiments, for the thyroglobulin detection method or the thyroglobulin detection kit, the acidic reagent also includes a surfactant.

[0116] As used herein, the term "surfactant" refers to a surface active compound that can form micelles and reduce the surface tension between two liquids. Surfactants are generally organic compounds. In the embodiments of the present invention, surfactants commonly used in the art can be used.

[0117] In some embodiments, the surfactant includes at least one of the following: a nonionic surfactant, a cationic surfactant, an anionic surfactant, and a zwitterionic surfactant.

[0118] In some embodiments, the surfactant comprises at least one of the following: hexadecyltrimethylammonium bromide, sodium lauryl sulfate, polysorbate, CHAPS.

[0119] Neutralization

[0120] In some embodiments, the method for detecting thyroglobulin further includes a neutralization step after the incubation, so that the pH value of the pretreated sample is 6.0-8.5; for example, it can be about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, etc.

[0121] In some embodiments, the neutralization step is performed before the detection, or the neutralization step is performed simultaneously with the detection. For example, the neutralization reagent can be added as an independent reagent to the solution obtained after incubation for neutralization; or the neutralization reagent can be added to the system together with the reagent for detection during the detection step for neutralization.

[0122] In some embodiments, the kit further comprises a neutralizing reagent; the neutralizing reagent comprises an alkaline substance or a buffer salt.

[0123] In some embodiments, the neutralizing agent includes the alkaline substance, and the concentration of the alkaline substance is 0.5% to 4% (w / v), for example, about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, etc.

[0124] In some embodiments, the neutralizing agent includes the buffer salt, the concentration of the buffer salt is 300-1000 mM, for example, it can be about 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, etc.; and / or, the pH value of the neutralizing agent is 6.0-9.0, for example, it can be about 6.0, 6.3, 6.5, 6.7, 6.8, 6.9, 7.0, 7.2, 7.3, 7.5, 7.8, 8.0, 8.5, 9.0, etc.

[0125] Method and reagent for detecting pre-treated samples

[0126] In some embodiments, the thyroglobulin in the pre-treated sample is detected by immunoassay. After the pre-treatment, TgAb is dissociated from thyroglobulin, and the thyroglobulin can be detected by a method commonly used in the art, which is not particularly limited in the present invention.

[0127] In some embodiments, the immunoassay comprises any one method selected from the group consisting of: enzyme-linked immunosorbent assay, immuno-western blot, immunoprecipitation assay, immunodiffusion assay, and fluorescent immunoassay. For example, the enzyme-linked immunosorbent assay comprises any one method selected from the group consisting of: indirect enzyme-linked immunosorbent assay, sandwich enzyme-linked immunosorbent assay, and competitive enzyme-linked immunosorbent assay.

[0128] In some embodiments, the detection reagent in the thyroglobulin detection kit is used to detect thyroglobulin by immunoassay. The detection reagent can be an immunoassay reagent for thyroglobulin commonly used in the art, for example, a related reagent in an existing kit, such as a detection reagent in a thyroglobulin (Tg) quantitative assay kit (CLIA) produced by Shenzhen Mindray Bio-Medical.

[0129] In some embodiments, the detection reagent includes a first antibody; the first antibody is an antibody or antigen-binding fragment that specifically binds to thyroglobulin. In some embodiments, the detection reagent also includes a second antibody, which is suitable for forming a sandwich immune complex of the first antibody-thyroglobulin-second antibody; thus, it is suitable for detecting thyroglobulin by a double antibody immune sandwich method.

[0130] In some embodiments, the first antibody and / or the second antibody is coupled to a detection label; the detection label includes at least one selected from the following group: a radioactive label, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, a quantum dot or an enzyme.

[0131] The following will list the embodiments of the present invention, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0132] Example 1-1

[0133] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-1, which includes reagents R1, R2 and R3.

[0134] Among them, R1 and R2 use reagents in commercially available kits, specifically, a thyroglobulin (Tg) quantitative assay kit (CLIA) (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), which includes:

[0135] Magnetic beads (Ra): Superparamagnetic particles coated with anti-thyroglobulin antibodies, suspended in buffer. Hereinafter referred to as R1.

[0136] Enzyme marker (Rb): anti-thyroglobulin antibody-alkaline phosphatase marker diluted in buffer, hereinafter referred to as R2.

[0137] R3 is obtained by preparation, as follows:

[0138] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and you will get the R3 reagent, which is refrigerated at 2-8°C for use. The composition of the R3 reagent is: 200mM methanesulfonic acid, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0139] Example 1-2

[0140] The thyroglobulin content in serum samples obtained clinically was measured.

