Triiodothyronine chemiluminescence immunoassay kit as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and particularly relates to a triiodothyronine chemiluminescence immunodetection kit as well as a preparation method and application thereof. According to the triiodothyronine chemiluminescence immunodetection kit provided by the invention, FT3 / T3 is detected on an electrochemical luminescence platform by adopting a double-antibody sandwich method for the first time, so that the detection sensitivity is effectively improved, the linear range of detection is increased, and the repeatability of detection is improved.

Description

Technical Field

[0001] The present application belongs to the technical field of immunoassay, and specifically relates to a triiodothyronine chemiluminescent immunoassay kit and a preparation method and application thereof. Background Art

[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 enzymatic removal of iodine at the 5' position from T4 (thyroxine). Therefore, the concentration of T3 in serum can better reflect the functional status of peripheral tissues rather than the secretory function of the thyroid gland.

[0003] Free T3 (FT3) is an unbound biologically active form that accounts for only 0.2-0.4% of total T3. The detection of FT3 is theoretically not affected by the concentration and binding properties of binding proteins, so there is no need for additional detection of items reflecting binding parameters (such as TBG). Therefore, the determination of FT3 is an important indicator for routine clinical assessment of thyroid function. The measurement results of FT3 can be used as a basis for differential diagnosis of thyroid diseases, and are used to distinguish different types of hyperthyroidism and diagnose T3 hyperthyroidism.

[0004] Currently, the existing methods for detecting FT3 content include: radioimmunoassay (RIA), chemiluminescence immunoassay, and isotope dilution / GC / MS. Among them, the radioimmunoassay is not easy to measure automatically, there is radioactive pollution, and it affects the health of operators; 131I has a short half-life and is not easy to store for a long time, and the results are not reproducible; the chemiluminescence immunoassay is expensive, has high requirements for the detection environment, has the risk of cross-reaction, and the instruments and equipment are complex and expensive; the sample pretreatment of the isotope dilution / GC / MS method is complex and time-consuming, the non-specific adsorption of the analyte to the membrane or device cannot be avoided, the degree of automation does not meet clinical requirements, and it is difficult to detect FT3 and FT4.

[0005] The detection process is generally that the FT3 / T3 in the sample and the biotin-labeled T3 derivative in the reagent compete for binding to the T3 antibody labeled with the luminescent marker, and then the immune complex of biotin T3 and the luminescent marker T3 antibody is captured by the added streptavidin-coated magnetic particles. After washing, the luminescent liquid is added to detect the signal value, and the concentration is calculated by the standard curve.

[0006] In the competition reaction, the amount of biotinylated antigen and antibody labeled with luminescent marker is limited, which makes the sensitivity of the competition method very limited by the affinity of the antibody. The affinity constant of T3 small molecule antibody is usually low, resulting in poor signal-to-noise ratio, low sensitivity and poor discrimination of low values ​​in the existing FT3 / T3 competition method. And because the detection of free thyroid-related hormones requires the endogenous balance between the bound state and the free state, high-value samples above the upper limit of linearity cannot be tested by dilution, and the linear range of the FT3 / T3 competition method is limited, resulting in the inability to produce values ​​for high-value samples.

[0007] In addition, since the binding site between the T3 small molecule compound and the corresponding antibody is a hole-like 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 and few antigen recognition sites for the T3 small molecule. Therefore, it is difficult to detect FT3 / T3 using the double antibody sandwich method. In immunological methodology, the competitive method is usually used to detect FT3 / T3.

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

[0009] Based on this, an embodiment of the present application provides a triiodothyronine chemiluminescent immunoassay kit and a preparation method and application thereof.

[0010] On the one hand, the present application provides a triiodothyronine chemiluminescent immunoassay kit, the detection kit comprising:

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

[0012] Streptavidin-solid phase carrier conjugate, antibody 1-biotin marker, antibody 2-chemiluminescent marker and triiodothyronine antigen calibrator, the antibody 1-biotin marker includes triiodothyronine antibody 1 labeled with a biotin marker, and the antibody 2-luminescent marker includes triiodothyronine antibody 2 labeled with a chemiluminescent marker. Both the antibody 1-biotin marker and the antibody 2-luminescent marker can specifically bind to the target analyte and have different antigen recognition epitopes.

[0013] In some embodiments, the solid phase carrier in the antibody 1-solid phase carrier conjugate includes magnetic beads.

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

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

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

[0017] In some of the embodiments, the antibody in the antibody 1-solid phase carrier conjugate and / or the antibody 2-chemiluminescent marker is a monoclonal antibody.

[0018] In some embodiments, the detection kit includes detection reagent 1, and the detection reagent 1 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, a surfactant 1, a preservative 1, a stabilizer 1 and a 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 includes one or both of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt) and polyoxyethylene lauryl 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 a phosphate buffer, a HEPES buffer and a MOPOS buffer.

[0025] In some embodiments, the diluent 1 includes 0.8wt% to 1.0wt% NaCl, 0.8wt% to 1.2wt% sucrose, 0.08v / v% to 0.12v / v% (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), 0.04v / v% to 0.06v / v% polyoxyethylene lauryl ether, 1.4wt% to 1.6wt% bovine serum albumin, 0.05v / v% to 0.5v / v% proclin300, and HEPES buffer with a pH value of 6.0 to 8.0 and a concentration of 0.01mol / L to 0.2mol / L.

[0026] In some embodiments, the concentration of the antibody 1-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 detection reagent 2, which includes antibody 2-chemiluminescent marker and diluent 2; or includes antibody 1-biotin marker, antibody 2-chemiluminescent marker and diluent 2.

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

[0029] In some embodiments, the salt ions 2 include 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 lauryl 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, mesna and tris(2-carbonylethyl)phosphine hydrochloride.

[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 a phosphate buffer, a MES buffer, a Tris buffer, and a HEPES buffer.

[0036] In some embodiments, the concentration of the antibody 1-biotin label is 0.5 μg / mL to 2.0 μg / mL.

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

[0038] In some embodiments, the diluent 2 comprises 0.8wt% to 1.0wt% NaCl, 0.08v / v% to 0.12v / v% Tween-20, 0.04wt% to 0.06wt% polyoxyethylene lauryl ether, 0.05v / v% to 0.5v / v% proclin300, 0.002wt% to 0.004wt% gentamicin sulfate, 1.8wt% to 2.2wt% bovine serum albumin, 4.8wt% to 5.2wt% trehalose, 4.8wt% to 5.2wt% mannitol, 80μg / mL to 120μg / mL MAK33 Poly, 80μg / mL~120μg / mL rabbit IgG, 80μg / mL~120μg / mL mouse IgG, 80μg / mL~120μg / mL bovine IgG, 80μg / mL~120μg / mL goat IgG, 0.8mg / mL~1.2mg / mL 8-aniline-1-naphthalenesulfonic acid, and PBS buffer with a pH of 7.3 to 7.5 and a concentration of 0.08mol / L to 0.12mol / L.

