Thyroid hormone detection kit and detection method

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a thyroid hormone detection kit and a thyroid hormone detection method. Wherein the thyroid hormone comprises at least one of triiodothyronine and tetraiodothyronine; the thyroid hormone detection kit comprises (i) a cyclodextrin reserve reagent; ii) an antibody capture reagent; iii) an antibody detection reagent; and, iv) a signalling agent. According to the kit and the detection method, the T3 and T4 detection signal intensity can be improved, the detection limit is reduced, and the accuracy and reliability of low-concentration sample detection are improved.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic technology, and in particular to thyroid hormone detection kits and detection methods. Background Technology

[0002] Thyroid hormones triiodothyronine (T3) and tetraiodothyronine (T4) are key endocrine regulatory molecules in the human body, and their serum concentration detection is of great significance for the diagnosis of thyroid diseases such as hyperthyroidism and hypothyroidism. Currently, in the field of immunoassay, large molecules are mostly detected using the double-antibody sandwich method. This method has advantages such as high specificity and simple operation. However, T3 and T4 have molecular weights of approximately 651 Da and 777 Da, respectively. As small molecules, they typically have only 1-2 effective epitopes. Due to the limited number of their own antigenic epitopes and their compact spatial structure, the traditional sandwich method faces significant technical bottlenecks: the distance between the antigenic epitopes of small molecules T3 and T4 is mostly between 2.4 and 8.8 Å, making it difficult to simultaneously form a stable "capture antibody-antigen-detection antibody" sandwich structure with two specific antibodies. Furthermore, steric hindrance easily occurs during antibody binding, leading to a decrease in the binding kinetic efficiency between antigen and antibody. Furthermore, due to the low formation rate of the sandwich complex, the signal intensity generated by subsequent enzymatic or fluorescent reactions is insufficient, resulting in problems such as a low slope of the standard curve and poor differentiation between positive and negative samples, affecting detection sensitivity and accuracy. In summary, T3 and T4, as small molecules, have weak binding affinity to antibodies. The low capture efficiency of antibodies for small molecules in sandwich assays leads to low electrochemiluminescence signals. On the one hand, the detection limit is insufficient for low-concentration samples; on the other hand, the low signal results in significant detection errors for low-concentration T3 and T4 samples, failing to accurately reflect the true concentration of the target analyte in the sample and affecting the reliability of clinical diagnosis. These problems are the core technical challenges faced in T3 and T4 detection.

[0003] The current solutions to alleviate the above problems are: (1) Using a competitive method to detect T3 and T4, but the sensitivity of this method is limited by antibody affinity and cross-reaction is easy to occur; some improved solutions increase epitopes by antigen modification, but the process is complicated and the cost is high, making it difficult to apply on a large scale. (2) Using signal enhancers, most signal enhancers currently use surfactants. Although surfactants can improve the dispersibility of the reaction system, their promoting effect on antibody-small molecule binding is limited, and at high concentrations, they are easy to destroy the spatial structure of the antibody and reduce the specificity of the immune response; although affinity ligands can enhance binding stability, their synthesis cost is high and they are easy to introduce non-specific binding sites, resulting in increased background signal. Summary of the Invention

[0004] Therefore, it is necessary to provide a thyroid hormone detection kit and detection method.

[0005] In a first aspect, a thyroid hormone detection kit is provided, wherein the thyroid hormone includes at least one selected from triiodothyronine and tetraiodothyronine; the thyroid hormone detection kit comprises (i) to (iv):

[0006] (i) A cyclodextrin stock reagent, wherein the cyclodextrin stock reagent contains at least one of cyclodextrin and cyclodextrin derivatives;

[0007] (ii) A capture antibody reagent, wherein the capture antibody reagent contains at least one of a capture antibody that specifically binds to triiodothyronine and a capture antibody that specifically binds to tetraiodothyronine;

[0008] (iii) A detection antibody reagent, said detection antibody reagent containing at least one of a detection antibody specifically binding to triiodothyronine and a detection antibody specifically binding to tetraiodothyronine; and,

[0009] (iv) Signal reagents.

[0010] In an optional embodiment, the cyclodextrin includes at least one of β-cyclodextrin, α-cyclodextrin, and γ-cyclodextrin.

[0011] In an optional embodiment, the cyclodextrin derivative includes at least one of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin.

[0012] In an optional embodiment, the working concentration of the cyclodextrin and cyclodextrin derivative is 5~50 mmol / L.

[0013] In an optional embodiment, the working concentration of the cyclodextrin and cyclodextrin derivative is 20-30 mmol / L.

[0014] In an optional embodiment, the signaling substance includes one or more of chemiluminescent reagents, fluorescent labels, colloids, quantum dots, radionuclides, and paramagnetic ions.

[0015] In an optional embodiment, the signaling substance includes an electrochemiluminescent reagent.

[0016] In an optional implementation, the signal is attached to the detection antibody.

[0017] In an optional embodiment, the detection reagent includes an antibody labeled with ruthenium tripyridine.

[0018] In an optional embodiment, the thyroid hormone detection kit further includes at least one of (v) to (vi):

[0019] (v) Electrochemiluminescence substrate reagents;

[0020] (vi) A detergent, wherein the detergent contains a surfactant and a buffer component;

[0021] (vii) At least one of calibrators and quality control products; wherein the concentration range of triiodothyronine in the calibrator is 0.1 to 10 nmol / L, and the concentration range of tetraiodothyronine in the calibrator is 10 to 200 nmol / L;

[0022] (ⅷ) Solid support.

[0023] In an optional embodiment, the kit includes an electrochemiluminescence reaction cell, wherein the capture antibody is immobilized on the surface of the working electrode of the reaction cell by covalent binding.

[0024] Secondly, a method for detecting thyroid hormones is provided, wherein the thyroid hormones include at least one of triiodothyronine and tetraiodothyronine.