[0141] 1. Detection method

[0142] Fresh serum samples obtained from the clinic were collected as samples to be tested, including 28 TgAb negative and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0143] Experimental group: Tg kit-1 provided in Example 1-1 was used. The sample to be tested was mixed evenly with R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 5 minutes; then a certain volume of neutralizing reagent (300 mM Tris buffer) was added to adjust the pH to 6.8, and a pre-treated sample was prepared.

[0144] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0145] Control group 1: The above 56 samples were tested using the Elecsys Tg II kit on the Roche Cobas e601 fully automatic chemiluminescence analyzer.

[0146] Control group 2: The test was performed in the same manner as the experimental group, except that the R3 reagent did not contain methanesulfonic acid.

[0147] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0148] 2. Experimental Results

[0149] With the TGMS test value as the X-axis and the test results of the experimental group, control group 1, and control group 2 as the Y-axis, a scatter plot was established to analyze the correlation. Figure 1 , Figure 2 and Figure 3 shown.

[0150] See also Figure 1 The K value of the methodological comparison between Tg kit-1 and TGMS in TgAb negative samples was 1.0748, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 1.086, and the correlation coefficient R 2 is 0.9806. Figure 3 When the R3 reagent does not contain methanesulfonic acid, the detection of TgAb positive samples is significantly interfered. This shows that the kit provided in Example 1-1 avoids the interference of TgAb in the sample by applying organic acid for pretreatment, and has a higher accuracy in the detection of Tg.

[0151] See also Figure 2 The K value of the methodological comparison between the commercially available Tg kit (Elecsys Tg II kit) and TGMS in TgAb negative samples was 1.0503, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 0.4422, and the correlation coefficient R 2 The value was 0.7850. This indicates that in TgAb-positive samples, the commercially available kits were significantly interfered by TgAb, and the accuracy of the test results was poor.

[0152] Example 1-3 Size Exclusion Chromatography Experiment

[0153] 1. Sample preparation:

[0154] Take a TgAb positive and a TgAb negative clinical serum sample, each sample is divided into two parts, and prepared into an untreated sample and a pre-treated sample respectively. In this embodiment, the R3 reagent in Example 1-1 is used as the pre-treatment solution, and 300mMTris buffer is used as the neutralization solution.

[0155] Untreated sample: Take 200 μL of pretreatment solution and 200 μL of neutralization solution, mix well, and add 100 μL of serum sample to obtain an untreated sample.

[0156] Pretreatment sample: Take 100 μL of serum sample, add 200 μL of pretreatment solution and mix well, incubate at 37°C for 6 min, then add 200 μL of neutralization solution and mix well to obtain a pretreatment sample.

[0157] 500 μL of each of the untreated sample and pretreated sample were separated by separation column, and UV absorption was measured. At the same time, the separation solution was recovered, and the Tg content in each recovered solution was measured using the Elecsys Tg II kit.

[0158] Size exclusion chromatography separation conditions:

[0159] Separation column: superose 6increase 10 / 300GL

[0160] Flow rate: 0.5mL / min

[0161] Separation buffer: PBS buffer

[0162] Recovery range: 5~20mL (0.5mL / portion)

[0163] Experimental results:

[0164] Results for TgAb-negative samples are Figure 4 The results of TgAb positive samples are shown in Figure 5 As shown. It is known that Tg is about 660kDa, and the complex of Tg and TgAb is ≥810kDa. Further analysis is as follows:

[0165] See also Figure 4 For TgAb-negative samples, Tg was detected in the elution fraction at 660 kDa in both untreated and pretreated samples.

[0166] See also Figure 5 For TgAb-positive samples, Tg can be detected in the elution fraction above 660 kDa for untreated samples, and Tg can be detected in the elution fraction at 660 kDa for pretreated samples. This indicates that for TgAb-positive samples, Tg binds to TgAb to form a complex, and after pretreatment, Tg is released from the complex.

[0167] Example 1-4 Addition recovery experiment

[0168] Sample preparation: Prepare some clinical serum samples with negative TgAb and positive TgAb as original samples; prepare some serum samples with negative TgAb and positive Tg concentration greater than 100ng / mL as spiked samples. Mix each solid sample with a spiked sample at a volume ratio of 8:2, equilibrate at room temperature for 24h, and use as spiked samples.

[0169] Determination method: Determine the Tg content in the original sample, spiked sample, and spiked sample according to the methods of the experimental group and control group 1 in Example 1-2, respectively. Calculate the spiked recovery rates of the two kits according to the following formula.

[0170] Calculation method:

[0171] Theoretical Tg value of the sample after addition = 0.8 × original sample Tg value + 0.2 × added sample Tg value

[0172] Addition recovery rate = (actual Tg value of sample after addition / theoretical Tg value of sample after addition) × 100%

[0173] Experimental results: As shown in Table 1 and Table 2. The Tg kit provided in Example 1-1 has an additive recovery rate of about 100% in both TgAb-negative and TgAb-positive samples, while the commercially available Elecsys Tg II kit has an additive recovery rate of about 100% in TgAb-negative samples and an additive recovery rate of <100% in TgAb-positive samples, with different samples showing inconsistent performance. This indicates that the Tg kit provided in Example 1-1 has better anti-interference ability in TgAb-positive samples than the commercially available Elecsys Tg II kit.