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

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

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

[0042] In some embodiments, the surfactant 3 includes Tween-20 and polyoxyethylene lauryl 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 a phosphate buffer, a Tris buffer and a HEPES buffer.

[0046] In some embodiments, the diluent 3 includes 0.04v / v%~0.06v / v% Tween-20, 1.4wt%~1.6wt% bovine serum albumin, 1.5wt%~2.5wt% trehalose, 1.5wt%~2.5wt% mannitol, 0.04v / v%~0.06v / v% proclin300 and HEPES buffer with a pH of 7.3~7.5 and a concentration of 0.04mol / L~0.06mol / L.

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

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

[0049] The streptavidin-solid phase carrier conjugate, the antibody 1-biotin marker, the antibody 2-chemiluminescent marker and the triiodothyronine antigen calibrator are provided respectively to prepare a detection kit.

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

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

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

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

[0054] In some embodiments, the blocking agent 1 includes 1.4wt% to 1.5wt% glycine, 0.8wt% to 1.2wt% sucrose, 0.04v / v% to 0.06v / v% Tween-20, 1.4wt% to 1.6wt% bovine serum albumin, 0.05v / v% to 0.5v / v% proclin300, and a PBS buffer having 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 includes 0.4wt% to 0.6wt% of poloxamer, 0.8wt% to 1.2wt% of sucrose, 0.04v / v% to 0.06v / v% of Tween-20, 1.4wt% to 1.6wt% of bovine serum albumin, 0.05v / v% to 0.5v / v% of proclin300, and a HEPES buffer having a pH of 7.3 to 7.5 and a concentration of 0.04mol / L to 0.06mol / L.

[0056] In some of the embodiments, the steps of preparing the streptavidin-solid phase carrier conjugate include: performing a second coupling reaction between streptavidin and the solid phase carrier, and sequentially adding the blocking agent 1 and the blocking agent 2 to the obtained second coupling reaction product for blocking, thereby preparing the streptavidin and the solid phase carrier.

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

[0058] In some embodiments, the conditions of the first coupling reaction include: the blocking temperature of the blocking agent 1 and the blocking agent 2 is 36° C. to 38° C., and the time is 20 h to 28 h.

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

[0060] The conditions of the third coupling reaction include: the blocking temperature of the blocking agent 1 and the blocking agent 2 is 36° C. to 38° C., and the time is 20 h to 28 h.

[0061] On the other hand, the present application provides a method for detecting triiodothyronine, which comprises the steps of using the triiodothyronine chemiluminescent immunoassay kit to detect triiodothyronine in a sample to be tested;

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

[0063] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. DETAILED DESCRIPTION

[0064] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0065] 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.

[0066] the term

[0067] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0068] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0069] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0070] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0071] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0072] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0073] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0074] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0075] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0076] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0077] In the present 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 documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0079] Free T3 (FT3) is an unbound biologically active form that accounts for only 0.2-0.4% of total T3. Theoretically, the detection of FT3 is not affected by the concentration and binding properties of the binding protein, so there is no need for additional detection of items reflecting binding parameters (such as TBG). Therefore, the determination of FT3 is an important indicator for routine clinical assessment of thyroid function. The measurement results of FT3 can be used as a basis for differential diagnosis of thyroid diseases, and are used to distinguish different types of hyperthyroidism and diagnose T3 hyperthyroidism.

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

[0081] The term “T3” stands for Total Triiodothyronine.

[0082] It can be understood that the FT3 and T3 mentioned below are abbreviations, and the full name is free triiodothyronine or total triiodothyronine.

[0083] In recent years, small molecule non-competitive methods have developed rapidly, including hapten open immune sandwich method, detection based on enzyme-labeled analyte-analyte antibody site replacement non-competitive method, sandwich method research based on other recognition materials, and double antibody sandwich method based on anti-immune complex antibody development. With the development of phage display technology and autonomous diversified library technology, the accurate identification of new antigenic epitopes of antigen-antibody complexes has been effectively solved, and anti-immune complex antibodies have been developed more efficiently and accurately; making it have a good application prospect in small molecule sandwich immunoassay. 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] The present application provides a method for detecting FT3 / T3 in a sample: a sandwich complex is formed by T3 monoclonal antibody 1, T3 monoclonal antibody 2 and FT3 / T3 antigen in the sample, and combined with a fully automatic electrochemiluminescence analyzer, the content of FT3 / T3 in the sample can be quickly, sensitively and accurately determined, while reducing operation time and reducing errors in human operation.

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

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

[0087] Streptavidin-solid phase carrier conjugate, antibody 1-biotin marker, antibody 2-chemiluminescent marker and triiodothyronine antigen calibrator, the antibody 1-biotin marker includes triiodothyronine antibody 1 labeled with a biotin marker, the antibody 2-luminescent marker includes triiodothyronine antibody 2 labeled with a chemiluminescent marker, the antibody 1-biotin marker and the antibody 2-luminescent marker can both specifically bind to the target analyte and have different antigen recognition epitopes. The present application provides a triiodothyronine chemiluminescent immunoassay kit, which uses a double antibody sandwich method to detect FT3 / T3 on an electrochemiluminescent platform for the first time, effectively improving the detection sensitivity and increasing the linear range of the detection, and improving the repeatability of the detection.

[0088] In some embodiments, the solid phase carrier in the antibody 1-solid phase carrier conjugate comprises 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 reactive groups modified on biotin include one or more of NHS ester group, amino group, maleimide group and hydrazide group.

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

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

[0093] In some embodiments, the detection kit includes detection 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, a surfactant 1, a preservative 1, a stabilizer 1 and a neutral buffer 1.

[0095] In some embodiments, the salt ions 1 include one or both of NaCl and KCl.

[0096] In some embodiments, surfactant 1 includes one or both of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt) and polyoxyethylene lauryl ether. The mass ratio of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt to polyoxyethylene lauryl ether is (1-3):1. For example, the ratio of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt to polyoxyethylene lauryl ether is 1:1, 2:1, 3:1 and 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, the neutral buffer 1 includes one or more of a phosphate buffer, a HEPES buffer, and a MOPOS buffer.

[0100] In some embodiments, the diluent 1 includes 0.8wt% to 1.0wt% NaCl, 0.8wt% to 1.2wt% sucrose, 0.08v / v% to 0.12v / v% (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), 0.04v / v% to 0.06v / v% polyoxyethylene lauryl ether, 1.4wt% to 1.6wt% bovine serum albumin, 0.05v / v% to 0.5v / v% proclin300, and HEPES buffer with a pH value of 6.0 to 8.0 and a concentration of 0.01mol / L to 0.2mol / L.

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

[0102] For example, the sucrose concentration is 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt% 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 lauryl ether, also known as Brij 35, has a concentration of 0.04 v / v%, 0.05 v / v%, 0.06 v / v% and any value in between.

[0105] For example, the concentration of bovine serum albumin is 1.4 wt%, 1.5 wt% or 1.6 wt% and any value therebetween.