[0025] The thyroid hormone detection method includes performing a binding reaction between a target antigen and an antibody in a reaction system containing at least one of cyclodextrin and cyclodextrin derivatives, then using a signaling substance to bind to a complex formed by the binding of at least one antibody and the target antigen, and detecting the signal of the signaling substance to obtain a detection result.

[0026] The target antigen includes an inclusion complex formed by the thyroid hormone and at least one of the cyclodextrin and cyclodextrin derivatives.

[0027] In an optional embodiment, the cyclodextrin includes at least one of β-cyclodextrin, α-cyclodextrin, and γ-cyclodextrin.

[0028] In an optional embodiment, the cyclodextrin derivative includes at least one of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin.

[0029] In an optional embodiment, the working concentration of the cyclodextrin and cyclodextrin derivative is 5~50 mmol / L.

[0030] In an optional embodiment, the working concentration of the cyclodextrin and cyclodextrin derivative is 20-30 mmol / L.

[0031] In an optional embodiment, the signaling substance includes one or more of chemiluminescent reagents, fluorescent labels, colloids, quantum dots, radionuclides, and paramagnetic ions.

[0032] In an optional embodiment, the signaling substance includes an electrochemiluminescent reagent.

[0033] In an optional implementation, the signal is attached to a detection antibody.

[0034] In an optional embodiment, the thyroid hormone detection method further includes using a washing reagent to remove unbound cyclodextrin and free antibodies. The washing step includes washing at least once with a washing reagent containing a surfactant, and then washing at least once with a washing reagent without a surfactant.

[0035] In an optional implementation, the product is first washed at least three times with a detergent containing a surfactant.

[0036] In an optional embodiment, the thyroid hormone detection method includes performing a binding reaction between a target antigen and a capture antibody in a reaction system containing cyclodextrin, then using a signaling substance to bind to the complex formed by the capture antibody and the detection antibody, and detecting the signal of the signaling substance to obtain a detection result.

[0037] In an optional embodiment, the thyroid hormone detection method includes incubating the sample in a reaction system containing the cyclodextrin and the capture antibody for 30-40 minutes.

[0038] In an optional embodiment, the pH of the reaction system of the cyclodextrin and the capture antibody is 7.2 to 7.6.

[0039] In an optional embodiment, the detection antibody is labeled with ruthenium tripyridine.

[0040] In a further optional embodiment, the electrochemiluminescence reaction conditions include an applied voltage of 1.0 to 1.2 V and an excitation time of 0.5 to 1 s.

[0041] This application addresses the core issue of low signal intensity for small molecules (T3 and T4) in immunoassay. It introduces cyclodextrin into the reaction system of the detection kit. As a macrocyclic compound derived from starch, cyclodextrin possesses a unique molecular structure with hydrophobic cavities and hydrophilic surfaces. Through multiple mechanisms, including hydrophobic interactions, van der Waals forces, and hydrogen bonds, it forms stable inclusion complexes with small molecules (T3 and T4), altering their spatial conformation, exposing more antibody-binding sites, and reducing steric hindrance, thus significantly improving the binding efficiency of antibodies for T3 and T4. In the optimized scheme, the reaction conditions, reagent ratios, and detection process of the kit are specifically optimized based on the characteristics of the electrochemiluminescence detection platform to achieve highly efficient signal amplification. Compared with existing surfactants and affinity ligands, cyclodextrin offers unique advantages such as good biocompatibility, no interference with the specificity of the immunoreaction, low cost, and no background signal interference, ultimately achieving a significant improvement in T3 and T4 detection signals, lowering the detection limit, and improving the accuracy and reliability of low-concentration sample detection. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0046] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0047] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0048] In this application, "separately independent", "each...independently selected" and "...separately independently selected" and "...independently selected" are interchangeable and should be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0049] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0050] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately, concurrently, or in turn with other steps or parts of the sub-steps or stages of other steps.

[0051] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0052] In this application, triiodothyronine, also referred to as "T3", is a type of thyroid hormone with a molecular weight of approximately 651.9 Da and is a small molecule antigen.

[0053] In this application, tetraiodothyronine is also referred to as "T4", also known as thyroxine, with a molecular weight of approximately 776.1 Da, and is a small molecule antigen.

[0054] In this application, the term "cyclodextrin (CD)" refers to a cyclic oligosaccharide formed by D-glucose units linked by α-1,4-glycosidic bonds, including α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0055] In this application, the term "electrochemiluminescence (ECL)" refers to the phenomenon of light emission produced by exciting substances through electrochemical methods, characterized by high sensitivity and strong specificity. The term "cyclodextrin derivative" refers to a compound with similar chemical properties obtained by modifying the groups on the cyclodextrin molecule through chemical modification methods while retaining the basic skeletal structure and function of the cyclodextrin; in optional embodiments, the cyclodextrin derivative is a compound obtained by modifying the hydroxyl groups of cyclodextrin.

[0056] In this application, the term "sandwich assay" refers to an immunoassay method in which capture antibodies and detection antibodies bind to different sites of the target antigen to form a "capture antibody-antigen-detection antibody" complex, thereby achieving quantitative detection of the target analyte.

[0057] In this application, the term "terpyridine ruthenium" refers to [Ru(bpy)3]. 2+ Tris(2,2'-bipyridyl)ruthenium(II) is a commonly used electrochemiluminescent label that can produce a stable luminescent signal under electrochemical excitation.

[0058] In this application, the term "immunoassay" refers to an in vitro diagnostic technique that utilizes the principle of specific binding between antigens and antibodies to qualitatively or quantitatively analyze target substances (antigens, antibodies, or haptens) in a sample by detecting this binding reaction. The antibodies used in immunoassay in this application refer to proteins that bind to specific antigens, broadly encompassing all proteins and protein fragments containing complementarity-determining regions (CDRs). These include polyclonal and monoclonal antibodies, as well as full-length antibodies or their antigen-binding fragments. Antigen-binding fragments include, but are not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody with VH and VL linked by a linker peptide), dsFv (disulfide-stabilized Fv fragments), and nanobodies, etc.