[0174] Table 1 Results of the addition and recovery experiment of the Tg kit provided in Example 1-1

[0175]

[0176] Table 2 Results of the spike recovery experiment of the commercially available Elecsys Tg II kit

[0177]

[0178]

[0179] Example 1-5 Sensitivity

[0180] The sensitivity of the kit provided in Example 1-1 was evaluated.

[0181] Sample preparation: According to the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", 5 blank limit (LoB) samples, 5 detection limit (LoD) samples and 5 quantification limit (LoQ) samples were prepared. The sample matrix was selected from multiple mixed serum samples, and the antigen used was human Tg antigen.

[0182] Determination method: On the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, using the Tg kit provided in Example 1-1, in accordance with the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", using 1 instrument system, 2 reagent batches, 3 test days, 5 blank limit / detection limit / quantification limit samples, and 4 repeated measurements for each sample, the number of test results for each reagent is ≥60, and the LOB, LOD, and LOQ are calculated.

[0183] Experimental results: The Tg kit provided in Example 1-1 has LOB=0.009ng / mL, LOD=0.015ng / mL, and LOQ=0.096ng / mL. That is, the LOQ of the Tg kit provided in Example 1-1 is less than 0.2ng / mL (the lowest level determined by clinical medicine is 0.2ng / ml), which meets the clinical application requirements of a high-sensitivity Tg kit.

[0184] Example 2-1

[0185] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-2, which includes reagents R1, R2 and R3.

[0186] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0187] R3 is obtained by preparation, as follows:

[0188] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and obtain the R3 reagent, which is refrigerated at 2-8°C for use. The composition of the R3 reagent is: 200mM methanesulfonic acid, 0.5% CHAPS, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0189] Example 2-2

[0190] The thyroglobulin content in serum samples obtained clinically was measured.

[0191] 1. Detection method

[0192] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0193] Experimental group: Tg kit-2 provided in Example 2-1 was used. The sample to be tested was mixed evenly with the R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 6 minutes; a certain volume of neutralizing reagent (0.5% sodium hydroxide) was then added to adjust the pH to 6.8, thus preparing a pre-treated sample.

[0194] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0195] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0196] 2. Experimental Results

[0197] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Figure 6 shown.

[0198] according to Figure 6 The results showed that the K value of the methodological comparison between Tg Kit-2 and TGMS in TgAb negative samples was 1.0734, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 1.0769, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 2-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0199] Example 2-3 Size Exclusion Chromatography Experiment

[0200] The size exclusion chromatography experiment was performed following the same procedure as in Example 1-3 except for the following differences:

[0201] In addition, one TgAb-positive and one TgAb-negative clinical serum sample were obtained; the R3 reagent in Example 2-1 was used as the pretreatment solution, and 300 mM Tris buffer was used as the neutralization solution.

[0202] Experimental results:

[0203] Results for TgAb-negative samples are Figure 7 As shown, for TgAb-negative samples, Tg can be detected in the elution fraction at 660 kDa in both untreated and pretreated samples.

[0204] Results for TgAb-positive samples Figure 8 As shown, Tg can be detected in the elution fraction above 660 kDa for the untreated sample, and Tg can be detected in the elution fraction at 660 kDa for the pretreated sample. This indicates that for TgAb-positive samples, Tg binds to TgAb to form a complex, and after pretreatment, Tg is released from the complex.

[0205] Example 2-4 Addition recovery experiment

[0206] Sample preparation: Prepare some clinical serum samples with negative TgAb and positive TgAb as original samples; prepare some serum samples with negative TgAb and positive Tg concentration greater than 100ng / mL as spiked samples. Mix each solid sample with a spiked sample at a volume ratio of 8:2, equilibrate at room temperature for 24h, and use as spiked samples.

[0207] Determination method: According to the method of the experimental group in Example 2-2, the Tg content in the original sample, the added sample, and the added sample was determined, and the added recovery rate was calculated (the calculation method is the same as that of Example 1-4).

[0208] Experimental results: as shown in Table 3. The recovery rates of the Tg kit provided in Example 2-1 in both TgAb negative and TgAb positive samples were about 100%, and the anti-interference ability of the kit when used for TgAb positive samples was significantly better than that of the commercially available Elecsys Tg II kit.

[0209] Table 3 Results of the addition and recovery experiment of the Tg kit provided in Example 2-1

[0210]

[0211] Example 2-5 Sensitivity

[0212] The sensitivity of the kit provided in Example 2-1 was evaluated.