[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, the pH value is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, and the HEPES buffer concentration is 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 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 embodiments, the concentration of the antibody 1-solid phase carrier conjugate or the streptavidin-solid phase carrier conjugate is 0.3 mg / mL to 0.9 mg / mL.

[0109] For example, the concentration of the antibody 1-solid phase carrier conjugate is 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 concentration of the streptavidin-solid phase carrier conjugate is 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 detection reagent 2, which includes antibody 2-chemiluminescent marker and diluent 2; or includes antibody 1-biotin marker, antibody 2-chemiluminescent marker and diluent 2.

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

[0113] In some embodiments, the salt ions 2 include one or more of NaCl, ammonium sulfate, and KCl.

[0114] In some embodiments, the surfactant 2 includes one or both of Tween-20 and polyoxyethylene lauryl ether. The mass ratio of Tween-20 to polyoxyethylene lauryl ether is (1-3):1. For example, the ratio of Tween-20 to polyoxyethylene lauryl ether is 1:1, 2:1, 3:1 and 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-anilino-1-naphthalenesulfonic acid, dithiothreitol, mesna and tris(2-carbonylethyl)phosphine hydrochloride.

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

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

[0120] In some embodiments, the concentration of the antibody 1-biotin label is 0.5 μg / mL to 2.0 μg / mL. For example, the concentration of the antibody 1-biotin 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, 2.0 μg / mL.

[0121] In some embodiments, the concentration of the antibody 2-luminescent marker is 0.5 μg / mL to 2.0 μg / mL. For example, the concentration of the antibody 2-luminescent marker 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 and any value in between.

[0122] In some embodiments, the diluent 2 includes 0.8wt% to 1.0wt% NaCl, 0.08v / v% to 0.12v / v% Tween-20, 0.04wt% to 0.06wt% polyoxyethylene lauryl ether, 0.05v / v% to 0.5v / v% proclin300, 0.002wt% to 0.004wt% gentamicin sulfate, 1.8wt% to 2.2wt% bovine serum albumin, 4.8wt% to 5.2wt% trehalose, 4.8wt% to 5.2wt% mannitol, 80μg / mL to 120μg / mL MAK33 Poly, 80μg / mL~120μg / mL rabbit IgG, 80μg / mL~120μg / mL mouse IgG, 80μg / mL~120μg / mL bovine IgG, 80μg / mL~120μg / mL goat IgG, 0.8mg / mL~1.2mg / mL 8-aniline-1-naphthalenesulfonic acid, and PBS buffer with a pH of 7.3 to 7.5 and a concentration of 0.08mol / L to 0.12mol / L.

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

[0124] For example, the concentration of Tween-20 is 0.08v / v%, 0.09v / v%, 0.10v / v%, 0.11v / v% or 0.12v / v% and any value in between. For example, polyoxyethylene lauryl ether, also known as Brij 35, has a concentration of 0.04v / v%, 0.05v / v% or 0.06v / v% and any value in between. For example, the concentration of bovine serum albumin is 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt% or 2.2wt% and any value in between. For example, the concentration of trehalose is 4.8wt%, 4.9wt%, 5.0wt%, 5.1wt% or 5.2wt% and any value in between. For example, the concentration of mannitol is 4.8wt%, 4.9wt%, 5.0wt%, 5.1wt% or 5.2wt% and any value in between. For example, the concentration of MAK33 Poly is 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 and any values ​​therebetween. For example, the concentration of rabbit IgG is 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 and any values ​​therebetween. For example, the concentration of mouse IgG is 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 and any values ​​therebetween. For example, the concentration of bovine IgG is 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 and any values ​​therebetween. For example, the concentration of goat IgG is 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 and any values ​​therebetween. For example, the concentration of 8-aniline-1-naphthalenesulfonic acid is 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL or 1.2 mg / mL and any values ​​therebetween. 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 detection reagent 3; detection reagent 3 includes triiodothyronine antigen calibrator and 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 ions 3 include one or both of NaCl and KCl.

[0128] In some embodiments, the surfactant 3 includes Tween-20 and polyoxyethylene lauryl ether. The mass ratio of Tween-20 to polyoxyethylene lauryl ether is (1-3):1. For example, the ratio of Tween-20 to polyoxyethylene lauryl ether is 1:1, 2:1, 3:1 and 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, the stabilizer 3 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose and mannitol.

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

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

[0133] For example, the concentration of Tween-20 is 0.04v / v%, 0.05v / v%, 0.06v / v% and any value in between. For example, the concentration of bovine serum albumin is 1.4wt%, 1.5wt% or 1.6wt% and any value in between. For example, the concentration of trehalose is 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt% or 2.5wt% and any value in between. For example, the concentration of mannitol is 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2.0wt%, 2.1wt%, 2.2wt%, 2.3wt%, 2.4wt% or 2.5wt% and any value in between. For example, the concentration of proclin300 is 0.04 v / v%, 0.05 v / v% or 0.06 v / v% and any value therebetween. For example, the pH value is 7.3, 7.4 or 7.5, and the concentration of PBS buffer is 0.04 mol / L, 0.05 mol / L or 0.06 mol / L and any value therebetween.

[0134] On the other hand, the present application provides a method for preparing a triiodothyronine chemiluminescent immunoassay kit, the preparation method comprising the following steps:

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

[0136] A streptavidin-solid phase carrier conjugate, an antibody 1-biotin marker, an antibody 2-chemiluminescent marker and a triiodothyronine antigen calibrator are provided respectively to prepare a detection kit.

[0137] In some of the embodiments, antibody 1 and a solid phase carrier undergo a first coupling reaction, and blocking agent 1 and blocking agent 2 are sequentially added to the obtained first coupling reaction product for blocking to prepare an antibody 1-solid phase carrier conjugate.

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

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

[0140] In some embodiments, the blocking agent 1 includes 1.4wt% to 1.5wt% glycine, 0.8wt% to 1.2wt% sucrose, 0.04v / v% to 0.06v / v% Tween-20, 1.4wt% to 1.6wt% bovine serum albumin, 0.05v / v% to 0.5v / v% proclin300, and a PBS buffer having a pH of 5.8 to 6.2 and a concentration of 0.04mol / L to 0.06mol / L.

[0141] For example, the concentration of glycine is 1.4wt%, 1.5wt% or 1.6wt% and any value in between. For example, the concentration of sucrose is 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt% or 1.2wt% and any value in between. For example, the concentration of Tween-20 is 0.04v / v%, 0.05v / v%, 0.06v / v% and any value in between. For example, the concentration of bovine serum albumin is 1.4wt%, 1.5wt% or 1.6wt% and any value in between. For example, the concentration of proclin300 is 0.05v / v%, 0.10v / v%, 0.15v / v%, 0.20v / v%, 0.25v / v%, 0.30v / v%, 0.35v / v%, 0.40v / v%, 0.45v / v% or 0.50v / v% and any value in between. For example, a PBS buffer having a pH of 5.8, 5.9, 6.0, 6.1 or 6.2 and a concentration of 0.04 mol / L, 0.05 mol / L or 0.06 mol / L.