[0059] In this application, the term "capture antibody" refers to an antibody immobilized or used to immobilize on a solid-phase support (such as a microplate, nitrocellulose membrane, magnetic beads, etc.) that is responsible for specifically binding to and "capturing" the target antigen from the sample.

[0060] In this application, the term "detection antibody" refers to an antibody that is free in the liquid phase, carries a signal or is used in subsequent steps to attach a signal, and is used to recognize another epitope of the antigen immobilized by the captured antibody and generate a detectable signal.

[0061] In this application, the term "working concentration" refers to the concentration of a reagent, solution, or substance at the start of a reaction in a reaction system.

[0062] In this application, the term "signaling substance" refers to a substance capable of providing a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can directly provide a signal, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; it can also indirectly provide a signal through a subsequent reaction involving the signaling substance, such as a signal generated by an electrochemiluminescence reaction or a signal generated by an enzyme-catalyzed chemiluminescence reaction. Chemiluminescent reagents can emit light directly or through an enzyme-catalyzed luminescence reaction or an electrochemical reaction. Examples of chemiluminescent reagents include, but are not limited to, at least one of luminol and its derivatives, luciferin, luteolin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives. Chemiluminescent reagents may also optionally include, but are not limited to, catalytic enzymes used in enzyme-catalyzed luminescence reactions, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase. Chemiluminescent reagents may also optionally include, but are not limited to, co-reactants in electrochemiluminescence reactions, such as, but not limited to, tripropylamine.

[0063] In one aspect, some embodiments provide a thyroid hormone detection kit, wherein the thyroid hormone includes at least one of triiodothyronine (T3) and tetraiodothyronine (T4).

[0064] This thyroid hormone testing kit includes (i) to (iv):

[0065] (i) Cyclodextrin stock reagent, wherein the cyclodextrin stock reagent contains at least one of cyclodextrin and cyclodextrin derivatives.

[0066] (ii) A capture antibody reagent containing at least one of a capture antibody specifically binding to T3 and a capture antibody specifically binding to T4. The capture antibody may or may not be attached to a solid-phase carrier. Optionally, the solid-phase carrier and the capture antibody in the kit may be packaged separately and attached only when needed; alternatively, the capture antibody may be pre-coated onto the solid-phase carrier. Those skilled in the art can immobilize the capture antibody to the solid-phase carrier using any conventional method known in the art, such as, but not limited to, covalent bonding, physical adsorption, or using specifically bound molecular pairs, such as, but not limited to, avidin-biotin, receptor-ligand, antibody-antigen, and complementary nucleic acid strands.

[0067] (iii) Detection antibody reagent, wherein the detection antibody reagent contains at least one of a detection antibody that specifically binds to T3 and a detection antibody that specifically binds to T4.

[0068] (iv) Signal reagent: The signal reagent can be packaged independently or pre-linked to the detection antibody. In the scheme where it is pre-linked to the detection antibody reagent, the detection antibody reagent and the signal reagent are the same reagent. Optionally, the signal reagent can also consist of multiple reagents. For example, when the signal reagent is a reagent that emits light based on an enzyme-catalyzed luminescence reaction or an electrochemical reaction, the signal reagent includes a catalytic enzyme and a luminescent substrate for the enzyme-catalyzed luminescence reaction; or the signal reagent includes a chemiluminescent reagent and co-reactants in the electrochemiluminescence reaction. When the signal reagent consists of multiple reagents, some components can be linked to the detection antibody. For example, in a signal reagent based on an enzyme-catalyzed luminescence reaction, the catalytic enzyme for the enzyme-catalyzed luminescence reaction is linked to the detection antibody, and the luminescent substrate is a separately packaged reagent; or in a signal reagent based on an electrochemiluminescence reaction, the chemiluminescent substance is linked to the detection antibody, and the co-reactants are separately packaged reagents.

[0069] In an optional embodiment, the cyclodextrin includes at least one of β-cyclodextrin, α-cyclodextrin, and γ-cyclodextrin.

[0070] In an optional embodiment, the cyclodextrin derivative includes at least one of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin.

[0071] In an optional embodiment, the working concentration of cyclodextrin and cyclodextrin derivatives is 5 to 50 mmol / L, for example, but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mmol / L, and more optionally, the working concentration is 20 to 30 mmol / L.

[0072] In an optional embodiment, the working concentration of cyclodextrin and cyclodextrin derivatives in the cyclodextrin stock reagent is 5 to 50 mmol / L, for example, but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mmol / L, and more preferably 40 to 60 mmol / L, and is directly mixed with the sample in equal proportion when used.

[0073] In an optional embodiment, the solvent for the cyclodextrin stock reagent includes a buffer component.

[0074] In optional embodiments, the signaling material includes one or more of chemiluminescent reagents, fluorescent labels, colloids, quantum dots, radionuclides, and paramagnetic ions.

[0075] In an optional implementation, the signaling agent includes an electrochemiluminescent reagent.

[0076] In an optional implementation, the signal is attached to the detection antibody.

[0077] In an optional implementation, the detection reagent includes an antibody labeled with ruthenium tripyridine.

[0078] In an optional embodiment, the thyroid hormone detection kit further includes at least one of (v) to (vi):

[0079] (v) Electrochemiluminescence substrate reagents.

[0080] (vi) Detergent, which contains surfactant and buffer components.

[0081] In an optional embodiment, the surfactant includes at least one of Tween-20, PEG6000, and TritonX-100.

[0082] In an optional embodiment, the washing reagent contains 0.01~0.1% w / v Tween-20, preferably 0.05% w / v Tween-20.

