Triiodothyronine chemiluminescence immunoassay kit and preparation method and application thereof

Abstract

The application belongs to the technical field of immune detection, and particularly relates to a triiodothyronine chemiluminescence immunoassay kit as well as a preparation method and application thereof. The application provides a triiodothyronine chemiluminescence immunoassay kit, which adopts a double-antibody sandwich method to detect FT3 / T3 on an electrochemiluminescence platform for the first time, effectively improves detection sensitivity, increases a linear range of detection, and improves detection repeatability.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology, specifically relating to a triiodothyronine chemiluminescent immunoassay kit and its preparation method and application. Background Technology

[0002] Triiodothyronine (3,5,3'-triiodothyronine, or T3) is an iodine-containing tyrosine derivative with a molecular weight of 651 Daltons and a half-life of 1.5 days. It is a thyroid hormone responsible for the metabolic regulation of various target organs. T3 is mainly produced in the liver by enzymatically removing the iodine at the 5' position from T4 (thyroxine). Therefore, serum T3 concentration is a better indicator of the functional status of peripheral tissues than the secretory function of the thyroid gland.

[0003] Free T3 (FT3) is an unbound, biologically active form of T3, comprising only 0.2-0.4% of total T3. Theoretically, FT3 detection is unaffected by the concentration and binding affinity of binding proteins, thus eliminating the need for additional tests reflecting binding parameters (such as TBG). Therefore, FT3 measurement is an important indicator for routine clinical assessment of thyroid function. FT3 results can serve as a basis for the differential diagnosis of thyroid diseases, distinguishing different types of hyperthyroidism and confirming T3 hyperthyroidism.

[0004] Currently available methods for detecting FT3 include radioimmunoassay (RIA), chemiluminescent immunoassay, and isotope dilution / GC / MS. Among these, RIA is difficult to automate, carries radiation pollution that could harm operators, and suffers from poor reproducibility due to the short half-life of 131I, which makes long-term storage challenging. Chemiluminescent immunoassay is costly, requires specific environmental conditions, carries the risk of cross-reactions, and involves complex and expensive equipment. Isotope dilution / GC / MS involves complex and time-consuming sample pretreatment, cannot avoid non-specific adsorption of analytes onto membranes or devices, and its automation level falls short of clinical requirements, making the detection of FT3 and FT4 difficult.

[0005] The detection process generally involves FT3 / T3 in the sample competing with biotin-labeled T3 derivatives in the reagent for binding to T3 antibodies labeled with luminescent markers. Then, the immune complex of biotin-T3 and luminescent T3 antibody is captured by magnetic microparticles coated with streptavidin. After washing, the sample is added to a luminescent solution for signal detection, and the concentration is calculated using a standard curve.

[0006] In competitive reactions, the amounts of biotinylated antigens and luminescently labeled antibodies are limited. This makes the sensitivity of competitive methods highly dependent on antibody affinity. T3 small molecule antibodies typically have low affinity constants, resulting in poor signal-to-noise ratios and low sensitivity in existing FT3 / T3 competitive methods, leading to poor differentiation of low values. Furthermore, since the detection of free thyroid-related hormones requires maintaining an endogenous balance between bound and free states, high-value samples above the upper linear limit cannot be tested by dilution. However, the limited linear range of the FT3 / T3 competitive method prevents the detection of high-value samples.

[0007] Furthermore, since the binding site of T3 small molecule compounds to their corresponding antibodies is a cavitation structure, most of the structure of the small molecule is covered after binding with the first antibody, resulting in insufficient binding space for the second antibody. In addition, T3 small molecule antigen recognition sites are few. Therefore, it is difficult to use the double antibody sandwich method to detect FT3 / T3. In immunomethodology, competitive methods are usually used to detect FT3 / T3.

[0008] The existing FT3 / T3 competitive immunoassay method has a poor signal-to-noise ratio, low sensitivity, and poor differentiation of low values, which cannot meet the clinical needs for accurate detection of low and high FT3 / T3 values. Summary of the Invention

[0009] Based on this, one embodiment of this application provides a triiodothyronine chemiluminescent immunoassay kit, its preparation method, and its application.

[0010] This application provides a chemiluminescent immunoassay kit for triiodothyronine, the kit comprising:

[0011] The antibody 1-solid-phase carrier conjugate, antibody 2-chemiluminescent label, and triiodothyronine antigen calibrator are provided. The antibody 1-solid-phase carrier conjugate comprises a solid-phase carrier coated with triiodothyronine antibody 1, and the antibody 2-luminescent label comprises triiodothyronine antibody 2 labeled with a chemiluminescent label. Both the antibody 1-solid-phase carrier conjugate and the antibody 2-luminescent label can specifically bind to the target analyte and have different antigen recognition epitopes.

[0012] The study includes a streptavidin-solid-phase carrier conjugate, antibody 1-biotin-labeled antibody, antibody 2-chemiluminescent-labeled antibody, and a triiodothyronine antigen calibrator. The antibody 1-biotin-labeled antibody comprises biotin-labeled triiodothyronine antibody 1, and the antibody 2-chemiluminescent-labeled antibody comprises chemiluminescent-labeled triiodothyronine antibody 2. Both antibody 1-biotin-labeled antibody and antibody 2-chemiluminescent-labeled antibody can specifically bind to the target analyte and have different antigenic recognition epitopes.

[0013] In some embodiments, the solid support in the antibody-solid support conjugate includes magnetic beads.

[0014] In some embodiments, the functional groups on the surface of the magnetic beads include one or more of carboxyl, amino, hydroxyl, epoxy, and p-toluenesulfonyl groups.

[0015] In some embodiments, the active groups modified on the biotin include one or more of NHS ester groups, amino groups, maleimide groups, and hydrazide groups.

[0016] In some embodiments, the chemiluminescent label includes one or more of Ru-NHS, horseradish peroxidase, alkaline phosphatase, acridine ester, luminol and its derivatives, isoluminol and its derivatives, adamantane and acridine compounds-NHS.

[0017] In some embodiments, the antibodies in the antibody 1-solid-phase carrier conjugate and / or the antibody 2-chemiluminescent label are monoclonal antibodies.

[0018] In some embodiments, the test kit includes test reagent 1, which includes the antibody 1-solid-phase carrier conjugate and diluent 1; or the streptavidin-solid-phase carrier conjugate and diluent 1.

[0019] In some embodiments, the diluent 1 includes salt ions 1, surfactant 1, preservative 1, stabilizer 1, and neutral buffer 1.

[0020] In some embodiments, the salt ion 1 includes one or both of NaCl and KCl.

[0021] In some embodiments, the surfactant 1 comprises one or both of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt) and polyoxyethylene lauroyl ether.

[0022] In some embodiments, the preservative 1 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT.

[0023] In some embodiments, the stabilizer 1 includes one or more of SA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose, and mannitol.

[0024] In some embodiments, the neutral buffer 1 includes one or more of phosphate buffer, HEPES buffer, and MOPOS buffer.

[0025] In some embodiments, the diluent 1 comprises 0.8 wt% to 1.0 wt% NaCl, 0.8 wt% to 1.2 wt% sucrose, 0.08 v / v% to 0.12 v / v% (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), 0.04 v / v% to 0.06 v / v% polyoxyethylene lauroyl ether, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and HEPES buffer with a pH of 6.0 to 8.0 and a concentration of 0.01 mol / L to 0.2 mol / L.

[0026] In some embodiments, the concentration of the antibody-solid-phase carrier conjugate or streptavidin-solid-phase carrier conjugate is 0.3 mg / mL to 0.9 mg / mL.

[0027] In some embodiments, the kit includes a detection reagent 2, which includes the antibody 2-chemiluminescent label and diluent 2; or includes the antibody 1-biotin label, the antibody 2-chemiluminescent label and diluent 2.

[0028] In some embodiments, the diluent 2 includes salt ions 2, surfactant 2, preservative 2, stabilizer 2, dissociating agent, blocking agent, and neutral buffer 2.

[0029] In some embodiments, the salt ion 2 includes one or more of NaCl, ammonium sulfate, and KCl.

[0030] In some embodiments, the surfactant 2 includes one or both of Tween-20 and polyoxyethylene lauroyl ether.

[0031] In some embodiments, the preservative 2 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT.

[0032] In some embodiments, the stabilizer 2 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, cystine, PEG, trehalose, and mannitol.

[0033] In some embodiments, the dissociating agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, messo, and tris(2-carbonylethyl)phosphohydrochloride.

[0034] In some embodiments, the blocking agent includes one or more of MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, and goat IgG.

[0035] In some embodiments, the neutral buffer 2 includes one or more of phosphate buffer, MES buffer, Tris buffer, and HEPES buffer.