[0142] In some embodiments, the blocking agent 2 includes 0.4wt% to 0.6wt% of poloxamer (i.e., F68), 0.8wt% to 1.2wt% of sucrose, 0.04v / v% to 0.06v / v% of Tween-20, 1.4wt% to 1.6wt% of bovine serum albumin, 0.05v / v% to 0.5v / v% of proclin300, and a HEPES buffer having a pH of 7.3 to 7.5 and a concentration of 0.04mol / L to 0.06mol / L.

[0143] For example, the concentration of poloxamer is 0.4wt%, 0.5wt% or 0.6wt% and any value in between. For example, the concentration of sucrose is 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt% or 1.2wt% and any value in between. For example, the concentration of Tween-20 is 0.04v / v%, 0.05v / v%, 0.06v / v% and any value in between. For example, the concentration of bovine serum albumin is 1.4wt%, 1.5wt% or 1.6wt% and any value in between. For example, the concentration of proclin300 is 0.05v / v%, 0.10v / v%, 0.15v / v%, 0.20v / v%, 0.25v / v%, 0.30v / v%, 0.35v / v%, 0.40v / v%, 0.45v / v% or 0.50v / v% and any value in between. 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 of the embodiments, the steps for preparing the streptavidin-solid phase carrier conjugate include: performing a second coupling reaction between streptavidin and the solid phase carrier, and sequentially adding blocking agent 1 and blocking agent 2 to the obtained second coupling reaction product for blocking, thereby preparing streptavidin and the solid phase carrier.

[0145] In some of the embodiments, the method for preparing the antibody 1-biotin marker includes: performing a third coupling reaction between the antibody 1 and the biotin marker to prepare the antibody 1-biotin marker.

[0146] In some embodiments, the conditions of the first coupling reaction include: the temperature of the blocking agent 1 and the 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 therebetween. The time is 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h and 28h and any value therebetween.

[0147] In some of the embodiments, the conditions of the second coupling reaction include: the mass ratio of antibody 2 to chemiluminescent marker is (28-32):1, the reaction conditions are 36° C.-38° C., and the reaction time is 1 h-3 h.

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

[0149] In some embodiments, the conditions of the third coupling reaction include: the temperature of the blocking agent 1 and the 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 therebetween. The time is 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h and 28h and any value therebetween.

[0150] Another aspect of the present application provides a method for detecting a target analyte, the method comprising the step of using the above-mentioned triiodothyronine chemiluminescent immunoassay kit to detect the target analyte in the sample to be tested. For example, the target analyte includes triiodothyronine.

[0151] In some embodiments, the sample to be tested is serum and plasma.

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

[0153] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0154] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0155] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean 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 the present application.

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

[0157] Example 1

[0158] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit, and the kit in this embodiment adopts a streptavidin (SA) and biotin (Biotin) system.

[0159] 1. Composition and preparation of triiodothyronine chemiluminescent immunoassay kit:

[0160] 1. Preparation of Streptavidin-coupled Magnetic Particle Solution

[0161] The streptavidin-coated magnetic beads were blocked with magnetic bead blocking solution 1 and magnetic bead blocking solution 2 at 37° C. for 24 h, respectively.

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

[0163] The components of magnetic bead blocking solution 2 include HEPES buffer with a pH of 7.4 and a concentration of 0.05 mol / L, and the buffer contains 0.5 wt % of poloxamer, 1 wt % of sucrose, 0.05 v % of polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt % of BSA, and 0.25 v / v % of proclin300.

[0164] The magnetic bead suspension is then subjected to magnetic separation using a magnetic separator, and after removing the supernatant, it is diluted with diluent 1. The diluent of the streptavidin-coupled magnetic particles is recorded as diluent 1.

[0165] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % of sodium chloride, 1 wt % of sucrose, 0.1 v / v % of chaps, 0.05 v / v % of Brij35 (polyoxyethylene lauryl ether), 1.5 wt % of BSA, and 0.25 v / v % of proclin300.

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

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

[0168] Among them, the T3 detection formula includes a dissociation agent, while the FT3 detection formula does not include a dissociation agent.

[0169] 3. Preparation of biotin-labeled T3 / FT3 monoclonal antibody 1

[0170] Take T3 / FT3 monoclonal antibody 1 in a pre-treated ultrafiltration tube, use pH 8.0 25mM Tris-HCl buffer ultrafiltration centrifugation for purification ultrafiltration 3 times, transfer the purified antibody to a collection tube, test the purified concentration and adjust it to 2mg / mL. Mix N-hydroxysuccinimide activated biotin and T3 / FT3 monoclonal antibody 1 at a molar ratio of 20:1 under 22 conditions for 1 hour, ultrafiltration or dialysis desalination to remove unreacted biotin, and obtain biotin-labeled T3 / FT3 monoclonal antibody 1.

[0171] 4. Preparation of T3 / FT3 monoclonal antibody 2 labeled with terpyridine ruthenium

[0172] Take T3 / FT3 monoclonal antibody 2 in a pre-treated ultrafiltration tube, use pH 8.0 25mM Tris-HCl buffer ultrafiltration centrifugation for purification ultrafiltration 3 times, transfer the purified antibody to a collection tube, test the purified concentration and adjust it to 2mg / mL. Mix the T3 / FT3 monoclonal antibody 2 solution with the acridine compound Ru-NHS ester solution at a mass ratio of 30:1, react at 37°C in the dark for 2 hours, then add glycine solution to terminate the reaction, dialyze the reaction solution to remove excess Ru-NHS ester, and obtain the terpyridine ruthenium labeled T3 / FT3 monoclonal antibody 2.

[0173] 5. Preparation of Calibrators and Quality Controls

[0174] Prepare the matrix for T3 / FT3 calibration and quality control. The dilution of the calibration and quality control is recorded as dilution 3.

[0175] Dilution solution 3 includes HEPES buffer with a pH of 7.4 and a concentration of 0.05 mol / L, and the buffer contains 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 calibrants and quality controls.

[0176] 2. How to use the triiodothyronine chemiluminescent immunoassay kit:

[0177] 1. Sample pretreatment: Take the sample to be tested (such as serum, plasma or artificially prepared sample), centrifuge it at 5000 rpm at 4°C for 5 minutes and set aside.

[0178] 2. Before using the reagent, put it into the Pumen Company's fully automatic electrochemiluminescence analyzer 30 minutes in advance to automatically mix the magnetic beads and keep them in a suspended state.

[0179] 3. Calibration and quality control of the test kit: Use the matching calibrators to perform calibration tests and determine the accuracy of the current system through the quality control results.

[0180] 4. Sample testing:

[0181] The triiodothyronine chemiluminescence immunoassay kit detects the T3 concentration and FT3 concentration in the sample. The specific test process 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 diluent 2 to obtain a biotin-labeled T3 monoclonal antibody 1 working solution.

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

[0185] The diluent 2 was used to dilute the terpyridine ruthenium labeled T3 monoclonal antibody 2 to a working concentration of 0.5 μg / mL to obtain a terpyridine ruthenium labeled T3 monoclonal antibody 2 working solution.