[0083] (vii) At least one of the calibrators and quality control products; the concentration range of T3 in the calibrators is 0.1~10 nmol / L, and the concentration range of T4 in the calibrators is 10~200 nmol / L;

[0084] (ⅷ) Solid support.

[0085] In one alternative embodiment, the thyroid hormone detection kit includes reagents (a) to (g):

[0086] (a) Cyclodextrin stock reagent, wherein the cyclodextrin stock reagent is at least one of β-cyclodextrin stock reagent, α-cyclodextrin stock reagent, γ-cyclodextrin stock reagent, and hydroxypropyl-β-cyclodextrin stock reagent; wherein the concentration of cyclodextrin or cyclodextrin derivative in the cyclodextrin stock reagent is independently 10 to 100 mmol / L, and the working concentration of cyclodextrin or cyclodextrin derivative is independently 5 to 50 mmol / L.

[0087] (b) A capture antibody reagent containing at least one of a capture antibody that specifically binds to T3 and a capture antibody that specifically binds to T4. In some embodiments, the capture antibody is at least one of a monoclonal antibody that specifically binds to T3 and a monoclonal antibody that specifically binds to T4.

[0088] (c) Detection antibody reagent, wherein the detection antibody reagent contains at least one of a detection antibody that specifically binds to T3 and a detection antibody that specifically binds to T4, and the detection antibody reagent is labeled with ruthenium terpyridine. Optionally, the ruthenium terpyridine is labeled with the detection antibody by an N-hydroxysuccinimide activation method.

[0089] In some embodiments, the detection antibody is at least one of a monoclonal antibody that specifically binds to T3 and a monoclonal antibody that specifically binds to T4, and is labeled with ruthenium tripyridine.

[0090] (d) Electrochemiluminescent substrate reagent containing a buffer component and tripropylamine.

[0091] In some embodiments, the electrochemiluminescent substrate reagent is a PBS buffer (pH 7.4) containing tripropylamine (TPA) at a concentration of 0.1–0.5 mol / L.

[0092] (e) The detergent contains surfactants and buffer components.

[0093] In some embodiments, the washing reagent is PBS buffer (pH 7.4) containing 0.05% w / v Tween-20.

[0094] (f) At least one of calibrators and quality control samples; the concentration range of T3 in the calibrators is 0.1~10 nmol / L, and the concentration range of T4 in the calibrators is 10~200 nmol / L. In an optional embodiment, the matrix of the calibrators and quality control samples is a serum matrix.

[0095] (g) Electrochemiluminescence reaction cell, where captured antibodies are immobilized on the surface of the working electrode of the reaction cell by covalent binding.

[0096] Secondly, some embodiments provide a method for detecting thyroid hormones, wherein the thyroid hormones include at least one of T3 and T4. This method involves performing a binding reaction between a target antigen and an antibody in a reaction system containing at least one of cyclodextrin and cyclodextrin derivatives, then using a signal agent bound to a complex formed by the binding of at least one antibody and the target antigen, and detecting the signal of the signal agent to obtain a detection result; the target antigen includes an inclusion complex formed by the thyroid hormone and at least one of cyclodextrin and cyclodextrin derivatives.

[0097] The detection principle of this method is as follows: after the antibody forms a complex with the target antigen, quantitative detection is achieved by detecting the signal generated by the signal bound to the complex, which is positively correlated with the concentrations of T3 and T4 in the sample. The promoting effect of cyclodextrin on antibody-small molecule binding is directly translated into a significant signal enhancement, effectively alleviating the problem of low signal in existing technologies. Taking the immunoassay of the double-antibody sandwich method combined with electrochemiluminescence as an example, the principle is as follows: after the formation of the "capture antibody-T3 / T4-cyclodextrin-detection antibody" complex, under electrochemical excitation, the ruthenium tripyridine on the detection antibody undergoes a redox reaction with tripropylamine in the substrate solution, generating a high-intensity electrochemiluminescence signal. The signal intensity is positively correlated with the concentrations of T3 / T4 in the sample, and quantitative detection is achieved by signal reading.

[0098] The principle behind cyclodextrin's signal enhancement is as follows: Cyclodextrin is a cyclic oligosaccharide formed by D-glucose units linked by α-1,4-glycosidic bonds. Its molecular structure has a cavity characteristic of being hydrophilic on the outside and hydrophobic on the inside. T3 and T4 molecules contain hydrophobic groups, such as aromatic ring structures, which can form stable inclusion complexes with the hydrophobic cavity of cyclodextrin through hydrophobic interactions and van der Waals forces. This inclusion complex can alter the spatial conformation of T3 and T4: after inclusion, the hydrophobic groups of the small molecules are encapsulated within the cyclodextrin cavity, exposing previously hidden antigenic epitopes and increasing effective binding sites for antibodies. This inclusion complex also reduces steric hindrance during binding: the hydrophilic exterior of cyclodextrin forms hydrogen bonds with the hydrophilic groups of the antibody molecule, bringing the small molecule closer to the antibody, reducing steric hindrance during binding, and increasing the binding rate and affinity. Finally, the formed inclusion complex can protect antibody activity: cyclodextrin has good biocompatibility, does not disrupt the spatial structure of the antibody, maintains the stability of the antibody in the reaction system, and further ensures capture efficiency.

[0099] In an optional embodiment, the cyclodextrin includes at least one of β-cyclodextrin, α-cyclodextrin, and γ-cyclodextrin.

[0100] In an optional embodiment, the cyclodextrin derivative includes at least one of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin.

[0101] In an optional embodiment, the working concentration of cyclodextrin and cyclodextrin derivatives is 5 to 50 mmol / L, for example, but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mmol / L, and more optionally, the working concentration is 20 to 30 mmol / L.

[0102] In optional embodiments, the signaling material includes one or more of chemiluminescent reagents, fluorescent labels, colloids, quantum dots, radionuclides, and paramagnetic ions.