[0036] In some embodiments, the concentration of the antibody-biotin-labeled antibody is 0.5 μg / mL to 2.0 μg / mL.

[0037] In some embodiments, the concentration of the antibody 2-luminescent label is 0.5 μg / mL to 2.0 μg / mL.

[0038] In some embodiments, the diluent 2 comprises 0.8 wt%–1.0 wt% NaCl, 0.08 v / v%–0.12 v / v% Tween-20, 0.04 wt%–0.06 wt% polyoxyethylene lauroyl ether, 0.05 v / v%–0.5 v / v% proclin 300, 0.002 wt%–0.004 wt% gentamicin sulfate, 1.8 wt%–2.2 wt% bovine serum albumin, 4.8 wt%–5.2 wt% trehalose, 4.8 wt%–5.2 wt% mannitol, and 80 μg / mL–120 μg / mL MAK33. Poly(hydroxychloroquine), 80 μg / mL–120 μg / mL rabbit IgG, 80 μg / mL–120 μg / mL mouse IgG, 80 μg / mL–120 μg / mL bovine IgG, 80 μg / mL–120 μg / mL goat IgG, 0.8 mg / mL–1.2 mg / mL 8-aniline-1-naphthalenesulfonic acid, and PBS buffer at pH 7.3–7.5 and concentrations of 0.08 mol / L–0.12 mol / L.

[0039] In some embodiments, the kit includes a detection reagent 3; the detection reagent 3 includes the triiodothyronine antigen calibrator and a diluent 3.

[0040] The diluent 3 includes salt ions 3, surfactant 3, preservative 3, stabilizer 3, and neutral buffer 3.

[0041] In some embodiments, the salt ion 3 includes one or both of NaCl and KCl.

[0042] In some embodiments, the surfactant 3 includes Tween-20 and polyoxyethylene lauroyl ether.

[0043] In some embodiments, the preservative 3 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT.

[0044] In some embodiments, the stabilizer 3 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose, and mannitol.

[0045] In some embodiments, the neutral buffer 3 includes one or more of phosphate buffer, Tris buffer, and HEPES buffer.

[0046] In some embodiments, the diluent 3 comprises 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 1.5 wt% to 2.5 wt% trehalose, 1.5 wt% to 2.5 wt% mannitol, 0.04 v / v% to 0.06 v / v% proclin 300, and HEPES buffer at a pH of 7.3 to 7.5 and a concentration of 0.04 mol / L to 0.06 mol / L.

[0047] In some embodiments, the preparation method includes the following steps:

[0048] Provide the antibody 1-solid-phase carrier conjugate, the antibody 2-chemiluminescent label, and the triiodothyronine antigen calibrator, respectively; or,

[0049] A detection kit is prepared by providing the streptavidin-solid-phase carrier conjugate, the antibody 1-biotin label, the antibody 2-chemiluminescent label, and the triiodothyronine antigen calibrator, respectively.

[0050] In some embodiments, the preparation steps of the antibody 1-solid-phase carrier conjugate include:

[0051] Antibody 1 and solid-phase carrier undergo a first coupling reaction. Blocking agent 1 and blocking agent 2 are added sequentially to the obtained first coupling reaction product for blocking, thereby preparing the antibody 1-solid-phase carrier conjugate.

[0052] The preparation steps of the antibody-2-chemiluminescent label include:

[0053] The antibody 2 solution was subjected to a second coupling reaction with the chemiluminescent label to prepare the antibody 2-chemiluminescent label.

[0054] In some embodiments, the blocking agent 1 comprises 1.4 wt% to 1.5 wt% glycine, 0.8 wt% to 1.2 wt% sucrose, 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and PBS buffer at a pH of 5.8 to 6.2 and a concentration of 0.04 to 0.06 mol / L.

[0055] Optionally, the blocking agent 2 comprises 0.4 wt% to 0.6 wt% poloxamer, 0.8 wt% to 1.2 wt% sucrose, 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and HEPES buffer at a pH of 7.3 to 7.5 and a concentration of 0.04 mol / L to 0.06 mol / L.

[0056] In some embodiments, the preparation steps of the streptavidin-solid-phase carrier conjugate include: a second coupling reaction of streptavidin and a solid-phase carrier, followed by the sequential addition of the blocking agent 1 and the blocking agent 2 to the resulting second coupling reaction product for blocking, thereby preparing the streptavidin and solid-phase carrier.

[0057] The method for preparing the antibody 1-biotin label includes: performing a third coupling reaction between antibody 1 and biotin label to prepare the antibody 1-biotin label.

[0058] In some embodiments, the conditions for the first coupling reaction include: the sealing agent 1 and the sealing agent 2 are sealed at a temperature of 36°C to 38°C for a time of 20h to 28h.

[0059] The conditions for the second coupling reaction include: the mass ratio of the antibody 2 to the chemiluminescent label is (28-32):1, the reaction conditions are 36℃-38℃, and the time is 1h-3h.

[0060] The conditions for the third coupling reaction include: the sealing temperature of the blocking agent 1 and the blocking agent 2 is 36℃~38℃, and the sealing time is 20h~28h.

[0061] This application also provides a method for detecting triiodothyronine, the method comprising the step of detecting triiodothyronine in a sample using the triiodothyronine chemiluminescent immunoassay kit.

[0062] In some embodiments, the sample to be tested is one or both of serum and plasma.

[0063] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Detailed Implementation

[0064] 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. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0065] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0066] the term

[0067] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0068] 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. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0069] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0070] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0071] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0072] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0073] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0074] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0075] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0076] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0077] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0078] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0079] Free T3 (FT3) is an unbound, biologically active form of T3, comprising only 0.2-0.4% of total T3. Theoretically, FT3 detection is unaffected by the concentration and binding affinity of binding proteins, thus eliminating the need for additional tests reflecting binding parameters (such as TBG). Therefore, FT3 measurement is an important indicator for routine clinical assessment of thyroid function. FT3 results can serve as a basis for the differential diagnosis of thyroid diseases, distinguishing different types of hyperthyroidism and confirming T3 hyperthyroidism.

[0080] The term "FT3" stands for Free Triiodothyronine.

[0081] The term "T3" stands for Total Triiodothyronine.

[0082] It is understood that FT3 and T3 mentioned below are abbreviations, which stand for free triiodothyronine or total triiodothyronine.

[0083] In recent years, non-competitive methods for small molecule immunoassays have developed rapidly, including open-cell immunoassays based on haptens, non-competitive methods based on enzyme-labeled analyte-analyte antibody site substitution, sandwich methods based on other recognition materials, and double-antibody sandwich methods developed based on anti-immune complex antibodies. With the development of phage display technology and autonomous diversified library technology, the accurate identification of new antigenic epitopes in antigen-antibody complexes has been effectively solved, leading to more efficient and accurate development of anti-immune complex antibodies; thus, they have a promising application prospect in small molecule sandwich immunoassays. This application selects highly sensitive and specific monoclonal antibodies developed based on antibody immune complexes and develops a highly sensitive and rapid immunoassay method for detecting FT3 / T3 on an electrochemiluminescence platform.

[0084] This application provides a method for determining FT3 / T3 in a sample: by forming a sandwich complex with T3 monoclonal antibody 1, T3 monoclonal antibody 2 and FT3 / T3 antigen in the sample, combined with a fully automated electrochemiluminescence analyzer, the content of FT3 / T3 in the sample can be determined quickly, sensitively and accurately, while reducing operation time and reducing human error.

[0085] In some embodiments, a chemiluminescent immunoassay kit for triiodothyronine is provided, the kit comprising:

[0086] The antibody 1-solid-phase carrier conjugate, antibody 2-chemiluminescent label, and triiodothyronine antigen calibrator, wherein the antibody 1-solid-phase carrier conjugate comprises a solid-phase carrier coated with triiodothyronine antibody 1, and the antibody 2-luminescent label comprises triiodothyronine antibody 2 labeled with a chemiluminescent label; both the antibody 1-solid-phase carrier conjugate and the antibody 2-luminescent label can specifically bind to the target analyte and have different antigen recognition epitopes; or,

[0087] This application provides a chemiluminescent immunoassay kit for triiodothyronine (FT3) using a streptavidin-solid-phase carrier conjugate, antibody 1 (biotin-labeled), antibody 2 (chemiluminescent), and a triiodothyronine antigen calibrator. The antibody 1 (biotin-labeled) comprises biotin-labeled triiodothyronine antibody 1, and the antibody 2 (chemiluminescent) comprises chemiluminescently labeled triiodothyronine antibody 2. Both antibody 1 (biotin-labeled) and antibody 2 (chemiluminescent) specifically bind to the target analyte and have different antigen recognition epitopes. This application is the first to use a double-antibody sandwich method on an electrochemiluminescence platform to detect FT3 / T3, effectively improving detection sensitivity, increasing the linear range, and enhancing repeatability.