[0186] The diluent 2 was used to dilute the terpyridine ruthenium labeled FT3 monoclonal antibody 2 to a working concentration of 0.4 μg / mL to obtain a terpyridine ruthenium labeled FT3 monoclonal antibody 2 working solution.

[0187] (2) After the user applies for the test, the system automatically draws 60 μL of biotin-labeled T3 monoclonal antibody 1 working solution, 60 μL of terpyridine ruthenium-labeled T3 monoclonal antibody 2 working solution, 40 μL of streptavidin-coated magnetic particles and 10 μL of the sample to be tested into a reaction cup; the system automatically draws 60 μL of biotin-labeled FT3 monoclonal antibody 1 working solution, 60 μL of terpyridine ruthenium-labeled FT3 monoclonal antibody 2 working solution, 40 μL of streptavidin-coated magnetic particles and 10 μL of the sample to be tested into another reaction cup. The mixture is automatically incubated at 37°C for 9 minutes to form an antigen-antibody sandwich complex, and then the entire complex is bound to the magnetic particles under the interaction of biotin and streptavidin.

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

[0189] Example 2

[0190] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The kit in this embodiment adopts an antibody directly coated magnetic bead system.

[0191] 1. Composition and preparation of triiodothyronine chemiluminescent immunoassay kit:

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

[0193] Select 3μm tosyl-modified magnetic beads, take 10mg of magnetic beads, wash 5 times with coating buffer 1 (0.1M boric acid buffer). Then add coating buffer 1 (0.1M boric acid buffer), coating buffer 2 (3M ammonium sulfate boric acid buffer), coating buffer 3 (0.5% proclin 300) and T3 / FT3 monoclonal antibody 1 to be coated, mix and react at 37℃ for 24. Then use magnetic bead blocking solution 1 and magnetic bead blocking solution 2 to block at 37℃ for 24. Finally, replace the volume with magnetic bead diluent and store at 4℃.

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

[0195] Magnetic bead blocking solution 2 includes HEPES buffer with a pH of 7.4 and a concentration of 0.05 mol / L, and the buffer contains 0.5 wt % of poloxamer, 1 wt % of sucrose, 0.05 v / v % of polyoxyethylene sorbitan monolaurate (TWEEN-20), 1.5 wt % of BSA, and 0.25 v / v % of proclin300.

[0196] Before use, a magnetic bead working solution with a concentration of 0.75 mg / mL was prepared using common magnetic bead diluent (0.1 M Tris, 1% wt% BSA, 0.05 v / v% polyoxyethylene sorbitan monolaurate (TWEEN-20), 0.05 v / v% proclin300).

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

[0198] The T3 / FT3 monoclonal antibody dilution is recorded as dilution 2, including a PBS buffer with a pH of 7.4 and a concentration of 0.1 mol / L, the buffer containing 0.9wt% NaCl, 0.1v / v% TW-20; 0.05wt% Brij35, 0.05v / v% Proclin300, 0.003wt% gentamicin sulfate, 2wt% BSA; 5wt% trehalose, 5wt% mannitol, 100μg / mL MAK33 Poly, 100μg / mL rabbit IgG, 100μg / mL mouse IgG, 100μg / mL bovine IgG, 100μg / mL goat IgG, and 1mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent.

[0199] The T3 test formula includes a dissociation agent, while the FT3 test formula does not include a dissociation agent.

[0200] 3. Preparation of T3 / FT3 monoclonal antibody 2 labeled with terpyridine ruthenium

[0201] T3 / FT3 monoclonal antibody 2 was placed in a pre-treated ultrafiltration tube, and ultrafiltration and centrifugation were performed for purification 3 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. The T3 / FT3 monoclonal antibody 2 solution was mixed with the acridine compound Ru-NHS ester solution at a mass ratio of 30:1, and reacted at 37°C in the dark for 2 hours, and then glycine solution was added to terminate the reaction. The reaction solution was dialyzed to remove excess Ru-NHS ester to obtain T3 / FT3 monoclonal antibody 2 labeled with terpyridine ruthenium.

[0202] 4. Preparation of Calibrators and Quality Controls

[0203] Prepare the matrix for T3 / FT3 calibration and quality control. The dilution of the calibration and quality control is recorded as dilution 3.

[0204] Dilution solution 3 includes HEPES buffer with a pH of 7.4 and a concentration of 0.05 mol / L, and the buffer contains 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 calibrants and quality controls.

[0205] 2. How to use the triiodothyronine chemiluminescent immunoassay kit:

[0206] 1. Sample pretreatment: Take the sample to be tested (such as serum, plasma or artificially prepared sample), centrifuge it at 5000 rpm at 4°C for 5 minutes and set aside.

[0207] 2. Before using the reagent, put it into the Pumen Company's fully automatic electrochemiluminescence analyzer 30 minutes in advance to automatically mix the magnetic beads and keep them in a suspended state.

[0208] 3. Calibration and quality control of the test kit: Use the matching calibrators to perform calibration tests and determine the accuracy of the current system through the quality control results.

[0209] 4. Sample testing:

[0210] The triiodothyronine chemiluminescence immunoassay kit detects the T3 concentration and FT3 concentration in the sample. The specific test process is as follows:

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

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

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

[0214] The diluent 2 was used to dilute the terpyridine ruthenium labeled T3 monoclonal antibody 2 to a working concentration of 0.5 μg / mL to obtain a terpyridine ruthenium labeled T3 monoclonal antibody 2 working solution.

[0215] The diluent 2 was used to dilute the terpyridine ruthenium labeled FT3 monoclonal antibody 2 to a working concentration of 0.5 μg / mL to obtain a terpyridine ruthenium labeled FT3 monoclonal antibody 2 working solution.

[0216] (2) After the user applies for the test, the system automatically draws 40μL of T3 magnetic particle working solution, 60μL of terpyridine ruthenium labeled T3 monoclonal antibody 2 working solution and 10μL of the sample to be tested into a reaction cup; the system automatically draws 40μL of FT3 magnetic particle working solution, 60μL of terpyridine ruthenium labeled FT3 monoclonal antibody 2 working solution and 10μL of the sample to be tested into another reaction cup. The mixture is automatically incubated at 37°C for 9 minutes to form an antigen-antibody sandwich complex, and then the entire complex is bound to the magnetic particles under the interaction of biotin and streptavidin.

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

[0218] Example 3

[0219] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0220] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % of sodium chloride, 1 wt % of sucrose, 0.12 v / v % of chaps, 0.06 v / v % of Brij35 (polyoxyethylene lauryl ether), 1.5 wt % of BSA, and 0.25 v / v % of proclin300.

[0221] Example 4

[0222] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0223] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % of sodium chloride, 1 wt % of sucrose, 0.08 v / v % of chaps, 0.04 v / v % of Brij35 (polyoxyethylene lauryl ether), 1.5 wt % of BSA, and 0.25 v / v % of proclin300.