[0103] In an optional implementation, the signaling agent includes an electrochemiluminescent reagent.

[0104] In an optional implementation, the signal is attached to the detection antibody.

[0105] In an optional embodiment, the thyroid hormone detection method further includes using a washing reagent to remove unbound cyclodextrin and free antibodies, employing a gradient washing method. The washing steps include washing at least once with a washing reagent containing a surfactant, followed by washing at least once with a washing reagent without a surfactant. This elution method helps remove unbound cyclodextrin and free detection antibodies, reducing nonspecific signals.

[0106] In an optional embodiment, the detergent is first washed at least three times with a detergent containing a surfactant. In an optional embodiment, the surfactant in the detergent includes at least one of Tween-20, PEG6000, and Triton X-100.

[0107] In an optional embodiment, the washing reagent contains 0.01~0.1% w / v Tween-20, preferably 0.05% w / v Tween-20.

[0108] In an optional embodiment, the thyroid hormone detection method includes performing a binding reaction between a target antigen and a capture antibody in a reaction system containing cyclodextrin, then using a signaling substance to bind to the complex formed by the capture antibody and the detection antibody, and obtaining a detection result from the signal of the detection signaling substance.

[0109] In an optional embodiment, the sample is incubated in a reaction system containing cyclodextrin and capture antibody for 30-40 minutes to ensure that the cyclodextrin fully encapsulates T3 or T4 before binding to the antibody.

[0110] In an optional embodiment, the reaction system of cyclodextrin and the capture antibody has a pH of 7.2–7.6. This pH range can be matched with the optimal pH (7.4) for the electrochemiluminescence reaction, avoiding the influence of pH differences on signal stability.

[0111] In an optional implementation, the detection antibody is labeled with ruthenium tripyridine ([Ru(bpy)3]). 2+ ).

[0112] In a further optional embodiment, the electrochemiluminescence reaction conditions include an applied voltage of 1.0–1.2 V and an excitation time of 0.5–1 s. These reaction conditions facilitate the full luminescence of ruthenium terpyridine while reducing background signal interference.

[0113] In an optional implementation, the thyroid hormone detection method provided in the second aspect is for non-diagnostic and non-therapeutic purposes.

[0114] In an optional implementation, the thyroid hormone detection method provided in the second aspect is implemented using the thyroid hormone detection kit provided in the first aspect.

[0115] The following are some examples.

[0116] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0117] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0118] The antibodies used in the following examples were purchased from Abcam and Sigma-Aldrich; the cyclodextrins were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. and Shanghai McLean Biochemical Technology Co., Ltd.

[0119] Example 1

[0120] Preparation and detection method of β-cyclodextrin-enhanced T3 detection kit:

[0121] (1) Kit preparation steps:

[0122] 1. Capture antibody coating reaction chamber: Dilute anti-T3 monoclonal antibody to 5 μg / mL with coating buffer (0.05 mol / L Tris-HCl, pH 9.6), add 100 μL to each well, and incubate at 4℃ for 12 h; discard the coating solution, and wash 3 times with washing buffer for 3 min each time;

[0123] 2. Blocking: Add blocking solution containing 3% bovine serum albumin (BSA) at 150 μL per well and incubate at 37 ℃ for 2 h; discard the blocking solution, wash 3 times, dry and seal for storage;

[0124] 3. Antibody preparation: The anti-T3 monoclonal antibody was labeled with ruthenium tripyridine using the N-hydroxysuccinimide activation method at a ratio of 1:5 (antibody:ruthenium tripyridine, molar ratio). After purification, it was diluted to 2 μg / mL with PBS buffer containing 1% BSA.

[0125] 4. Preparation of cyclodextrin solution: Weigh β-cyclodextrin, dissolve it in PBS buffer (pH 7.4) to prepare a cyclodextrin solution with a concentration of 20 mmol / L, filter to remove bacteria and store at 4°C;

[0126] 5. Substrate preparation: Dissolve tripropylamine in PBS buffer (pH 7.4) to prepare a substrate solution with a concentration of 0.3 mol / L;

[0127] 6. Reagent kit assembly: Package the coated reaction cell, detection antibody solution, β-cyclodextrin solution, calibrator, quality control sample, washing solution, and substrate solution in the specified proportions to obtain the finished reagent kit.

[0128] (2) Detection steps:

[0129] 1. Take 50 μL each of the calibrator, quality control sample, and test sample, and add them to the coating reaction cell respectively;

[0130] 2. Add 50 μL of β-cyclodextrin solution (final concentration 10 mmol / L) to each well and incubate at 37 ℃ for 35 min;

[0131] 3. Wash three times with washing buffer, add 100 μL of detection antibody solution to each well, and incubate at 37 ℃ for 25 min;

[0132] 4. Wash 4 times with washing buffer using a gradient washing method. First, wash 3 times with PBS buffer containing 0.05% w / v Tween-20, then wash once with pure PBS buffer to remove unbound cyclodextrin and free detection antibody, reducing non-specific signal. Then add 150 μL of substrate solution to each well.

[0133] 5. Place the reaction cell into the electrochemiluminescence detector (Roche Elecsys 2010), set the excitation voltage to 1.1 V and the excitation time to 0.8 s, and read the luminescence signal value (RLU).

[0134] 6. Plot a standard curve with calibrator concentration on the x-axis and signal value on the y-axis, and calculate the T3 concentration in the sample.