[0213] Sample preparation: According to the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", 5 blank limit (LoB) samples, 5 detection limit (LoD) samples and 5 quantification limit (LoQ) samples were prepared. The sample matrix was selected from multiple mixed serum samples, and the antigen used was human Tg antigen.

[0214] Determination method: On the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, using the Tg kit provided in Example 2-1, in accordance with the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", using 1 instrument system, 2 reagent batches, 3 test days, 5 blank limit / detection limit / quantification limit samples, and 4 repeated measurements for each sample, the number of test results for each reagent is ≥60, and the LOB, LOD, and LOQ are calculated.

[0215] Experimental results: The Tg kit provided in Example 2-1 has LOB=0.010ng / mL, LOD=0.015ng / mL, and LOQ=0.092ng / mL. That is, the LOQ of the Tg kit provided in Example 2-1 is less than 0.2ng / mL (the lowest level determined by clinical medicine is 0.2ng / ml), which meets the clinical application requirements of a high-sensitivity Tg kit.

[0216] Example 3-1

[0217] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-3, which includes reagents R1, R2 and R3.

[0218] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0219] R3 is obtained by preparation, as follows:

[0220] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and you will get the R3 reagent, which is refrigerated at 2-8℃ for later use. The composition of the R3 reagent is: 200mM phosphoric acid, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0221] Example 3-2

[0222] The thyroglobulin content in serum samples obtained clinically was measured.

[0223] 1. Detection method

[0224] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0225] Experimental group: Tg kit-3 provided in Example 3-1 was used. The sample to be tested was mixed evenly with the R3 reagent in a volume ratio of 1:1, and incubated at 37°C for 3 minutes; a certain volume of neutralizing reagent (4% (w / v) sodium carbonate) was added to adjust the pH to 7.0, and a pre-treated sample was prepared.

[0226] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0227] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0228] 2. Experimental Results

[0229] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Fig. 9 shown.

[0230] according to Fig. 9 The results showed that the K value of the methodological comparison between Tg Kit-3 and TGMS in TgAb negative samples was 1.047, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 1.0656, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 3-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0231] Example 3-3 Size Exclusion Chromatography Experiment

[0232] The size exclusion chromatography experiment was performed following the same procedure as in Example 1-3 except for the following differences:

[0233] In addition, one TgAb-positive and one TgAb-negative clinical serum sample were obtained; the R3 reagent in Example 3-1 was used as the pretreatment solution, and 300 mM Tris buffer was used as the neutralization solution.

[0234] Experimental results:

[0235] Results for TgAb-negative samples are Fig.10 As shown, for TgAb-negative samples, Tg can be detected in the elution fraction at 660 kDa in both untreated and pretreated samples.

[0236] Results for TgAb-positive samples Fig.11 As shown, Tg can be detected in the elution fraction above 660 kDa for the untreated sample, and Tg can be detected in the elution fraction at 660 kDa for the pretreated sample. This indicates that for TgAb-positive samples, Tg binds to TgAb to form a complex, and after pretreatment, Tg is released from the complex.

[0237] Example 3-4 Addition recovery experiment

[0238] Sample preparation: Prepare some clinical serum samples with negative TgAb and positive TgAb as original samples; prepare some serum samples with negative TgAb and positive Tg concentration greater than 100ng / mL as spiked samples. Mix each solid sample with a spiked sample at a volume ratio of 8:2, equilibrate at room temperature for 24h, and use as spiked samples.

[0239] Determination method: According to the method of the experimental group in Example 3-2, the Tg content in the original sample, the added sample, and the added sample was determined, and the added recovery rate was calculated (the calculation method is the same as that of Example 1-4).

[0240] Experimental results: as shown in Table 4. The recovery rates of the Tg kit provided in Example 3-1 in both TgAb negative and TgAb positive samples were about 100%, and the anti-interference ability of the kit when used for TgAb positive samples was significantly better than that of the commercially available Elecsys Tg II kit.

[0241] Table 4 Results of the addition and recovery experiment of the Tg kit provided in Example 3-1

[0242]

[0243] Example 3-5 Sensitivity

[0244] The sensitivity of the kit provided in Example 3-1 was evaluated.

[0245] Sample preparation: According to the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", 5 blank limit (LoB) samples, 5 detection limit (LoD) samples and 5 quantification limit (LoQ) samples were prepared. The sample matrix was selected from multiple mixed serum samples, and the antigen used was human Tg antigen.

[0246] Determination method: On the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, using the Tg kit provided in Example 3-1, in accordance with the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", using 1 instrument system, 2 reagent batches, 3 test days, 5 blank limit / detection limit / quantification limit samples, and 4 repeated measurements for each sample, the number of test results for each reagent is ≥60, and the LOB, LOD, and LOQ are calculated.