[0224] Example 5

[0225] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0226] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % of sodium chloride, 1 wt % of sucrose, 0.08 v / v % of chaps, 0.06 v / v % of Brij35 (polyoxyethylene lauryl ether), 1.5 wt % of BSA, and 0.25 v / v % of proclin300.

[0227] Example 6

[0228] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0229] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % sodium chloride, 1 wt % sucrose, 0.12 v / v % chaps, 0.04 v / v % Brij35 (polyoxyethylene lauryl ether), 1.5 wt % BSA, and 0.25 v / v % proclin300.

[0230] Example 7

[0231] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0232] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with a HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % sodium chloride, 1 wt % sucrose, 0.12 v / v % chaps, 1.5 wt % BSA, and 0.25 v / v % proclin300.

[0233] Example 8

[0234] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0235] The components of dilution solution 1 include resuspending to a concentration of 0.75 mg / mL with HEPES buffer having a pH of 7.4 and a concentration of 0.05 mol / L, as well as 0.9 wt % sodium chloride, 1 wt % sucrose, 0.06 v / v % Brij35, 1.5 wt % BSA, and 0.25 v / v % proclin300.

[0236] Example 9

[0237] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0238] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, including a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 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. Among them, the T3 detection formula contains 1 mg / mL of 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, and the FT3 detection formula does not contain a dissociation agent.

[0239] Example 10

[0240] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0241] The T3 / FT3 monoclonal antibody dilution is recorded as dilution 2, including a PBS buffer with a pH of 7.4 and a concentration of 0.1 mol / L. The buffer 2 contains 0.9wt% NaCl, 0.08v / v% TWEEN-20, 0.04wt% Brij35, 0.05v / v% Proclin 300, 0.003wt% gentamicin sulfate, 2wt% BSA, 5wt% trehalose, 5wt% mannitol, 100μg / mL MAK33 Poly, 100μg / mL rabbit IgG, 100μg / mL mouse IgG, 100μg / mL bovine IgG, 100μg / mL goat IgG, and 1mg / 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 include a dissociation agent.

[0242] Embodiment 11

[0243] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0244] The T3 / FT3 monoclonal antibody dilution is recorded as dilution 2, including a PBS buffer with a pH of 7.4 and a concentration of 0.1 mol / L. The buffer 2 contains 0.9wt% NaCl, 0.08v / v% TWEEN-20, 0.06wt% Brij35, 0.05v / v% Proclin 300, 0.003wt% gentamicin sulfate, 2wt% BSA, 5wt% trehalose, 5wt% mannitol, 100μg / mL MAK33 Poly, 100μg / mL rabbit IgG, 100μg / mL mouse IgG, 100μg / mL bovine IgG, 100μg / mL goat IgG, and 1mg / 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 include a dissociation agent.

[0245] Example 12

[0246] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0247] The T3 / FT3 monoclonal antibody dilution is recorded as dilution 2, including a PBS buffer with a pH of 7.4 and a concentration of 0.1 mol / L. The buffer 2 contains 0.9wt% NaCl, 0.12v / v% TWEEN-20, 0.04wt% Brij35, 0.05v / v% Proclin 300, 0.003wt% gentamicin sulfate, 2wt% BSA, 5wt% trehalose, 5wt% mannitol, 100μg / mL MAK33 Poly, 100μg / mL rabbit IgG, 100μg / mL mouse IgG, 100μg / mL bovine IgG, 100μg / mL goat IgG, and 1mg / 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 include a dissociation agent.

[0248] Embodiment 13

[0249] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0250] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, including a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 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. Among them, 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent is included in the T3 detection formula, and the FT3 detection formula does not include a dissociation agent.

[0251] Embodiment 14

[0252] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0253] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, including a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 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 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. Among them, the T3 detection formula contains 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, and the FT3 detection formula does not contain a dissociation agent.

[0254] Embodiment 15

[0255] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0256] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, comprising a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 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. Among them, the T3 detection formula contains 1 mg / mL dithiothreitol dissociation agent, and the FT3 detection formula does not contain a dissociation agent.

[0257] Example 16

[0258] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0259] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, including a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 contains 0.9wt% NaCl, 0.1v / v% TWEEN-20, 0.05wt% Brij35, 0.05v / v% Proclin 300, 0.003wt% gentamicin sulfate, 2wt% BSA, 5wt% trehalose, 5wt% mannitol, 100μg / mL MAK33 Poly, 500μg / mL MAK33 Poly, and 1mg / mL mesna dissociating agent. Among them, the T3 detection formula contains 1mg / mL mesna dissociating agent, and the FT3 detection formula does not contain a dissociating agent.

[0260] Embodiment 17

[0261] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0262] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, including a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 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. Among them, the T3 detection formula contains 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, and the FT3 detection formula does not contain a dissociation agent.

[0263] Embodiment 18

[0264] This embodiment provides a triiodothyronine chemiluminescent immunoassay kit. The detection kit of this embodiment is substantially the same as that of embodiment 1, and the main differences are as follows:

[0265] The T3 / FT3 monoclonal antibody diluent is designated as diluent 2, comprising a PBS buffer having a pH of 7.4 and a concentration of 0.1 mol / L, wherein the buffer 2 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. Among them, the T3 detection formula contains 1 mg / mL 8-aniline-1-naphthalenesulfonic acid (ANS) dissociation agent, and the FT3 detection formula does not contain a dissociation agent.

[0266] Performance Testing

[0267] Working principle of the kit: The kit is equipped with a fully automatic electrochemiluminescence instrument; it realizes automatic sample addition, incubation, washing and detection steps; it avoids the result deviation caused by human operation. After the serum or plasma sample is fully mixed with the first and second reagents, the FT3 / T3 in the sample combines with the monoclonal antibody 1 and monoclonal antibody 2 in the first and second reagents to form a sandwich structure complex, and is fixed to the magnetic particles of the first reagent. Then the reaction mixture is washed away by magnetic absorption to remove the components that are not bound to the magnetic particles, and a continuous and stable light signal is emitted through the electrochemiluminescence system of the fully automatic electrochemiluminescence instrument; the light signal calculates the concentration of the sample through the standard curve; the signal value is proportional to the concentration.

[0268] 1. Detection limit determination

[0269] 1. Samples to be tested: Use matrix to dilute antigen and prepare 5 samples close to the LoD concentration value, where the LoD concentration value is set as: T3: 0.08 ng / mL, FT3: 0.08 pg / mL.

[0270] The above-mentioned prepared samples, in addition to the corresponding test substance (ie, FT3 / T3), also contain the following components: Tris buffer of Proclin-300, the concentration of Tris buffer is 0.01 mol / L, and the mass percentage content of Proclin-300 is 0.05%.

[0271] 2. Use the triiodothyronine chemiluminescent immunoassay kit of each embodiment to detect the sample to be tested in step 1. Specifically, 5 samples close to the LoD concentration value are tested using 2 batches of reagents for 5 days, with each sample repeated 8 times per day. After multiple batches of verification of the above multiple samples, the test results can meet the LoD.