[0135] Example 2

[0136] Preparation and detection method of hydroxypropyl-β-cyclodextrin enhanced T4 assay kit:

[0137] (1) Kit preparation steps:

[0138] 1. Capture antibody coating reaction chamber: Dilute anti-T4 monoclonal antibody to 6 μg / mL with coating buffer (0.05 mol / L Tris-HCl, pH 9.6), add 100 μL to each well, and incubate at 4℃ for 14 h; discard the coating solution and wash 3 times;

[0139] 2. Blocking: Add blocking solution containing 4% BSA, 150 μL per well, and incubate at 37 ℃ for 2 h; wash and dry for storage;

[0140] 3. Antibody preparation: Using the same labeling method as in Example 1, ruthenium tripyridine was labeled onto the anti-T4 monoclonal antibody at a labeling ratio of 1:6, and then purified and diluted to 2.5 μg / mL;

[0141] 4. Preparation of cyclodextrin solution: Weigh hydroxypropyl-β-cyclodextrin, dissolve it in PBS buffer (pH 7.4) to prepare a 30 mmol / L solution, and filter to sterilize;

[0142] 5. Substrate preparation: PBS buffer (pH 7.4) with a tripropylamine concentration of 0.4 mol / L;

[0143] 6. Reagent kit assembly: Same as in Example 1.

[0144] (2) Detection steps:

[0145] 1. Sample and calibrator processing: Add 50 μL of sample / calibrator to the reaction cell, add 50 μL of hydroxypropyl-β-cyclodextrin solution (final concentration 15 mmol / L), and incubate at 37℃ for 40 min;

[0146] 2. Subsequent steps (antibody incubation, washing, substrate addition, signal reading) are the same as in Example 1, with an excitation voltage of 1.2V and an excitation time of 1s.

[0147] Examples 3-16

[0148] Examples 3-16 are examples of the preparation and detection methods of cyclodextrin-enhanced T3 detection kits. The same preparation method as in Example 1 was used. The only difference from Example 1 was the type of cyclodextrin used, the concentration of the cyclodextrin solution in the kit, and the working concentration of cyclodextrin in the detection step. The cyclodextrin type, the concentration of the cyclodextrin solution in the kit, and the working concentration of cyclodextrin in the detection step in Examples 3-16 are shown in Table 1.

[0149] Table 1

[0150]

[0151] Example 17

[0152] Example 17 is an example of the preparation and detection of a β-cyclodextrin-enhanced T4 detection kit. The only difference from Example 1 is that in the kit preparation steps, step 1 involves coating with T4 monoclonal antibody.

[0153] Example 18

[0154] Example 18 is an example of the preparation and detection of a hydroxypropyl-β-cyclodextrin-enhanced T3 detection kit. The only difference from Example 2 is that in the kit preparation steps, step 1 involves coating with T3 monoclonal antibody.

[0155] Comparative Example 1

[0156] T3 standard kit without signal enhancer:

[0157] The preparation method is the same as in Example 1, except that cyclodextrin and other enhancers are not added to the reaction system, the incubation time in the detection step is 30 min, and other parameters are the same as in Example 1.

[0158] Comparative Example 2

[0159] T4 standard kit without signal enhancer:

[0160] The preparation method is the same as in Example 17, except that cyclodextrin and other enhancers are not added to the reaction system, the incubation time in the detection step is 30 min, and other parameters are the same as in Example 2.

[0161] Comparative Example 3

[0162] Surfactant-enhanced T3 kit:

[0163] The preparation method is the same as in Example 1, except that the cyclodextrin solution is replaced with 0.1% Tween-20 solution, and the detection steps remain unchanged.

[0164] Comparative Example 4

[0165] Surfactant-enhanced T4 kit:

[0166] The preparation method is the same as in Example 17, except that the cyclodextrin solution is replaced with 0.1% Tween-20 solution, and the detection steps remain unchanged.

[0167] Comparative Example 5

[0168] Affinity-enhanced T3 kit:

[0169] The preparation method is the same as in Example 1, except that the cyclodextrin solution is replaced with a 0.05 mg / mL biotin-streptavidin derivative solution, and the detection steps remain unchanged.

[0170] Comparative Example 6

[0171] Affinity-enhanced T4 kit:

[0172] The preparation method is the same as in Example 17, except that the cyclodextrin solution is replaced with a 0.05 mg / mL biotin-streptavidin derivative solution, and the detection steps remain unchanged.

[0173] Comparative Example 7

[0174] T3 kit for cucurbit[6]uril (CB[6]) enhancer:

[0175] The preparation method is the same as in Example 1, except that the cyclodextrin solution is replaced with 20 mmol / L cucurbita[6]urea (CB[6]) solution, and the detection steps remain the same.

[0176] Comparative Example 8

[0177] T4 kit for cucurbit[6]uril (CB[6]) enhancer:

[0178] The preparation method is the same as in Example 17, except that the cyclodextrin solution is replaced with 20 mmol / L cucurbita[6]urea (CB[6]) solution, and the detection steps remain the same.

[0179] Comparative Example 9

[0180] T3 kit for cyclophane (CP) enhancers:

[0181] The preparation method is the same as in Example 1, except that the cyclodextrin solution is replaced with a 20 mmol / L cycloaromatic (CP) solution, and the detection steps remain unchanged.

[0182] Comparative Example 10

[0183] T4 kit for cyclophane (CP) enhancers:

[0184] The preparation method is the same as in Example 17, except that the cyclodextrin solution is replaced with a 20 mmol / L cycloaromatic (CP) solution, and the detection steps remain unchanged.

[0185] Comparative Example 11

[0186] T3 kit for crown ether (18-crown-6, 18-Crown-6) enhancers:

[0187] The preparation method is the same as in Example 1, except that the cyclodextrin solution is replaced with a 20 mmol / L crown ether (18-crown-6) solution, and the detection steps remain unchanged.

[0188] Comparative Example 12

[0189] T4 kit for crown ether (18-crown-6, 18-Crown-6) enhancers:

[0190] The preparation method is the same as in Example 17, except that the cyclodextrin solution is replaced with a 20 mmol / L crown ether (18-crown-6) solution, and the detection steps remain unchanged.