[0088] In some embodiments, the solid support in the antibody 1-solid support conjugate includes magnetic beads.

[0089] In some embodiments, the functional groups modified on the surface of the magnetic beads include one or more of carboxyl, amino, hydroxyl, epoxy, and p-toluenesulfonyl groups.

[0090] In some embodiments, the active groups modified on the biotin include one or more of NHS ester groups, amino groups, maleimide groups, and hydrazide groups.

[0091] In some embodiments, the chemiluminescent label includes one or more of Ru-NHS, horseradish peroxidase, alkaline phosphatase, acridine ester, luminol and its derivatives, isoluminol and its derivatives, adamantane and acridine compounds-NHS.

[0092] In some embodiments, the antibodies in antibody 1-solid-phase carrier conjugate and / or antibody 2-chemiluminescent label are monoclonal antibodies.

[0093] In some embodiments, the test kit includes test reagent 1, which includes antibody 1-solid-phase carrier conjugate and diluent 1; or streptavidin-solid-phase carrier conjugate and diluent 1.

[0094] In some embodiments, the diluent 1 includes salt ions 1, surfactant 1, preservative 1, stabilizer 1, and neutral buffer 1.

[0095] In some embodiments, salt ion 1 includes one or both of NaCl and KCl.

[0096] In some embodiments, surfactant 1 comprises one or both of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt) and polyethylene lauroyl ether. The mass ratio of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt to polyethylene lauroyl ether is (1-3):1. For example, the ratio of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt to polyethylene lauroyl ether is 1:1, 2:1, 3:1, or any value in between.

[0097] In some embodiments, preservative 1 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT.

[0098] In some embodiments, stabilizer 1 includes one or more of SA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose, and mannitol.

[0099] In some embodiments, neutral buffer 1 includes one or more of phosphate buffer, HEPES buffer, and MOPOS buffer.

[0100] In some embodiments, diluent 1 comprises 0.8 wt% to 1.0 wt% NaCl, 0.8 wt% to 1.2 wt% sucrose, 0.08 v / v% to 0.12 v / v% (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), 0.04 v / v% to 0.06 v / v% polyoxyethylene lauroyl ether, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and HEPES buffer with a pH of 6.0 to 8.0 and a concentration of 0.01 mol / L to 0.2 mol / L.

[0101] For example, the concentration of NaCl can be 0.8 wt%, 0.9 wt%, or 1.0 wt%, or any value in between.

[0102] For example, sucrose concentrations of 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, and any value in between.

[0103] For example, (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), also known as CHAPS, has a concentration of 0.08 v / v%, 0.09 v / v%, 0.10 v / v%, 0.11 v / v%, or 0.12 v / v%, and any value in between.

[0104] For example, polyoxyethylene lauroyl ether, also known as Brij 35, has concentrations of 0.04 v / v%, 0.05 v / v%, 0.06 v / v%, and any intermediate value.

[0105] For example, bovine serum albumin concentrations of 1.4 wt%, 1.5 wt%, or 1.6 wt%, or any value in between.

[0106] For example, the concentration of proclin300 is 0.05 v / v%, 0.10 v / v%, 0.15 v / v%, 0.20 v / v%, 0.25 v / v%, 0.30 v / v%, 0.35 v / v%, 0.40 v / v%, 0.45 v / v%, or 0.50 v / v%, and any value in between.

[0107] For example, pH values ​​of 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, and 8.0, with HEPES buffer concentrations of 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, and 0.05 mol / L. , 0.06mol / L, 0.07mol / L, 0.08mol / L, 0.09mol / L, 0.10mol / L, 0.11mol / L, 0.12mol / L, 0.13 mol / L, 0.14mol / L, 0.15mol / L, 0.16mol / L, 0.17mol / L, 0.18mol / L, 0.19mol / L or 0.20mol / L.

[0108] In some of these embodiments, the concentration of antibody 1-solid-phase carrier conjugate or streptavidin-solid-phase carrier conjugate is 0.3 mg / mL to 0.9 mg / mL.

[0109] For example, the concentrations of antibody-solid-phase carrier conjugates are 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, and 0.9 mg / mL.

[0110] For example, the concentrations of streptavidin-solid-phase carrier conjugates are 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, and 0.9 mg / mL.

[0111] In some embodiments, the kit includes a detection reagent 2, which includes antibody 2-chemiluminescent label and diluent 2; or includes antibody 1-biotin label, antibody 2-chemiluminescent label and diluent 2.

[0112] In some embodiments, the diluent 2 includes salt ions 2, surfactant 2, preservative 2, stabilizer 2, dissociating agent, blocking agent, and neutral buffer 2.

[0113] In some embodiments, salt ion 2 includes one or more of NaCl, ammonium sulfate, and KCl.

[0114] In some embodiments, surfactant 2 includes one or both of Tween-20 and polyethylene lauroyl ether. The mass ratio of Tween-20 to polyethylene lauroyl ether is (1–3):1. For example, the ratio of Tween-20 to polyethylene lauroyl ether is 1:1, 2:1, 3:1, or any value in between.

[0115] In some embodiments, preservative 2 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT.

[0116] In some embodiments, stabilizer 2 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, cystine, PEG, trehalose, and mannitol.

[0117] In some embodiments, the dissociating agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, messo, and tris(2-carbonylethyl)phosphohydrochloride.

[0118] In some embodiments, the blocking agent includes one or more of MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, and goat IgG.

[0119] In some embodiments, neutral buffer 2 includes one or more of phosphate buffer, MES buffer, Tris buffer, and HEPES buffer.

[0120] In some embodiments, the concentration of antibody-1-biotinylated marker is 0.5 μg / mL to 2.0 μg / mL. For example, the concentrations of antibody-1-biotinylated marker are 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL, and 2.0 μg / mL.

[0121] In some embodiments, the concentration of antibody 2-luminescent label is 0.5 μg / mL to 2.0 μg / mL. For example, the concentration of antibody 2-luminescent label is 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL, or 2.0 μg / mL, or any value in between.

[0122] In some embodiments, diluent 2 comprises 0.8 wt%–1.0 wt% NaCl, 0.08 v / v%–0.12 v / v% Tween-20, 0.04 wt%–0.06 wt% polyoxyethylene lauroyl ether, 0.05 v / v%–0.5 v / v% proclin 300, 0.002 wt%–0.004 wt% gentamicin sulfate, 1.8 wt%–2.2 wt% bovine serum albumin, 4.8 wt%–5.2 wt% trehalose, 4.8 wt%–5.2 wt% mannitol, and 80 μg / mL–120 μg / mL MAK33. Poly(hydroxychloroquine), 80 μg / mL–120 μg / mL rabbit IgG, 80 μg / mL–120 μg / mL mouse IgG, 80 μg / mL–120 μg / mL bovine IgG, 80 μg / mL–120 μg / mL goat IgG, 0.8 mg / mL–1.2 mg / mL 8-aniline-1-naphthalenesulfonic acid, and PBS buffer at pH 7.3–7.5 and concentrations of 0.08 mol / L–0.12 mol / L.

[0123] For example, the concentration of NaCl can be 0.8 wt%, 0.9 wt%, or 1.0 wt%, or any value in between.

[0124] For example, Tween-20 concentrations are 0.08 v / v%, 0.09 v / v%, 0.10 v / v%, 0.11 v / v%, or 0.12 v / v%, or any value in between. Polyethylene lauroyl ether, also known as Brij 35, has concentrations of 0.04 v / v%, 0.05 v / v%, or 0.06 v / v%, or any value in between. Bovine serum albumin concentrations are 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, or 2.2 wt%, or any value in between. Trehalose concentrations are 4.8 wt%, 4.9 wt%, 5.0 wt%, 5.1 wt%, or 5.2 wt%, or any value in between. Mannitol concentrations are 4.8 wt%, 4.9 wt%, 5.0 wt%, 5.1 wt%, or 5.2 wt%, or any value in between. For example, the concentrations of MAK33 Poly are 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, or 120 μg / mL, or any value in between. For example, the concentrations of rabbit IgG are 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, or 120 μg / mL, or any value in between. For example, the concentrations of mouse IgG are 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, or 120 μg / mL, or any value in between. For example, bovine IgG concentrations can be 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, or 120 μg / mL, or any value in between. Similarly, goat IgG concentrations can be 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, or 120 μg / mL, or any value in between. Likewise, 8-aniline-1-naphthalenesulfonic acid concentrations can be 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, or 1.2 mg / mL, or any value in between. For example, the pH value is 7.3, 7.4, or 7.5, and the PBS buffer concentration is any value among 0.08 mol / L, 0.09 mol / L, 0.10 mol / L, 0.11 mol / L, or 0.12 mol / L.