[0247] Experimental results: The Tg kit provided in Example 3-1 has LOB=0.015ng / mL, LOD=0.017ng / mL, and LOQ=0.091ng / mL. That is, the LOQ of the Tg kit provided in Example 3-1 is less than 0.2ng / mL (the lowest level determined by clinical medicine is 0.2ng / ml), which meets the clinical application requirements of a high-sensitivity Tg kit.

[0248] Example 4-1

[0249] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-4, which includes reagents R1, R2 and R3.

[0250] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0251] R3 is obtained by preparation, as follows:

[0252] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and obtain the R3 reagent, which is refrigerated at 2-8°C for use. The composition of the R3 reagent is: 200mM phosphoric acid, 0.3% CTAB, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0253] Example 4-2

[0254] The thyroglobulin content in serum samples obtained clinically was measured.

[0255] 1. Detection method

[0256] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0257] Experimental group: Tg kit-4 provided in Example 4-1 was used. The sample to be tested was mixed evenly with R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 5 minutes; then a certain volume of neutralizing reagent (300mM Tris buffer) was added to adjust the pH to 7.0, and a pre-treated sample was prepared.

[0258] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0259] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0260] 2. Experimental Results

[0261] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Fig.12 shown.

[0262] according to Fig.12 The results showed that the K value of the methodological comparison between Tg Kit-4 and TGMS in TgAb negative samples was 1.0678, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 1.0808, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 4-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0263] Example 4-3 Size Exclusion Chromatography Experiment

[0264] The size exclusion chromatography experiment was performed following the same procedure as in Example 1-3 except for the following differences:

[0265] In addition, one TgAb-positive and one TgAb-negative clinical serum sample were obtained; the R3 reagent in Example 4-1 was used as the pretreatment solution, and 300 mM Tris buffer was used as the neutralization solution.

[0266] Experimental results:

[0267] Results for TgAb-negative samples are Fig.13 As shown, for TgAb-negative samples, Tg can be detected in the elution fraction at 660 kDa in both untreated and pretreated samples.

[0268] Results for TgAb-positive samples are as follows Fig.14 As shown, Tg can be detected in the elution fraction above 660 kDa for the untreated sample, and Tg can be detected in the elution fraction at 660 kDa for the pretreated sample. This indicates that for TgAb-positive samples, Tg binds to TgAb to form a complex, and after pretreatment, Tg is released from the complex.

[0269] Example 4-4 Addition recovery experiment

[0270] Sample preparation: Prepare some clinical serum samples with negative TgAb and positive TgAb as original samples; prepare some serum samples with negative TgAb and positive Tg concentration greater than 100ng / mL as spiked samples. Mix each solid sample with a spiked sample at a volume ratio of 8:2, equilibrate at room temperature for 24h, and use as spiked samples.

[0271] Determination method: According to the method of the experimental group in Example 4-2, the Tg content in the original sample, the added sample, and the added sample was determined, and the added recovery rate was calculated (the calculation method is the same as that of Example 1-4).

[0272] Experimental results: as shown in Table 5. The recovery rates of the Tg kit provided in Example 4-1 in both TgAb negative and TgAb positive samples were about 100%, and the anti-interference ability of the kit when used for TgAb positive samples was significantly better than that of the commercially available Elecsys Tg II kit.

[0273] Table 5 Results of the addition and recovery experiment of the Tg kit provided in Example 4-1

[0274]

[0275]

[0276] Example 4-5 Sensitivity

[0277] The sensitivity of the kit provided in Example 4-1 was evaluated.

[0278] Sample preparation: According to the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", 5 blank limit (LoB) samples, 5 detection limit (LoD) samples and 5 quantification limit (LoQ) samples were prepared. The sample matrix was selected from multiple mixed serum samples, and the antigen used was human Tg antigen.

[0279] Determination method: On the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, using the Tg kit provided in Example 4-1, in accordance with the "Pharmaceutical Industry Standard of the People's Republic of China YY / T 1789.3-2022", using 1 instrument system, 2 reagent batches, 3 test days, 5 blank limit / detection limit / quantification limit samples, and 4 repeated measurements for each sample, the number of test results for each reagent is ≥60, and the LOB, LOD, and LOQ are calculated.

[0280] Experimental results: The Tg kit provided in Example 4-1 has LOB=0.008ng / mL, LOD=0.011ng / mL, and LOQ=0.093ng / mL. That is, the LOQ of the Tg kit provided in Example 4-1 is less than 0.2ng / mL (the lowest level determined by clinical medicine is 0.2ng / ml), which meets the clinical application requirements of a high-sensitivity Tg kit.

[0281] Example 5-1

[0282] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-5, which includes reagents R1, R2 and R3.