[0191] Example 1

[0192] Comparison of the effects of different cyclodextrins and their working concentrations:

[0193] The detection performance of the kits and detection methods used in Examples 1, 3-16, and Example 3 were compared. The signal values ​​of the samples were those of the same concentration of T3 calibrator (5 nmol / L). The results are shown in Table 2.

[0194] Table 2. Detection effects of different cyclodextrin types and concentrations

[0195]

[0196] Conclusion: When the concentration of cyclodextrin is 20-30 mmol / L, the signal enhancement rate can reach 128.9%-135.8%. With further increase in concentration, the signal enhancement rate tends to level off. Moreover, β-cyclodextrin has a better enhancement effect than α-cyclodextrin and γ-cyclodextrin.

[0197] Example 2

[0198] Comparison of the effects of different reinforcing agents:

[0199] The detection performance of the kits and detection methods used in Examples 1 and 2, and Examples 17 and 18 was compared to detect T3 and T4. The detection samples were the signal values ​​of the same concentration of T3 calibrator (0.5 nmol / L) and T4 calibrator (20 nmol / L). The results are shown in Table 3.

[0200] Table 3

[0201]

[0202] Note: The higher the specificity coefficient in Table 3, the stronger the specificity of the immune response and the smaller the non-specific interference.

[0203] in conclusion:

[0204] 1. Signal enhancement effect: Example 1 / 17 (β-cyclodextrin) > Example 2 / 18 (hydroxypropyl-β-cyclodextrin) > Comparative Example 5 / 6 (affinity ligand) > Comparative Example 3 / 4 (surfactant) > Comparative Example 7 / 8 (cucurbita[6]urea) > Comparative Example 9 / 10 (cycloaromatic hydrocarbon) > Comparative Example 11 / 12 (crown ether) > Comparative Example 1 (no enhancer). The signal enhancement rate of cyclodextrin is 2.5 to 3.2 times that of surfactant and 1.9 to 2.1 times that of affinity ligand.

[0205] 2. Specificity: The specificity coefficients of Examples 1 / 17 and 2 / 18 (T3: 18.2~18.3; T4: 51.0~51.3) were significantly higher than those of Comparative Examples 3 / 4 (T3: 6.7; T4: 17.0), Comparative Examples 5 / 6 (T3: 6.6; T4: 16.5), and Comparative Examples 7-12 (T3: 5.8~9.3; T4: 15.9~16.4), indicating that cyclodextrin does not introduce non-specific binding, has good biocompatibility, and does not interfere with the specificity of the immune response.

[0206] 3. Detection effect at low concentrations: In Examples 1 / 17, the signal values ​​for low concentrations of T3 (0.5 nmol / L) and T4 (20 nmol / L) reached 5860 RLU and 16320 RLU, respectively, which are much higher than the 2556 RLU and 7050 RLU of Comparative Examples 1 and 2. This can effectively reduce the detection limit and improve the detection accuracy of low concentration samples.

[0207] Example 3

[0208] Results of reagent kit precision and stability tests:

[0209] 1. Precision testing: The quality control samples at each concentration were tested 10 times using the kit from Example 1 to obtain the intra-batch and inter-batch coefficients of variation. The results are shown in Table 4.

[0210] Table 4. Precision test results (n=10) of the kit in Example 1

[0211]

[0212] Note: CV: Coefficient of Variation, an indicator reflecting the precision of the test results. The smaller the CV, the higher the precision.

[0213] 2. Stability test: The kit prepared in Example 1 was stored at 4 °C for 0 days, 30 days, 60 days and 90 days respectively. Then the final concentration control sample was tested to obtain the signal value and signal retention rate. The results are shown in Table 5.

[0214] Table 5. Stability test of the kit in Example 1 (stored at 4°C)

[0215]

[0216] Conclusion: The kit of Example 1 has an intra-batch CV ≤ 3.2% and an inter-batch CV ≤ 4.5%, which meets the precision requirements for clinical testing (CV ≤ 10%). After storage at 4°C for 90 days, the signal retention rate is still ≥ 92.6%, indicating good stability.

[0217] Example of effect 4

[0218] Results of the reagent kit's limit of detection and linear range tests:

[0219] 1. Limit of Detection Test: The blank samples were tested 20 times using the kits from Examples 1 and 17, and the mean signal value (RLU) was calculated. and standard deviation (SD), The dose-response curves were used to calculate the corresponding concentration values, and the results are shown in Table 6.

[0220] Table 6. Limit of Detection Tests for Kits in Examples 1 and 17

[0221]

[0222] Conclusion: The limit of detection (LOB) of the kit in Example 1 is 0.08 nmol / L, which meets the clinical limit of detection requirement (LOB≤0.6 nmol / L); the limit of detection (LOB) of the kit in Example 17 is 9.69 nmol / L, which meets the clinical limit of detection requirement (LOB≤12.872 nmol / L).

[0223] 2. Linearity Range Test: Five concentrations of samples were prepared according to the preset linear range. The kit from Example 1 was used to perform three parallel measurements on each concentration sample. The average result was fitted to the theoretical concentration using the least squares method to obtain a linear correlation coefficient r. The results are shown in Table 7.

[0224] Table 7. Limit of detection test of the kit in Example 1 (unit: nmol / L)

[0225]

[0226] Conclusion: The kit in Example 1 showed a linear correlation coefficient (r) of no more than 0.9900 within the range of 0.21-9.18 nmol / L, and the linear range was verified, meeting the clinical linear range requirements (1.22~9.16 nmol / L).

[0227] In summary, the kit and detection method using the kit provided in the above embodiments have the following advantages:

[0228] 1. Significantly improves detection signal: Through the inclusion effect of cyclodextrin with T3 and T4, the antibody capture efficiency of small molecules is improved, and the electrochemiluminescence signal enhancement rate reaches more than 128.9%, which effectively alleviates the core technical problem of low signal when the sandwich method is used to detect small molecules.