[0125] In some embodiments, the kit includes a test reagent 3; the test reagent 3 includes a triiodothyronine antigen calibrator and a diluent 3.

[0126] The diluent 3 includes salt ions 3, surfactant 3, preservative 3, stabilizer 3, and neutral buffer 3.

[0127] In some embodiments, the salt ion 3 includes one or both of NaCl and KCl.

[0128] In some embodiments, surfactant 3 comprises Tween-20 and polyethylene lauroyl ether. The mass ratio of Tween-20 to polyethylene lauroyl ether is (1–3):1. For example, the ratio of Tween-20 to polyethylene lauroyl ether is 1:1, 2:1, 3:1, or any value in between.

[0129] In some embodiments, preservative 3 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT.

[0130] In some embodiments, stabilizer 3 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose, and mannitol.

[0131] In some embodiments, neutral buffer 3 includes one or more of phosphate buffer, Tris buffer, and HEPES buffer.

[0132] In some embodiments, diluent 3 comprises 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 1.5 wt% to 2.5 wt% trehalose, 1.5 wt% to 2.5 wt% mannitol, 0.04 v / v% to 0.06 v / v% proclin 300, and HEPES buffer at a pH of 7.3 to 7.5 and a concentration of 0.04 mol / L to 0.06 mol / L.

[0133] For example, Tween-20 concentrations are 0.04 v / v%, 0.05 v / v%, 0.06 v / v%, and any intermediate value. For example, bovine serum albumin concentrations are 1.4 wt%, 1.5 wt%, or 1.6 wt%, and any intermediate value. For example, trehalose concentrations are 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, or 2.5 wt%, and any intermediate value. For example, mannitol concentrations are 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, or 2.5 wt%, and any intermediate value. For example, proclin 300 concentrations can be 0.04 v / v%, 0.05 v / v%, or 0.06 v / v%, or any value in between. For example, pH values ​​can be 7.3, 7.4, or 7.5, and PBS buffer concentrations can be 0.04 mol / L, 0.05 mol / L, or 0.06 mol / L, or any value in between.

[0134] This application also provides a method for preparing a chemiluminescent immunoassay kit for triiodothyronine, the method comprising the following steps:

[0135] Antibody 1-solid phase carrier conjugate, antibody 2-chemiluminescent label, and triiodothyronine antigen calibrator are provided respectively.

[0136] A detection kit was prepared by providing streptavidin-solid-phase carrier conjugate, antibody 1-biotin label, antibody 2-chemiluminescent label, and triiodothyronine antigen calibrator.

[0137] In some embodiments, antibody 1 and solid-phase support undergo a first coupling reaction, and blocking agent 1 and blocking agent 2 are added sequentially to the resulting first coupling reaction product for blocking, thereby preparing antibody 1-solid-phase support conjugate.

[0138] In some embodiments, the preparation steps of antibody 2-chemiluminescent label include:

[0139] The antibody 2 solution was subjected to a second coupling reaction with the chemiluminescent label to prepare the antibody 2-chemiluminescent label.

[0140] In some embodiments, blocking agent 1 comprises 1.4 wt% to 1.5 wt% glycine, 0.8 wt% to 1.2 wt% sucrose, 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and PBS buffer at a pH of 5.8 to 6.2 and a concentration of 0.04 mol / L to 0.06 mol / L.

[0141] For example, glycine concentrations can be 1.4 wt%, 1.5 wt%, or 1.6 wt%, or any intermediate value. Sucrose concentrations can be 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, or 1.2 wt%, or any intermediate value. Tween-20 concentrations can be 0.04 v / v%, 0.05 v / v%, 0.06 v / v%, or any intermediate value. Bovine serum albumin concentrations can be 1.4 wt%, 1.5 wt%, or 1.6 wt%, or any intermediate value. Proclin 300 concentrations can be 0.05 v / v%, 0.10 v / v%, 0.15 v / v%, 0.20 v / v%, 0.25 v / v%, 0.30 v / v%, 0.35 v / v%, 0.40 v / v%, 0.45 v / v%, or 0.50 v / v%, or any intermediate value. For example, PBS buffers with pH values ​​of 5.8, 5.9, 6.0, 6.1, or 6.2, and concentrations of 0.04 mol / L, 0.05 mol / L, or 0.06 mol / L.

[0142] In some embodiments, the blocking agent 2 comprises 0.4 wt% to 0.6 wt% poloxamer (i.e., F68), 0.8 wt% to 1.2 wt% sucrose, 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and HEPES buffer at a pH of 7.3 to 7.5 and a concentration of 0.04 mol / L to 0.06 mol / L.

[0143] For example, poloxamer concentrations are 0.4 wt%, 0.5 wt%, or 0.6 wt%, or any intermediate value. For example, sucrose concentrations are 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, or 1.2 wt%, or any intermediate value. For example, Tween-20 concentrations are 0.04 v / v%, 0.05 v / v%, 0.06 v / v%, or any intermediate value. For example, bovine serum albumin concentrations are 1.4 wt%, 1.5 wt%, or 1.6 wt%, or any intermediate value. For example, proclin 300 concentrations are 0.05 v / v%, 0.10 v / v%, 0.15 v / v%, 0.20 v / v%, 0.25 v / v%, 0.30 v / v%, 0.35 v / v%, 0.40 v / v%, 0.45 v / v%, or 0.50 v / v%, or any intermediate value. For example, the pH value is 7.3, 7.4, or 7.5, and the HEPES buffer concentration is any value among 0.04 mol / L, 0.05 mol / L, or 0.06 mol / L.

[0144] In some embodiments, the preparation steps of the streptavidin-solid-phase carrier conjugate include: a second coupling reaction between streptavidin and a solid-phase carrier, followed by the sequential addition of blocking agent 1 and blocking agent 2 to the resulting second coupling reaction product for blocking, thereby preparing streptavidin and a solid-phase carrier.

[0145] In some embodiments, the preparation method of antibody 1-biotin label includes: antibody 1 and biotin label undergoing a third coupling reaction to prepare antibody 1-biotin label.

[0146] In some embodiments, the conditions for the first coupling reaction include: the sealing temperature of blocking agent 1 and blocking agent 2 is 36°C to 38°C, and the time is 20h to 28h. For example, the temperature is 36°C, 36.5°C, 37°C, 37.5°C, or 38°C, and any value in between. The time is 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, and 28h, and any value in between.

[0147] In some embodiments, the conditions for the second coupling reaction include: a mass ratio of antibody 2 to chemiluminescent label of (28-32):1, a reaction temperature of 36°C-38°C, and a reaction time of 1-3 hours.

[0148] For example, the mass ratio is 28:1, 29:1, 30:1, 31:1 or 32:1, the reaction temperature is 36℃, 36.5℃, 37℃, 37.5℃ or 38℃, and the time is 1h, 2h or 3h.

[0149] In some embodiments, the conditions for the third coupling reaction include: sealing the blocking agent 1 and blocking agent 2 at a temperature of 36°C to 38°C for a time of 20h to 28h. For example, the temperature can be 36°C, 36.5°C, 37°C, 37.5°C, or 38°C, or any value in between. The time can be 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, or 28h, or any value in between.

[0150] This application also provides a method for detecting a target analyte, the method comprising the step of detecting the target analyte in a sample using the aforementioned triiodothyronine chemiluminescent immunoassay kit. For example, the target analyte may include triiodothyronine.

[0151] In some of these embodiments, the samples to be tested are serum and plasma.

[0152] This application achieves further performance improvement through special designs such as a special magnetic bead blocking process, a special magnetic bead blocking solution formulation, a special surfactant design for the antibody diluent, and the addition of T3 free antibody to the antibody diluent.

[0153] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, 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.

[0154] In the specific embodiments described below, the measurement parameters involving 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.

[0155] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0156] The application provides a method for determining FT3 / T3 in a sample: by forming a sandwich complex with T3 monoclonal antibody 1, T3 monoclonal antibody 2 and FT3 / T3 antigen in the sample, combined with a fully automated electrochemiluminescence analyzer, the content of FT3 / T3 in the sample can be determined quickly, sensitively and accurately, while reducing operation time and reducing human error.

[0157] Example 1

[0158] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine, which uses a streptavidin (SA) and biotin system.

[0159] I. Composition and preparation of the triiodothyronine chemiluminescent immunoassay kit:

[0160] 1. Preparation of streptavidin-coupled magnetic microparticle solution

[0161] Streptavidin-coated magnetic beads were blocked at 37°C for 24 hours using magnetic bead blocking solution 1 and magnetic bead blocking solution 2, respectively.