[0283] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0284] R3 is obtained by preparation, as follows:

[0285] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and you will get the R3 reagent, which is refrigerated at 2-8°C for use. The composition of the R3 reagent is: 250mM trichloromethanesulfonic acid, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0286] Example 5-2

[0287] The thyroglobulin content in serum samples obtained clinically was measured.

[0288] 1. Detection method

[0289] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0290] Experimental group: Tg kit-5 provided in Example 5-1 was used. The sample to be tested was mixed evenly with R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 5 minutes; then a certain volume of neutralizing reagent (300mM Tris buffer) was added to adjust the pH to 7.0, and a pre-treated sample was prepared.

[0291] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0292] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0293] 2. Experimental Results

[0294] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Fig.15 shown.

[0295] according to Fig.15 The results showed that the K value of the methodological comparison between Tg Kit-5 and TGMS in TgAb negative samples was 1.006, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 0.9822, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 5-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0296] Example 6-1

[0297] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-6, which includes reagents R1, R2 and R3.

[0298] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0299] R3 is obtained by preparation, as follows:

[0300] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and you will get the R3 reagent, which is refrigerated at 2-8℃ for later use. The composition of the R3 reagent is: 250mM trichloroacetic acid, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0301] Example 6-2

[0302] The thyroglobulin content in serum samples obtained clinically was measured.

[0303] 1. Detection method

[0304] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0305] Experimental group: Tg kit-6 provided in Example 6-1 was used. The sample to be tested was mixed evenly with R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 5 minutes; then a certain volume of neutralizing reagent (300mM Tris buffer) was added to adjust the pH to 7.0, and a pre-treated sample was prepared.

[0306] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0307] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0308] 2. Experimental Results

[0309] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Fig.16 shown.

[0310] according to Fig.16 The results showed that the K value of the methodological comparison between Tg Kit-6 and TGMS in TgAb negative samples was 0.9955, and the correlation coefficient R 2The K value of the method comparison in TgAb positive samples was 0.978, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 6-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0311] Example 7-1

[0312] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-7, which includes reagents R1, R2 and R3.

[0313] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0314] R3 is obtained by preparation, as follows:

[0315] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and you will get the R3 reagent, which is refrigerated at 2-8℃ for later use. The composition of the R3 reagent is: 300mM monomethyl phosphate, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0316] Example 7-2

[0317] The thyroglobulin content in serum samples obtained clinically was measured.

[0318] 1. Detection method

[0319] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0320] Experimental group: Tg kit-7 provided in Example 7-1 was used. The sample to be tested was mixed evenly with R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 5 minutes; then a certain volume of neutralizing reagent (300mM Tris buffer) was added to adjust the pH to 7.0, and a pre-treated sample was prepared.

[0321] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0322] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0323] 2. Experimental Results

[0324] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Fig.17 shown.

[0325] according to Fig.17 The results showed that the K value of the methodological comparison between Tg Kit-7 and TGMS in TgAb negative samples was 0.9805, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 0.9962, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 7-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0326] Example 8-1

[0327] This embodiment provides a thyroglobulin quantitative detection kit, referred to as Tg kit-8, which includes reagents R1, R2 and R3.

[0328] Wherein, R1 and R2 are the same as R1 and R2 in Example 1-1.

[0329] R3 is obtained by preparation, as follows:

[0330] Add pure water to the liquid preparation container, place it on a magnetic stirrer to stir, and then add all the materials in sequence according to the R3 reagent formula. Each time, add the next material after the previous material is fully dissolved. After all the materials are added, continue to stir until they are fully dissolved, and finally adjust the volume to the target volume, filter with a 0.22μm filter membrane, and you will get the R3 reagent, which is refrigerated at 2-8℃ for later use. The composition of the R3 reagent is: 250mM hydrobromic acid, 16mM citric acid, 0.7M urea, 1.6% maltose, pH 1.5.

[0331] Example 8-2

[0332] The thyroglobulin content in serum samples obtained clinically was measured.

[0333] 1. Detection method

[0334] The samples to be tested are the same as those in Example 1-2, namely, fresh serum samples from the clinic, including 28 TgAb negative samples and 28 TgAb positive samples. The tests were performed according to the following group methods.

[0335] Experimental group: Tg kit-8 provided in Example 8-1 was used. The sample to be tested was mixed evenly with R3 reagent at a volume ratio of 1:1, and incubated at 37°C for 5 minutes; then a certain volume of neutralizing reagent (300mM Tris buffer) was added to adjust the pH to 7.0, and a pre-treated sample was prepared.