[0229] 2. Optimized signal enhancement effect: Compared with surfactants and affinity ligands, cyclodextrin has a more significant signal enhancement effect, which is more than 2.5 times that of surfactants, and does not interfere with the specificity of immune response. Non-specific signal is low, and the specificity coefficient is increased to more than 18.

[0230] 3. Reduced detection limit: The detection signal of low concentration samples (T3 0.3 nmol / L, T4 20 nmol / L) is significantly enhanced, and the detection limit is reduced to T3 0.1 nmol / L and T4 10 nmol / L, meeting the clinical needs for low concentration sample detection.

[0231] 4. Simple operation and low cost: Cyclodextrin does not require complex synthesis, can be directly purchased commercially, and is easy to add. It does not require additional detection steps and does not increase the production cost of the reagent kit.

[0232] 5. Adapted to electrochemiluminescence platform: Through targeted process adjustments, the enhancement effect of cyclodextrin is ensured to match the electrochemiluminescence detection principle, resulting in good signal stability and precision and stability that meet clinical testing requirements.

[0233] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0234] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A thyroid hormone detection kit, characterized in that, The thyroid hormone includes at least one of triiodothyronine and tetraiodothyronine; the thyroid hormone detection kit includes (i) to (iv): (i) A cyclodextrin stock reagent, wherein the cyclodextrin stock reagent contains at least one of cyclodextrin and cyclodextrin derivatives; (ii) A capture antibody reagent, wherein the capture antibody reagent contains at least one of a capture antibody that specifically binds to triiodothyronine and a capture antibody that specifically binds to tetraiodothyronine; (iii) A detection antibody reagent, wherein the detection antibody reagent contains at least one of a detection antibody that specifically binds to triiodothyronine and a detection antibody that specifically binds to tetraiodothyronine; and, (iv) Signal reagents.

2. The thyroid hormone detection kit according to claim 1, characterized in that, The cyclodextrin includes at least one of β-cyclodextrin, α-cyclodextrin and γ-cyclodextrin; Optionally, the cyclodextrin derivative includes at least one of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin.

3. The thyroid hormone detection kit according to claim 1, characterized in that, The working concentration of the cyclodextrin and cyclodextrin derivatives is 5~50 mmol / L; Optionally, the working concentration of the cyclodextrin and cyclodextrin derivative is 20-30 mmol / L.

4. The thyroid hormone detection kit according to claim 1, characterized in that, The signaling material includes one or more of the following: chemiluminescent reagents, fluorescent labels, colloids, quantum dots, radionuclides, and paramagnetic ions; Optionally, the signaling substance includes an electrochemiluminescent reagent; Optionally, the signal is connected to the detection antibody; Optionally, the detection reagent includes an antibody labeled with ruthenium tripyridine.

5. The thyroid hormone detection kit according to any one of claims 1 to 4, characterized in that, It also includes at least one of (v) to (vi): (v) Electrochemiluminescence substrate reagents; (vi) A detergent, wherein the detergent contains a surfactant and a buffer component; (vii) At least one of calibrators and quality control products; wherein the concentration range of triiodothyronine in the calibrator is 0.1 to 10 nmol / L, and the concentration range of tetraiodothyronine in the calibrator is 10 to 200 nmol / L; (ⅷ) Solid support; Optionally, the kit includes an electrochemiluminescence reaction cell, wherein the capture antibody is immobilized on the surface of the working electrode of the reaction cell by covalent binding.

6. A method for detecting thyroid hormones, characterized in that, The thyroid hormones include at least one of triiodothyronine and tetraiodothyronine. The thyroid hormone detection method includes performing a binding reaction between a target antigen and an antibody in a reaction system containing at least one of cyclodextrin and cyclodextrin derivatives, then using a signaling substance to bind to a complex formed by the binding of at least one antibody and the target antigen, and detecting the signal of the signaling substance to obtain a detection result. The target antigen includes an inclusion complex formed by the thyroid hormone and at least one of the cyclodextrin and cyclodextrin derivatives.

7. The method for detecting thyroid hormones according to claim 6, characterized in that, The cyclodextrin includes at least one of β-cyclodextrin, α-cyclodextrin and γ-cyclodextrin; Optionally, the cyclodextrin derivative includes at least one of hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin; Optionally, the working concentration of the cyclodextrin and cyclodextrin derivatives is 5~50 mmol / L; Optionally, the working concentration of the cyclodextrin and cyclodextrin derivative is 20-30 mmol / L.

8. The method for detecting thyroid hormones according to claim 6, characterized in that, The signaling material includes one or more of the following: chemiluminescent reagents, fluorescent labels, colloids, quantum dots, radionuclides, and paramagnetic ions; Optionally, the signaling substance includes an electrochemiluminescent reagent; Optionally, the signal is connected to a detection antibody.

9. The method for detecting thyroid hormones according to claim 6, characterized in that, The thyroid hormone detection method further includes using a washing reagent to remove unbound cyclodextrin and free antibodies. The washing step includes washing at least once with a washing reagent containing a surfactant, and then washing at least once with a washing reagent without a surfactant. Optionally, wash at least three times with a detergent containing surfactant.

10. The method for detecting thyroid hormones according to any one of claims 6 to 9, characterized in that, The thyroid hormone detection method includes performing a binding reaction between a target antigen and a capture antibody in a reaction system containing cyclodextrin, then using a signaling substance to bind to the complex formed by the capture antibody and the detection antibody, and detecting the signal of the signaling substance to obtain the detection result; Optionally, the sample is incubated in a reaction system containing the cyclodextrin and the capture antibody for 30-40 min; Optionally, the pH of the reaction system of the cyclodextrin and the capture antibody is 7.2 to 7.6; Optionally, the detection antibody is labeled with ruthenium tripyridine; Further optionally, the electrochemiluminescence reaction conditions include an applied voltage of 1.0–1.2 V and an excitation time of 0.5–1 s.

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