[0162] The magnetic bead blocking solution 1 comprises a PBS buffer with a pH of 6.0 and a concentration of 0.05 mol / L. The buffer contains 1.5 wt% glycine, 1 wt% sucrose, 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt% BSA, and 0.25 v / v% proclin300.

[0163] The magnetic bead blocking solution 2 consists of HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, which contains 0.5 wt% poloxamer, 1 wt% sucrose, 0.05 v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0164] The magnetic bead suspension was then magnetically separated using a magnetic separator. After removing the supernatant, it was diluted with diluent 1. The diluent for the streptavidin-coupled magnetic microparticles is denoted as diluent 1.

[0165] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.1 v / v% chaps, 0.05 v / v% Brij35 (polyethylene lauroyl ether), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0166] 2. Preparation of T3 / FT3 monoclonal antibody dilution buffer

[0167] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, consists of a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.1 v / v% TWEEN-20, 0.05 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent.

[0168] The T3 test formulation contains a dissociation agent, while the FT3 test formulation does not.

[0169] 3. Preparation of Biotin-labeled T3 / FT3 Monoclonal Antibody 1

[0170] T3 / FT3 monoclonal antibody 1 was placed in a pre-treated ultrafiltration tube and purified by ultrafiltration and centrifugation three times using pH 8.0 25mM Tris-HCl buffer. The purified antibody was transferred to a collection tube, and the concentration was tested and adjusted to 2 mg / mL. N-hydroxysuccinimide-activated biotin was mixed with T3 / FT3 monoclonal antibody 1 at a molar ratio of 20:1 at 22°C for 1 hour. Unreacted biotin was removed by ultrafiltration or dialysis to obtain biotin-labeled T3 / FT3 monoclonal antibody 1.

[0171] 4. Preparation of ruthenium tripyridine-labeled T3 / FT3 monoclonal antibody 2

[0172] T3 / FT3 monoclonal antibody 2 was placed in a pre-treated ultrafiltration tube and purified by ultrafiltration three times using pH 8.0 25mM Tris-HCl buffer and centrifugation. The purified antibody was transferred to a collection tube, and the concentration was tested and adjusted to 2 mg / mL. The T3 / FT3 monoclonal antibody 2 solution was mixed with an acridine compound Ru-NHS ester solution at a mass ratio of 30:1 and reacted at 37°C in the dark for 2 hours. The reaction was then terminated by adding glycine solution. The reaction solution was dialyzed to remove excess Ru-NHS ester, yielding ruthenium tripyridine-labeled T3 / FT3 monoclonal antibody 2.

[0173] 5. Preparation of calibrators and quality control samples

[0174] Prepare the matrix for T3 / FT3 calibration quality control samples. The diluent for the calibrator and quality control samples is denoted as diluent 3.

[0175] Diluent 3 includes HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and a buffer containing 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt% BSA, 2 wt% trehalose, 2 wt% mannitol, and 0.05 v / v% proclin 300. This matrix is ​​used to dilute T3 / FT3 antigen to prepare calibrators and quality control samples.

[0176] II. Instructions for use of the Triiodothyronine Chemiluminescent Immunoassay Kit:

[0177] 1. Sample pretreatment: Take the sample to be tested (e.g., serum, plasma or artificially prepared sample), centrifuge at 4℃ and 5000rpm for 5min and then set aside.

[0178] 2. Before using the reagent, place it in the Pumen Company's fully automatic electrochemiluminescence analyzer 30 minutes in advance to automatically mix the magnetic bead particles and keep them in suspension.

[0179] 3. Calibration and quality control of the reagent kit: Perform calibration tests using the matching calibrators, and determine the accuracy of the current system through the results of quality control.

[0180] 4. Sample testing:

[0181] The triiodothyronine chemiluminescent immunoassay kit was used to detect the concentrations of T3 and FT3 in the samples. The specific testing procedure is as follows:

[0182] (1) Preparation of antibody working solution:

[0183] The biotin-labeled T3 monoclonal antibody 1 was diluted to a working concentration of 0.7 μg / mL using the above-mentioned diluent 2 to obtain the working solution of biotin-labeled T3 monoclonal antibody 1.

[0184] The biotin-labeled FT3 monoclonal antibody 1 was diluted to a working concentration of 0.5 μg / mL using the above-mentioned diluent 2 to obtain the working solution of biotin-labeled FT3 monoclonal antibody 1.

[0185] The ruthenium tripyridine-labeled T3 monoclonal antibody 2 was diluted to a working concentration of 0.5 μg / mL using the above-mentioned diluent 2 to obtain the working solution of ruthenium tripyridine-labeled T3 monoclonal antibody 2.

[0186] The FT3 monoclonal antibody 2 labeled with ruthenium tripyridine was diluted to a working concentration of 0.4 μg / mL using the above-mentioned diluent 2 to obtain the working solution of the FT3 monoclonal antibody 2 labeled with ruthenium tripyridine.

[0187] (2) After the user requests a test, the system automatically aspirates 60 μL of biotin-labeled T3 monoclonal antibody 1 working solution, 60 μL of ruthenium tripyridine-labeled T3 monoclonal antibody 2 working solution, 40 μL of streptavidin-coated magnetic microparticles, and 10 μL of the test sample and adds them together to a reaction vessel; the system also automatically aspirates 60 μL of biotin-labeled FT3 monoclonal antibody 1 working solution, 60 μL of ruthenium tripyridine-labeled FT3 monoclonal antibody 2 working solution, 40 μL of streptavidin-coated magnetic microparticles, and 10 μL of the test sample and adds them together to another reaction vessel. The mixture is automatically incubated at 37°C for 9 minutes to form an antigen-antibody sandwich complex. The entire complex then binds to the magnetic microparticles through the interaction of biotin and streptavidin.

[0188] (3) After incubation, the system automatically draws the reaction mixture into the measuring cell. The magnetic particles are adsorbed onto the electrode by the magnet. Unbound substances are washed away. After voltage is applied to the electrode, chemiluminescence is generated. The generated light signal is detected by the photomultiplier tube. The detection result is automatically obtained by the instrument from the calibration curve (this calibration curve is obtained by the instrument through two-point calibration of the main calibration curve obtained by scanning).

[0189] Example 2

[0190] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine, which uses an antibody-coated magnetic bead system.

[0191] I. Composition and preparation of the triiodothyronine chemiluminescent immunoassay kit:

[0192] 1. Preparation of magnetic microparticles coated with T3 / FT3 monoclonal antibody 1

[0193] Select toluenesulfonyl-modified magnetic beads with a particle size of 3 μm. Take 10 mg of magnetic beads and wash 5 times with coating buffer 1 (0.1 M borate buffer). Then add coating buffer 1 (0.1 M borate buffer), coating buffer 2 (3 M ammonium sulfate borate buffer), coating buffer 3 (0.5% proclin 300), and the T3 / FT3 monoclonal antibody to be coated. Mix and coat at 37℃ for 24 hours. Then block with magnetic bead blocking buffer 1 and magnetic bead blocking buffer 2 at 37℃ for 24 hours. Finally, replace with an equal volume of magnetic bead dilution buffer and store at 4℃.

[0194] The magnetic bead blocking solution 1 includes a PBS buffer with a pH of 6.0 and a concentration of 0.05 mol / L. The buffer contains 1.5 wt% glycine, 1 wt% sucrose, 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt% BSA, and 0.25 v / v% proclin300.

[0195] The magnetic bead blocking solution 2 includes a 0.05 mol / L HEPES buffer at pH 7.4, containing 0.5 wt% poloxamer, 1 wt% sucrose, 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0196] Before use, prepare a magnetic bead working solution with a concentration of 0.75 mg / mL using ordinary magnetic bead diluent (0.1 M Tris, 1% wt% BSA, 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 0.05 v / v% proclin 300).

[0197] 2. Preparation of T3 / FT3 monoclonal antibody dilution buffer

[0198] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, consists of a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.1 v / v% TW-20, 0.05 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent.

[0199] The T3 test formulation contains a dissociation agent, while the FT3 test formulation does not.

[0200] 3. Preparation of ruthenium tripyridine-labeled T3 / FT3 monoclonal antibody 2

[0201] T3 / FT3 monoclonal antibody 2 was placed in a pre-treated ultrafiltration tube and purified by ultrafiltration three times using pH 8.0 25mM Tris-HCl buffer and centrifugation. The purified antibody was transferred to a collection tube, and the concentration was tested and adjusted to 2 mg / mL. The T3 / FT3 monoclonal antibody 2 solution was mixed with an acridine compound Ru-NHS ester solution at a mass ratio of 30:1 and reacted at 37°C in the dark for 2 hours. The reaction was then terminated by adding glycine solution. The reaction solution was dialyzed to remove excess Ru-NHS ester, yielding ruthenium tripyridine-labeled T3 / FT3 monoclonal antibody 2.