[0336] Then, the R2 reagent and the R3 reagent were used to detect the Tg concentration in the sample to be treated by double antibody sandwich enzyme-linked immunosorbent assay. The specific operation steps include: loading the R2 reagent and the R3 reagent on the Mindray CL-6000i fully automatic chemiluminescence immunoassay analyzer, detecting the Tg concentration in the pre-treated sample, and calculating the Tg concentration in the sample to be tested.

[0337] Mass spectrometry group: Tg mass spectrometry (Thyroglobulin Mass Spectrometry, TGMS) was performed on the above 56 samples.

[0338] 2. Experimental Results

[0339] With the TGMS test value as the X-axis and the test results of the experimental group as the Y-axis, a scatter plot was established to analyze the correlation. Fig.18 shown.

[0340] according to Fig.18 The results showed that the K value of the methodological comparison between Tg Kit-8 and TGMS in TgAb negative samples was 1.0058, and the correlation coefficient R 2 The K value of the method comparison in TgAb positive samples was 0.9946, and the correlation coefficient R 2 This indicates that by using the kit provided in Example 8-1, the interference of TgAb in the sample is avoided, and the detection of Tg has a higher accuracy.

[0341] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for detecting thyroglobulin, characterized in that: The following steps are involved: Providing a sample to be tested, and pre-treating the sample to be tested to obtain a pre-treated sample; The thyroglobulin in the pre-treated sample is detected; wherein, The pretreatment includes mixing the sample to be tested with an acidic reagent and incubating the mixture; The acidic agent includes an organic acid or a salt thereof.

2. The method for detecting thyroglobulin according to claim 1, characterized in that: The organic acid comprises at least one of the following groups: sulfonic acid, halogenated sulfonic acid; Preferably, the organic acid comprises at least one of the following groups: C1-C6 alkyl sulfonic acid, C1-C6 halogenated alkyl sulfonic acid, C6-C12 aryl sulfonic acid, C6-C12 halogenated aryl sulfonic acid; Preferably, the halogenated group is selected from at least one of fluorine substitution, chlorine substitution and bromine substitution; Preferably, the organic acid comprises at least one of the following group: methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, trifluoromethanesulfonic acid, trichloromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid.

3. The method for detecting thyroglobulin according to claim 1, characterized in that: The organic acid includes a halogenated carboxylic acid; Preferably, the halogenated carboxylic acid comprises a C1-C6 halogenated alkyl carboxylic acid; Preferably, the halogenated group is selected from at least one of fluorine substitution, chlorine substitution and bromine substitution; Preferably, the organic acid comprises at least one of the following group: tribromoacetic acid, trifluoroacetic acid, trichloroacetic acid, trifluoropropionic acid, trifluorobutyric acid, monochloroacetic acid, dichloroacetic acid.

4. The method for detecting thyroglobulin according to claim 1, characterized in that: The organic acid comprises at least one of the following groups: C1-C6 monoalkyl phosphate, C1-C6 dialkyl phosphate; Preferably, the organic acid comprises at least one of the following group: monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, diethyl phosphate, mono-n-butyl phosphate, and di-n-butyl phosphate.

5. The method for detecting thyroglobulin according to any one of claims 1 to 4, characterized in that: The pH of the acidic reagent is ≤ 2; and / or, In the acidic reagent, the concentration of the organic acid or its salt is 0.1-1M.

6. The method for detecting thyroglobulin according to any one of claims 1 to 4, characterized in that: The acidic reagent also includes a surfactant; Preferably, the surfactant comprises at least one of the following: a nonionic surfactant, a cationic surfactant, an anionic surfactant, and a zwitterionic surfactant; Preferably, the surfactant comprises at least one of the following: hexadecyltrimethylammonium bromide, sodium lauryl sulfate, polysorbate, CHAPS.

7. The method for detecting thyroglobulin according to any one of claims 1 to 4, characterized in that: A neutralization step is also included after the incubation to make the pH value of the pretreated sample be 6.0 to 8.5; Preferably, the neutralization step is performed before the detection, or the neutralization step is performed simultaneously with the detection.

8. The method for detecting thyroglobulin according to any one of claims 1 to 4, characterized in that: detecting thyroglobulin in the pre-treated sample by immunoassay; Preferably, the immunoassay comprises any one method selected from the group consisting of enzyme-linked immunosorbent assay, immunowestern blot, immunoprecipitation assay, immunodiffusion assay and fluorescent immunoassay; Preferably, the ELISA comprises any one method selected from the group consisting of indirect ELISA, sandwich ELISA, and competitive ELISA.

9. A method for detecting thyroglobulin, characterized in that: The following steps are involved: Providing a sample to be tested, and pre-treating the sample to be tested to obtain a pre-treated sample; The thyroglobulin in the pre-treated sample is detected; wherein, The pretreatment includes mixing the sample to be tested with an acidic reagent and incubating the mixture; the acidic reagent includes an inorganic acid or a salt thereof; The inorganic acid comprises at least one of the following group: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid.