[0202] 4. Preparation of calibrators and quality control samples

[0203] Prepare the matrix for T3 / FT3 calibration quality control samples. The diluent for the calibrators and quality control samples is denoted as diluent 3.

[0204] Diluent 3 includes HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and a buffer containing 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt% BSA, 2 wt% trehalose, 2 wt% mannitol, and 0.05 v / v% proclin 300. This matrix is ​​used to dilute T3 / FT3 antigen to prepare calibrators and quality control samples.

[0205] II. Instructions for use of the Triiodothyronine Chemiluminescent Immunoassay Kit:

[0206] 1. Sample pretreatment: Take the sample to be tested (e.g., serum, plasma or artificially prepared sample), centrifuge at 4℃ and 5000rpm for 5min and then set aside.

[0207] 2. Before using the reagent, place it in the Pumen Company's fully automatic electrochemiluminescence analyzer 30 minutes in advance to automatically mix the magnetic bead particles and keep them in suspension.

[0208] 3. Calibration and quality control of the reagent kit: Perform calibration tests using the matching calibrators, and determine the accuracy of the current system through the results of quality control.

[0209] 4. Sample testing:

[0210] The triiodothyronine chemiluminescent immunoassay kit was used to detect the concentrations of T3 and FT3 in the samples. The specific testing procedure is as follows:

[0211] (1) Preparation of antibody working solution:

[0212] The magnetic microparticles coated with T3 monoclonal antibody 1 were diluted to a working concentration of 0.75 mg / mL using the above-mentioned diluent 2 to obtain the T3 magnetic microparticle working solution.

[0213] The magnetic microparticles coated with FT3 monoclonal antibody 1 were diluted to a working concentration of 0.75 mg / mL using the above-mentioned diluent 2 to obtain the FT3 magnetic microparticle working solution.

[0214] The ruthenium tripyridine-labeled T3 monoclonal antibody 2 was diluted to a working concentration of 0.5 μg / mL using the above-mentioned diluent 2 to obtain the working solution of ruthenium tripyridine-labeled T3 monoclonal antibody 2.

[0215] The FT3 monoclonal antibody 2 labeled with ruthenium tripyridine was diluted to a working concentration of 0.5 μg / mL using the above-mentioned diluent 2 to obtain the working solution of the FT3 monoclonal antibody 2 labeled with ruthenium tripyridine.

[0216] (2) After the user requests testing, the system automatically aspirates 40 μL of T3 magnetic microparticle working solution, 60 μL of ruthenium terpyridine-labeled T3 monoclonal antibody 2 working solution, and 10 μL of the test sample and adds them together to a reaction vessel; the system also automatically aspirates 40 μL of FT3 magnetic microparticle working solution, 60 μL of ruthenium terpyridine-labeled FT3 monoclonal antibody 2 working solution, and 10 μL of the test sample and adds them together to another reaction vessel. The mixture is automatically incubated at 37°C for 9 minutes to form an antigen-antibody sandwich complex. The entire complex then binds to the magnetic microparticles through the interaction of biotin and streptavidin.

[0217] (3) After incubation, the system automatically draws the reaction mixture into the measuring cell. The magnetic particles are adsorbed onto the electrode by the magnet. Unbound substances are washed away. After voltage is applied to the electrode, chemiluminescence is generated. The generated light signal is detected by the photomultiplier tube. The detection result is automatically obtained by the instrument from the calibration curve (this calibration curve is obtained by the instrument through two-point calibration of the main calibration curve obtained by scanning).

[0218] Example 3

[0219] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0220] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.12 v / v% chaps, 0.06 v / v% Brij35 (polyethylene lauroyl ether), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0221] Example 4

[0222] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0223] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.08 v / v% chaps, 0.04 v / v% Brij35 (polyethylene lauroyl ether), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0224] Example 5

[0225] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0226] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.08 v / v% chaps, 0.06 v / v% Brij35 (polyethylene lauroyl ether), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0227] Example 6

[0228] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0229] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.12 v / v% chaps, 0.04 v / v% Brij35 (polyethylene lauroyl ether), 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0230] Example 7

[0231] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0232] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.12 v / v% chaps, 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0233] Example 8

[0234] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0235] The components of diluent 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer at pH 7.4 and a concentration of 0.05 mol / L, and 0.9 wt% sodium chloride, 1 wt% sucrose, 0.06 v / v% Brij 35, 1.5 wt% BSA, and 0.25 v / v% proclin 300.

[0236] Example 9

[0237] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0238] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.12 v / v% TWEEN-20, 0.06 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, and 100 μg / mL goat IgG. The T3 assay formulation includes 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) as a dissociation agent, while the FT3 assay formulation does not contain a dissociation agent.

[0239] Example 10

[0240] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0241] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, consists of a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.08 v / v% TWEEN-20, 0.04 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation contains 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) as a dissociating agent, while the FT3 assay formulation does not contain a dissociating agent.

[0242] Example 11

[0243] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0244] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, consists of a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.08 v / v% TWEEN-20, 0.06 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation contains 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) as a dissociating agent, while the FT3 assay formulation does not contain a dissociating agent.

[0245] Example 12

[0246] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0247] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, consists of 0.1 mol / L PBS buffer at pH 7.4. Buffer 2 contains 0.9 wt% NaCl, 0.12 v / v% TWEEN-20, 0.04 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation contains 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) as a dissociating agent, while the FT3 assay formulation does not contain a dissociating agent.

[0248] Example 13

[0249] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0250] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.12 v / v% TWEEN-20, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation includes 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, while the FT3 assay formulation does not contain any dissociation agent.

[0251] Example 14

[0252] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0253] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.06 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 100 μg / mL rabbit IgG, 100 μg / mL mouse IgG, 100 μg / mL bovine IgG, 100 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation includes 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, while the FT3 assay formulation does not contain any dissociation agent.

[0254] Example 15

[0255] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0256] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.1 v / v% TWEEN-20, 0.05 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 500 μg / mL rabbit IgG, and 1 mg / mL dithiothreitol dissociation agent. The T3 assay formulation includes 1 mg / mL dithiothreitol dissociation agent, while the FT3 assay formulation does not.

[0257] Example 16

[0258] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0259] The T3 / FT3 monoclonal antibody dilution buffer, designated as Dilution Buffer 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.1 v / v% TWEEN-20, 0.05 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 100 μg / mL MAK33 Poly, 500 μg / mL MAK33 Poly, and 1 mg / mL mesna dissociation agent. The T3 assay formulation includes 1 mg / mL mesna dissociation agent, while the FT3 assay formulation does not.

[0260] Example 17

[0261] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0262] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.1 v / v% TWEEN-20, 0.05 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 300 μg / mL MAK33 Poly, 200 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation includes 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, while the FT3 assay formulation does not contain any dissociation agent.

[0263] Example 18

[0264] This embodiment provides a chemiluminescent immunoassay kit for triiodothyronine. The kit in this embodiment is largely the same as that in Example 1, with the main differences being as follows:

[0265] The T3 / FT3 monoclonal antibody diluent, designated as Diluent 2, comprises a 0.1 mol / L PBS buffer at pH 7.4. This buffer contains 0.9 wt% NaCl, 0.1 v / v% TWEEN-20, 0.05 wt% Brij35, 0.05 v / v% Proclin 300, 0.003 wt% gentamicin sulfate, 2 wt% BSA, 5 wt% trehalose, 5 wt% mannitol, 300 μg / mL rabbit IgG, 200 μg / mL goat IgG, and 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent. The T3 assay formulation includes 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, while the FT3 assay formulation does not contain any dissociation agent.

[0266] Performance testing

[0267] Reagent Kit Working Principle: The reagent kit is paired with a fully automated electrochemiluminescence analyzer, automating sample addition, incubation, washing, and detection steps, thus avoiding result deviations caused by human operation. After the serum or plasma sample is thoroughly mixed with the first and second reagents, the FT3 / T3 in the sample binds to monoclonal antibody 1 and monoclonal antibody 2 in the first and second reagents to form a sandwich complex, which is then immobilized onto the magnetic microparticles of the first reagent. The reaction mixture is then magnetically washed away to remove components not bound to the magnetic microparticles. The fully automated electrochemiluminescence analyzer emits a continuous and stable light signal. The concentration of the sample is calculated using a standard curve based on the light signal; the signal value is directly proportional to the concentration.

[0268] I. Limit of Detection

[0269] 1. Samples to be tested: The antigen was diluted with a matrix to prepare 5 samples with LoD concentration values ​​close to the original concentration (LoD). The LoD concentration values ​​were set as follows: T3: 0.08 ng / mL, FT3: 0.08 pg / mL.

[0270] Each of the above-mentioned prepared samples, in addition to containing the corresponding analyte (i.e., FT3 / T3), also contains the following components: Tris buffer for Proclin-300, with a concentration of 0.01 mol / L and a mass percentage of 0.05% for Proclin-300.

[0271] 2. The triiodothyronine chemiluminescent immunoassay kits from the above embodiments were used to detect the samples to be tested in step 1. Specifically, five samples with LoD concentrations close to the above values ​​were tested using two batches of reagents over five days, with each sample repeated eight times per day. After multiple batches of verification with the above samples, the test results all met the LoD requirements.

[0272] For details, please refer to "Performance Evaluation Methods for In Vitro Diagnostic Testing Systems, Part 3: Limit of Detection and Limit of Quantification" in the People's Republic of China Pharmaceutical Industry Standard YY1789.3-2022.

[0273] The detection limits are shown in Table 1.

[0274] As shown in Table 1, the detection limits of the kits in Examples 1 and 3-6 are lower than those in Examples 7-8, indicating that the synergistic effect of chapters and Brij35 in diluent 1 helps to lower the detection limit and improve the detection sensitivity of the kits. In particular, the detection limits of the kits in Examples 1 and 3-4 are relatively close and lower than those in Examples 5-6, further demonstrating that a mass ratio of 2 of chapters to Brij35 in diluent 1 is more conducive to lowering the detection limit of the kits.

[0275] The detection limits of the kits in Examples 1 and 9-12 are lower than those in Examples 13-14, indicating that the synergistic effect of TWEEN-20 and Brij35 in diluent 2 helps to lower the detection limit and improve the detection sensitivity of the kits. Furthermore, the detection limits of the kits in Examples 1 and 9-10 are relatively close and lower than those in Examples 11-12, further demonstrating that a mass ratio of TWEEN-20 to Brij35 of 2 in diluent 2 is more conducive to lowering the detection limit of the kits.

[0276] The detection limit of the kit in Example 17 was lower than that in Examples 15-16, indicating that the synergistic effect of MAK33 Poly and rabbit IgG in diluent 2 is beneficial to reducing the detection limit and improving the detection sensitivity of the kit. The detection limit of the kit in Example 1 was lower than that in Examples 17-18, indicating that in the kit of Example 1, the synergistic effect of various blocking agents in diluent 2 is more beneficial to reducing the detection limit of the kit.

[0277] In summary, the triiodothyronine chemiluminescent immunoassay kit of this application has a detection limit of no more than 0.08 ng / mL for T3 detection and no more than 0.08 pg / mL for FT3 detection, which is superior to the detection limits of conventional triiodothyronine assay kits (0.20 ng / mL for T3 and 0.28 pg / mL for FT3). The triiodothyronine chemiluminescent immunoassay kit of this application also exhibits high sensitivity.

[0278] Table 1. Detection limits of the triiodothyronine chemiluminescent immunoassay kits of each embodiment.

[0279] Example T3 (ng / mL) FT3 (pg / mL) 1 0.062 0.064 2 0.074 0.078 3 0.063 0.066 4 0.064 0.067 5 0.071 0.074 6 0.070 0.073 7 0.095 0.110 8 0.093 0.108 9 0.064 0.065 10 0.065 0.068 11 0.072 0.075 12 0.071 0.074 13 0.089 0.099 14 0.088 0.102 15 0.072 0.075 16 0.069 0.069 17 0.065 0.066 18 0.070 0.072

[0280] II. Linear Correlation Measurement

[0281] 1. Sample to be tested:

[0282] In the FT3 detection, sample X1 was prepared with an FT3 concentration of 0.069 pg / mL, and sample X7 was prepared with an FT3 concentration of 54.856 pg / mL. Samples X1 and X7 were mixed according to the proportions in Table 2 to obtain samples X2 to X6.

[0283] In the T3 test, X1 sample: a sample prepared with a T3 concentration of 0.056 ng / mL, and X7 sample: a sample prepared with a clinical T3 concentration of 10.450 ng / mL; X1 and X7 samples are mixed according to the proportions in Table 3 to obtain X2 to X6 samples.

[0284] Each of the above-mentioned prepared samples, in addition to containing the corresponding analyte (i.e., FT3 / T3), also contains the following components: Tris buffer for Proclin-300, with a concentration of 0.01 mol / L and a mass percentage of 0.05% for Proclin-300.

[0285] 2. Detection kit

[0286] The linear correlation was determined using the triiodothyronine chemiluminescent immunoassay kit from Example 1.

[0287] The test results are shown in Tables 2 and 3.

[0288] As shown in Table 2, the linear range of FT3 detection for the triiodothyronine chemiluminescent immunoassay kit of this application is 0.10 pg / mL to 50 pg / mL, which is wider than that of conventional triiodothyronine assay kits (FT3 detection linear range is 0.28 pg / mL to 32 pg / mL).

[0289] As shown in Table 3, the linear range of T3 detection for the triiodothyronine chemiluminescent immunoassay kit of this application is 0.08–10.00 ng / mL, which is wider than that of conventional triiodothyronine assay kits (the linear range of T3 detection is 0.40 ng / mL–6.50 ng / mL).

[0290] Table 2. Linear correlation results of FT3 detection in the triiodothyronine chemiluminescent immunoassay kit of this application.

[0291]

[0292] Table 3. Linear correlation results of T3 detection in the triiodothyronine chemiluminescent immunoassay kit of this application.

[0293]

[0294] III. Repeatability Test

[0295] 1. Sample to be tested:

[0296] In the FT3 test, low-value sample 1, medium-value sample 2, and high-value sample 3 were clinical blood samples whose FT3 concentrations met the requirements of 1.100±0.200pg / mL, 3.500±0.500pg / mL, and 40.00±4.000pg / mL, respectively.

[0297] In the T3 test, low-value sample 1, medium-value sample 2, and high-value sample 3 were clinical blood samples whose clinical T3 concentrations met the requirements of 0.500±0.100ng / mL, 1.500±0.300ng / mL, and 6.400±0.600ng / mL, respectively.

[0298] 2. Test kit

[0299] The linear correlation was determined using the triiodothyronine chemiluminescent immunoassay kit from Example 1.

[0300] The test results are shown in Tables 4 and 5.

[0301] As shown in Tables 4 and 5, the intra-assay repeatability of the triiodothyronine chemiluminescent immunoassay kit of this application is ≤5% for FT3 detection and ≤5% for T3 detection.

[0302] Table 4. Repeatability results of T3 detection for the triiodothyronine chemiluminescent immunoassay kit of this application.

[0303]

[0304] Table 5. Repeatability results of FT3 detection in the triiodothyronine chemiluminescent immunoassay kit of this application.

[0305]

[0306] In summary, the chemiluminescent immunoassay kit for triiodothyronine of this application showed the following results in FT3 detection: the limit of detection was improved from no more than 0.28 pg / mL (the limit of detection of conventional triiodothyronine assay kits) to no more than 0.08 pg / mL; the linear range was expanded from 0.28 pg / mL to 32 pg / mL to 0.10 pg / mL to 50 pg / mL; intra-assay repeatability was ≤5%; the reaction time was shortened to 9 min; and the sample volume was reduced from 15 μL (the standard limit of detection for triiodothyronine assay kits). The sample volume of the kit was reduced to 10 μL. In T3 detection, the detection limit was improved from no more than 0.20 ng / mL (the detection limit of a conventional triiodothyronine assay kit) to no more than 0.08 ng / mL, the linear range expanded from 0.40 ng / mL to 6.50 ng / mL to 0.08 to 10.00 ng / mL, the intra-assay repeatability was ≤5%, the reaction time was shortened to 9 min, and the sample volume was reduced from 30 μL (the sample volume of a conventional triiodothyronine assay kit) to 10 μL. The triiodothyronine chemiluminescent immunoassay kit of this application effectively solves the problems of poor sensitivity and narrow linear range in competitive methods for detecting FT3 / T3.