10. The method for detecting thyroglobulin according to claim 9, characterized in that: The pH of the acidic reagent is ≤ 2; and / or, In the acidic reagent, the concentration of the inorganic acid or its salt is 0.1-1M.

11. The method for detecting thyroglobulin according to any one of claims 9 to 10, characterized in that: The acidic reagent also includes a surfactant; Preferably, the surfactant comprises at least one of the following: a nonionic surfactant, a cationic surfactant, an anionic surfactant, and a zwitterionic surfactant; Preferably, the surfactant comprises at least one of the following: hexadecyltrimethylammonium bromide, sodium lauryl sulfate, polysorbate, CHAPS.

12. The method for detecting thyroglobulin according to any one of claims 9 to 10, characterized in that: A neutralization step is also included after the incubation to make the pH value of the pretreated sample be 6.0 to 8.5; Preferably, the neutralization step is performed before the detection, or the neutralization step is performed simultaneously with the detection.

13. The method for detecting thyroglobulin according to any one of claims 9 to 10, characterized in that: detecting thyroglobulin in the pre-treated sample by immunoassay; Preferably, the immunoassay comprises any one method selected from the group consisting of enzyme-linked immunosorbent assay, immunowestern blot, immunoprecipitation assay, immunodiffusion assay and fluorescent immunoassay; Preferably, the ELISA comprises any one method selected from the group consisting of indirect ELISA, sandwich ELISA, and competitive ELISA.

14. Use of an acidic reagent in the preparation of a thyroglobulin detection product, wherein: The acidic reagent comprises at least one of the following groups: an organic acid or a salt thereof, an inorganic acid or a salt thereof; The inorganic acid comprises at least one of the following group: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid.

15. A thyroglobulin detection kit, characterized in that: It includes pre-treatment reagents and detection reagents; among which, The pretreatment reagent includes an acidic reagent; the acidic reagent includes at least one of the following groups: an organic acid or a salt thereof, an inorganic acid or a salt thereof; the inorganic acid includes at least one of the following groups: phosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid, perbromic acid, periodic acid, and nitric acid; The detection reagent is used for detecting thyroglobulin.

16. The thyroglobulin detection kit according to claim 15, characterized in that: The organic acid comprises at least one of the following: (i) sulfonic acid, halogenated sulfonic acid; the halogenated acid is preferably at least one of fluorine substitution, chlorine substitution and bromine substitution; preferably, C1-C6 alkyl sulfonic acid, C1-C6 halogenated alkyl sulfonic acid, C6-C12 aryl sulfonic acid, C6-C12 halogenated aryl sulfonic acid; more preferably, methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, trifluoromethanesulfonic acid, trichloromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid; (ii) halogenated carboxylic acid; the halogenated carboxylic acid is preferably at least one of fluorine substitution, chlorine substitution and bromine substitution; preferably, a C1-C6 halogenated alkyl carboxylic acid; more preferably, tribromoacetic acid, trifluoroacetic acid, trichloroacetic acid, trifluoropropionic acid, trifluorobutyric acid, monochloroacetic acid, dichloroacetic acid; (iii) C1-C6 monoalkyl phosphates, C1-C6 dialkyl phosphates; preferably, monomethyl phosphate, dimethyl phosphate, monoethyl phosphate, diethyl phosphate, mono-n-butyl phosphate, di-n-butyl phosphate.

17. The thyroglobulin detection kit according to claim 15 or 16, characterized in that: The pH of the acidic reagent is ≤ 2; and / or, In the acidic reagent, the concentration of the organic acid or its salt and the inorganic acid or its salt is 0.1-1M.

18. The thyroglobulin detection kit according to claim 15 or 16, characterized in that: The kit further comprises a neutralizing agent; the neutralizing agent comprises an alkaline substance or a buffer salt; Preferably, the neutralizing agent comprises the alkaline substance, and the concentration of the alkaline substance is 0.5% to 4% (w / v); Preferably, the neutralizing agent comprises the buffer salt, the concentration of the buffer salt is 300-1000 mM; and / or the pH value of the neutralizing agent is 6.0-9.

0.

19. The thyroglobulin detection kit according to claim 15 or 16, characterized in that: The detection reagent comprises a first antibody; the first antibody is an antibody or antigen-binding fragment that specifically binds to thyroglobulin; Preferably, the detection reagent further comprises a second antibody, which is suitable for forming a sandwich immune complex of the first antibody-thyroglobulin-second antibody; Preferably, the first antibody and / or the second antibody is coupled with a detection label; the detection label comprises at least one selected from the following group: a radioactive label, a fluorescent label, a chemiluminescent label, biotin, colloidal gold, an electrochemiluminescent label, a quantum dot or an enzyme.

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