[0307] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A chemiluminescent immunoassay kit for triiodothyronine, characterized in that, The detection kit includes: The antibody 1-solid-phase carrier conjugate, antibody 2-chemiluminescent label, and triiodothyronine antigen calibrator are provided. The antibody 1-solid-phase carrier conjugate comprises a solid-phase carrier coated with triiodothyronine antibody 1, and the antibody 2-chemiluminescent label comprises triiodothyronine antibody 2 labeled with a chemiluminescent label. Both the antibody 1-solid-phase carrier conjugate and the antibody 2-chemiluminescent label can specifically bind to the target analyte and have different antigen recognition epitopes; or, The assay kit comprises a streptavidin-solid-phase carrier conjugate, antibody 1-biotin-labeled antibody, antibody 2-chemiluminescent-labeled antibody, and a triiodothyronine antigen calibrator. The antibody 1-biotin-labeled antibody includes biotin-labeled triiodothyronine antibody 1, and the antibody 2-chemiluminescent-labeled antibody includes chemiluminescent-labeled triiodothyronine antibody 2. Both antibody 1-biotin-labeled antibody and antibody 2-chemiluminescent-labeled antibody can specifically bind to the target analyte and have different antigen recognition epitopes. The assay kit includes assay reagent 1, which comprises the antibody 1-solid-phase carrier conjugate and diluent 1; or the streptavidin-solid-phase carrier conjugate and diluent 1. The diluent 1 comprises 0.8 wt%~1.0 wt% NaCl, 0.8 wt%~1.2 wt% sucrose, 0.08 v / v%~0.12 v / v% (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), 0.04 v / v%~0.06 v / v% polyoxyethylene lauroyl ether, 1.4 wt%~1.6 wt% bovine serum albumin, 0.05 v / v%~0.5 v / v% proclin 300, and HEPES buffer with a pH of 6.0~8.0 and a concentration of 0.01 mol / L~0.2 mol / L. The mass ratio of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt) to the polyoxyethylene lauroyl ether is 2; The solid support includes magnetic beads; The test kit further includes test reagent 2, which comprises the antibody 2-chemiluminescent label and diluent 2; or comprises the antibody 1-biotin label, the antibody 2-chemiluminescent label and diluent 2; The diluent 2 includes salt ions 2, surfactant 2, preservative 2, stabilizer 2, dissociating agent, blocking agent, and neutral buffer solution 2; The surfactant 2 includes Tween-20 and polyoxyethylene lauroyl ether; The chemiluminescent label includes Ru-NHS.

2. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that, The test kit satisfies one or more of the following conditions (1) to (3): (1) The functional groups of the surface modification of the magnetic beads include one or more of carboxyl, amino, hydroxyl, epoxy and p-toluenesulfonyl groups; (2) The active groups modified on the biotin include one or more of the following: NHS ester group, amino group, maleimide group and hydrazide group; (3) The antibodies in the antibody 1-solid phase carrier conjugate and / or the antibody 2-chemiluminescent label are monoclonal antibodies.

3. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that, The concentration of the antibody-solid-phase carrier conjugate or streptavidin-solid-phase carrier conjugate is 0.3 mg / mL to 0.9 mg / mL.

4. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that, The salt ion 2 includes one or more of NaCl, ammonium sulfate, and KCl; The preservative 2 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT; The stabilizer 2 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, cystine, PEG, trehalose, and mannitol; The dissociation agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, messodium, and tris(2-carbonylethyl) phosphate hydrochloride; The blocking agent includes one or more of MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, and goat IgG; The neutral buffer 2 includes one or more of phosphate buffer, MES buffer, Tris buffer, and HEPES buffer; The concentration of the antibody-biotin-labeled antibody is 0.5 μg / mL to 2.0 μg / mL; The concentration of the antibody-2 chemiluminescent label is 0.5 μg / mL to 2.0 μg / mL.

5. The triiodothyronine chemiluminescent immunoassay kit according to claim 4, characterized in that, The diluent 2 comprises 0.8 wt%~1.0 wt% NaCl, 0.08 v / v%~0.12 v / v% Tween-20, 0.04 wt%~0.06 wt% polyoxyethylene lauroyl ether, 0.05 v / v%~0.5 v / v% Proclin 300, 0.002 wt%~0.004 wt% gentamicin sulfate, 1.8 wt%~2.2 wt% bovine serum albumin, 4.8 wt%~5.2 wt% trehalose, 4.8 wt%~5.2 wt% mannitol, 80 μg / mL~120 μg / mL MAK33 Poly, and 80 μg / mL~120 μg / mL Rabbit IgG, 80 μg / mL~120 μg / mL mouse IgG, 80 μg / mL~120 μg / mL bovine IgG, 80 μg / mL~120 μg / mL goat IgG, 0.8 mg / mL~1.2 mg / mL 8-aniline-1-naphthalenesulfonic acid, and PBS buffer at pH 7.3~7.5 and concentration of 0.08 mol / L~0.12 mol / L.

6. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that, The kit includes test reagent 3; the test reagent 3 includes the triiodothyronine antigen calibrator and diluent 3; The diluent 3 includes salt ions 3, surfactant 3, preservative 3, stabilizer 3, and neutral buffer 3; The salt ion 3 includes one or both of NaCl and KCl; The surfactant 3 includes one or both of Tween-20 and polyoxyethylene lauroyl ether; The preservative 3 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT; The stabilizer 3 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose, and mannitol; The neutral buffer 3 includes one or more of phosphate buffer, Tris buffer, and HEPES buffer.

7. The triiodothyronine chemiluminescent immunoassay kit according to claim 6, characterized in that, The diluent 3 comprises 0.04 v / v%~0.06 v / v% Tween-20, 1.4 wt%~1.6 wt% bovine serum albumin, 1.5 wt%~2.5 wt% trehalose, 1.5 wt%~2.5 wt% mannitol, 0.04 v / v%~0.06 v / v% proclin 300, and HEPES buffer at a pH of 7.3~7.5 and a concentration of 0.04 mol / L~0.06 mol / L.

8. The method for preparing the triiodothyronine chemiluminescent immunoassay kit according to any one of claims 1 to 7, characterized in that, The preparation method includes the following steps: Provide the antibody 1-solid-phase carrier conjugate, the antibody 2-chemiluminescent label, and the triiodothyronine antigen calibrator, respectively; or, A detection kit is prepared by providing the streptavidin-solid-phase carrier conjugate, the antibody 1-biotin label, the antibody 2-chemiluminescent label, and the triiodothyronine antigen calibrator, respectively.

9. The preparation method according to claim 8, characterized in that, The preparation steps of the antibody-solid-phase carrier conjugate include: Antibody 1 and a solid-phase support undergo a first coupling reaction. Blocking agent 1 and blocking agent 2 are sequentially added to the resulting first coupling reaction product for blocking, thereby preparing the antibody 1-solid-phase support conjugate; and / or The preparation steps of the antibody-2-chemiluminescent label include: The antibody 2 solution was subjected to a second coupling reaction with the chemiluminescent label to prepare the antibody 2-chemiluminescent label. The blocking agent 1 comprises 1.4 wt%~1.5 wt% glycine, 0.8 wt%~1.2 wt% sucrose, 0.04 v / v%~0.06 v / v% Tween-20, 1.4 wt%~1.6 wt% bovine serum albumin, 0.05 v / v%~0.5 v / v% proclin 300, and PBS buffer with a pH of 5.8~6.2 and a concentration of 0.04~0.06 mol / L. The blocking agent 2 comprises 0.4 wt% to 0.6 wt% poloxamer, 0.8 wt% to 1.2 wt% sucrose, 0.04 v / v% to 0.06 v / v% Tween-20, 1.4 wt% to 1.6 wt% bovine serum albumin, 0.05 v / v% to 0.5 v / v% proclin 300, and HEPES buffer at a pH of 7.3 to 7.5 and a concentration of 0.04 mol / L to 0.06 mol / L.

10. The preparation method according to claim 9, characterized in that, The preparation steps of the streptavidin-solid-phase carrier conjugate include: streptavidin and solid-phase carrier undergoing a third coupling reaction, and blocking agent 1 and blocking agent 2 are added sequentially to the obtained third coupling reaction product for blocking, thereby preparing the streptavidin-solid-phase carrier conjugate. The preparation method of the antibody 1-biotin label includes: performing a fourth coupling reaction between antibody 1 and biotin label to prepare the antibody 1-biotin label.

11. The preparation method according to claim 9, characterized in that, The conditions for the first coupling reaction include: the sealing temperature of the blocking agent 1 and the blocking agent 2 is 36℃~38℃, and the sealing time is 20h~28h; The conditions for the second coupling reaction include: the mass ratio of the antibody 2 to the chemiluminescent label is (28~32):1, the reaction conditions are 36℃~38℃, and the time is 1h~3h.

12. The preparation method according to claim 10, characterized in that, The conditions for the third coupling reaction include: the sealing temperature of the blocking agent 1 and the blocking agent 2 is 36℃~38℃, and the sealing time is 20h~28h.

13. A method for detecting triiodothyronine (TTI) for non-diagnostic purposes, characterized in that, The detection method includes the step of detecting triiodothyronine in the sample to be tested using the triiodothyronine chemiluminescent immunoassay kit according to any one of claims 1 to 7; The sample to be tested includes one or both of serum and plasma.

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