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

[0273] The detection limit results are shown in Table 1.

[0274] As can be seen from Table 1, the detection limits of the kits of Example 1 and Example 3-Example 6 are lower than those of Example 7-Example 8, indicating that the synergistic cooperation of chaps and Brij35 in diluent 1 is conducive to reducing the detection limit of the kit and improving the detection sensitivity of the kit. Among them, the detection limits of the kits of Example 1 and Example 3-Example 4 are relatively close and lower than those of Example 5-Example 6, further indicating that the mass ratio of chaps to Brij35 in diluent 1 is 2, which is more conducive to reducing the detection limit of the kit.

[0275] The detection limits of the kits of Example 1 and Example 9 to Example 12 are lower than those of Example 13 to Example 14, indicating that the synergistic cooperation of TWEEN-20 and Brij35 in diluent 2 is beneficial to reducing the detection limit of the kit and improving the detection sensitivity of the kit. Among them, the detection limits of the kits of Example 1 and Example 9 to Example 10 are relatively close and lower than those of Example 11 to Example 12, further indicating that the mass ratio of TWEEN-20 to Brij35 in diluent 2 is 2, which is more conducive to reducing the detection limit of the kit.

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

[0277] In summary, the detection limit of the triiodothyronine chemiluminescent immunoassay kit of the present application for T3 detection is no more than 0.08 ng / mL, and the detection limit for FT3 detection is no more than 0.08 pg / mL, which is better than the detection limit of the conventional triiodothyronine detection kit (T3 is 0.20 ng / mL, FT3 is 0.28 pg / mL). The sensitivity of the triiodothyronine chemiluminescent immunoassay kit of the present application is high.

[0278] Table 1 Detection limit of the triiodothyronine chemiluminescent immunoassay kit 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] 2. Linear Correlation Determination

[0281] 1. Samples to be tested:

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

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

[0284] The above-mentioned prepared samples, in addition to the corresponding test substance (ie, FT3 / T3), also contain the following components: Tris buffer of Proclin-300, the concentration of Tris buffer is 0.01 mol / L, and the mass percentage content of Proclin-300 is 0.05%.

[0285] 2. Detection kit

[0286] The triiodothyronine chemiluminescent immunoassay kit of Example 1 was used to perform linear correlation determination.

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

[0288] As can be seen from Table 2, the FT3 detection linear range of the triiodothyronine chemiluminescent immunoassay kit of the present application is 0.10 pg / mL to 50 pg / mL, which is wider than that of the conventional triiodothyronine detection kit (FT3 detection linear range is 0.28 pg / mL to 32 pg / mL).

[0289] As can be seen from Table 3, the linear range of T3 detection of the triiodothyronine chemiluminescent immunoassay kit of the present application is 0.08-10.00 ng / mL, which is wider than that of the conventional triiodothyronine detection kit (the linear range of T3 detection is 0.40 ng / mL-6.50 ng / mL).

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

[0291]

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

[0293]

[0294] 3. Repeatability Determination

[0295] 1. Samples to be tested:

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

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

[0298] 2. Detection kit

[0299] The triiodothyronine chemiluminescent immunoassay kit of Example 1 was used to perform linear correlation determination.

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

[0301] It can be seen from Table 4 and Table 5 that the intra-batch repeatability of the triiodothyronine chemiluminescent immunoassay kit of the present application for FT3 detection is ≤5%, and the intra-batch repeatability for T3 detection is ≤5%.

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

[0303]

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

[0305]

[0306] In summary, the triiodothyronine chemiluminescent immunoassay kit of the present application shows in the FT3 test that the detection limit is increased from no more than 0.28 pg / mL (the detection limit of the conventional triiodothyronine detection kit) to no more than 0.08 pg / mL, the linear range is expanded from 0.28 pg / mL to 32 pg / mL to 0.10 pg / mL to 50 pg / mL, the intra-batch repeatability is ≤5%, the reaction time is shortened to 9 min, and the sample volume is increased from 15 μL (the detection limit of the conventional triiodothyronine detection kit) to 0.10 pg / mL to 50 pg / mL. The sample volume of the conventional triiodothyronine detection kit) was reduced to 10 μL; the results of the T3 test showed that the detection limit was increased from no more than 0.20 ng / mL (the detection limit of the conventional triiodothyronine detection kit) to no more than 0.08 ng / mL, the linear range was expanded from 0.40 ng / mL to 6.50 ng / mL to 0.08 to 10.00 ng / mL, the intra-batch repeatability was ≤5%, the reaction time was shortened to 9 min, and the sample volume was reduced from 30 μL (the sample volume of the conventional triiodothyronine detection kit) to 10 μL. The triiodothyronine chemiluminescent immunoassay kit of the present application solves the problems of poor sensitivity and narrow linear range in the competitive detection of FT3 / T3.

[0307] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A chemiluminescent immunoassay kit for triiodothyronine, characterized in that: The detection kit comprises: Antibody 1-solid phase carrier conjugate, antibody 2-chemiluminescent marker and triiodothyronine antigen calibrator, wherein the antibody 1-solid phase carrier conjugate comprises a solid phase carrier coated with triiodothyronine antibody 1, the antibody 2-luminescent marker comprises triiodothyronine antibody 2 labeled with a chemiluminescent marker, and both the antibody 1-solid phase carrier conjugate and the antibody 2-luminescent marker can specifically bind to a target analyte and have different antigen recognition epitopes; or, The invention relates to a streptavidin-solid phase carrier conjugate, an antibody 1-biotin marker, an antibody 2-chemiluminescent marker and a triiodothyronine antigen calibrator. The antibody 1-biotin marker comprises a triiodothyronine antibody 1 labeled with a biotin marker, the antibody 2-luminescent marker comprises a triiodothyronine antibody 2 labeled with a chemiluminescent marker, and both the antibody 1-biotin marker and the antibody 2-luminescent marker can specifically bind to a target analyte and have different antigen recognition epitopes.

2. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that: The detection kit meets one or more of the following conditions (1) to (4): (1) The solid phase carrier in the antibody 1-solid phase carrier conjugate comprises magnetic beads; Optionally, the surface modified functional groups of the magnetic beads include one or more of carboxyl, amino, hydroxyl, epoxy and p-toluenesulfonyl groups; (2) The modified active groups on the biotin include one or more of NHS ester group, amino group, maleimide group and hydrazide group; (3) the chemiluminescent marker includes one or more of Ru-NHS, horseradish peroxidase, alkaline phosphatase, acridinium ester, luminol and its derivatives, isoluminol and its derivatives, adamantane and acridinium compound-NHS; and, (4) The antibody in the antibody 1-solid phase carrier conjugate and / or the antibody 2-chemiluminescent marker is a monoclonal antibody.

3. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that: The detection kit comprises a detection reagent 1, wherein the detection reagent 1 comprises the antibody 1-solid phase carrier conjugate and a diluent 1; or the streptavidin-solid phase carrier conjugate and a diluent 1; Optionally, the diluent 1 includes salt ions 1, a surfactant 1, a preservative 1, a stabilizer 1 and a neutral buffer 1; Optionally, the salt ion 1 includes one or both of NaCl and KCl; Optionally, the surfactant 1 includes one or both of (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt) and polyoxyethylene lauryl ether; Optionally, the preservative 1 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Optionally, the stabilizer 1 includes one or more of SA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose and mannitol; Optionally, the neutral buffer 1 includes one or more of a phosphate buffer, a HEPES buffer and a MOPOS buffer; Optionally, the diluent 1 comprises 0.8wt% to 1.0wt% NaCl, 0.8wt% to 1.2wt% sucrose, 0.08v / v% to 0.12v / v% (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt), 0.04v / v% to 0.06v / v% polyoxyethylene lauryl ether, 1.4wt% to 1.6wt% bovine serum albumin, 0.05v / v% to 0.5v / v% proclin300, and HEPES buffer having a pH value of 6.0 to 8.0 and a concentration of 0.01mol / L to 0.2mol / L; Optionally, the concentration of the antibody 1-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 kit comprises a detection reagent 2, wherein the detection reagent 2 comprises the antibody 2-chemiluminescent marker and diluent 2; or comprises the antibody 1-biotin marker, the antibody 2-chemiluminescent marker and diluent 2; Optionally, the diluent 2 comprises salt ions 2, surfactant 2, preservative 2, stabilizer 2, dissociation agent, blocking agent and neutral buffer 2; Optionally, the salt ion 2 includes one or more of NaCl, ammonium sulfate and KCl; Optionally, the surfactant 2 includes one or both of Tween-20 and polyoxyethylene lauryl ether; Optionally, the preservative 2 comprises one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Optionally, the stabilizer 2 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, cystine, PEG, trehalose and mannitol; Optionally, the dissociating agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, mesna and tris(2-carbonylethyl)phosphine hydrochloride; Optionally, the blocking agent comprises one or more of MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG and goat IgG; Optionally, the neutral buffer 2 includes one or more of a phosphate buffer, a MES buffer, a Tris buffer and a HEPES buffer; Optionally, the concentration of the antibody 1-biotin label is 0.5 μg / mL to 2.0 μg / mL; Optionally, the concentration of the antibody 2-luminescent marker is 0.5 μg / mL to 2.0 μg / mL; Optionally, the diluent 2 comprises 0.8wt% to 1.0wt% NaCl, 0.08v / v% to 0.12v / v% Tween-20, 0.04wt% to 0.06wt% polyoxyethylene lauryl ether, 0.05v / v% to 0.5v / v% proclin300, 0.002wt% to 0.004wt% gentamicin sulfate, 1.8wt% to 2.2wt% bovine serum albumin, 4.8wt% to 5.2wt% trehalose, 4.8wt% to 5.2wt% mannitol, 80μg / mL to 120μg / mL MAK33 Poly, 80μg / mL~120μg / mL rabbit IgG, 80μg / mL~120μg / mL mouse IgG, 80μg / mL~120μg / mL bovine IgG, 80μg / mL~120μg / mL goat IgG, 0.8mg / mL~1.2mg / mL 8-aniline-1-naphthalenesulfonic acid, and PBS buffer with a pH of 7.3 to 7.5 and a concentration of 0.08mol / L to 0.12mol / L.

5. The triiodothyronine chemiluminescent immunoassay kit according to claim 1, characterized in that: The kit includes a detection reagent 3; the detection reagent 3 includes the triiodothyronine antigen calibrator and a diluent 3; The diluent 3 includes salt ions 3, surfactant 3, preservative 3, stabilizer 3 and neutral buffer 3; Optionally, the salt ions 3 include one or both of NaCl and KCl; Optionally, the surfactant 3 includes Tween-20 and polyoxyethylene lauryl ether; Optionally, the preservative 3 includes one or more of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Optionally, the stabilizer 3 includes one or more of BSA, HSA, casein, gelatin, sucrose, glycerol, glycine, trehalose and mannitol; Optionally, the neutral buffer 3 includes one or more of a phosphate buffer, a Tris buffer and a HEPES buffer; Optionally, the diluent 3 includes 0.04v / v% to 0.06v / v% Tween-20, 1.4wt% to 1.6wt% bovine serum albumin, 1.5wt% to 2.5wt% trehalose, 1.5wt% to 2.5wt% mannitol, 0.04v / v% to 0.06v / v% proclin 300 and HEPES buffer with a pH of 7.3 to 7.5 and a concentration of 0.04mol / L to 0.06mol / L.

6. The method for preparing the triiodothyronine chemiluminescent immunoassay kit according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: providing the antibody 1-solid phase carrier conjugate, the antibody 2-chemiluminescent marker and the triiodothyronine antigen calibrator respectively; or, The streptavidin-solid phase carrier conjugate, the antibody 1-biotin marker, the antibody 2-chemiluminescent marker and the triiodothyronine antigen calibrator are provided respectively to prepare a detection kit.

7. The preparation method according to claim 6, characterized in that: The steps for preparing the antibody 1-solid phase carrier conjugate include: Antibody 1 and the solid phase carrier undergo a first coupling reaction, and blocking agent 1 and blocking agent 2 are sequentially added to the obtained first coupling reaction product for blocking to prepare the antibody 1-solid phase carrier conjugate; and / or The preparation steps of the antibody 2-chemiluminescent marker include: Conducting a second coupling reaction between the antibody 2 solution and the chemiluminescent marker to prepare the antibody 2-chemiluminescent marker; Optionally, the blocking agent 1 includes 1.4wt% to 1.5wt% glycine, 0.8wt% to 1.2wt% sucrose, 0.04v / v% to 0.06v / v% Tween-20, 1.4wt% to 1.6wt% bovine serum albumin, 0.05v / v% to 0.5v / v% proclin300, and a PBS buffer having a pH of 5.8 to 6.2 and a concentration of 0.04 to 0.06 mol / L; Optionally, the blocking agent 2 includes 0.4wt% to 0.6wt% of poloxamer, 0.8wt% to 1.2wt% of sucrose, 0.04v / v% to 0.06v / v% of Tween-20, 1.4wt% to 1.6wt% of bovine serum albumin, 0.05v / v% to 0.5v / v% of proclin300, and a HEPES buffer having a pH of 7.3 to 7.5 and a concentration of 0.04mol / L to 0.06mol / L.

8. The preparation method according to claim 7, characterized in that: The steps of preparing the streptavidin-solid phase carrier conjugate include: performing a second coupling reaction on the streptavidin and the solid phase carrier, and sequentially adding the blocking agent 1 and the blocking agent 2 to the obtained second coupling reaction product for blocking, thereby preparing the streptavidin and the solid phase carrier; The method for preparing the antibody 1-biotin marker comprises: performing a third coupling reaction between the antibody 1 and the biotin marker to prepare the antibody 1-biotin marker.

9. The preparation method according to any one of claims 7 to 8, characterized in that: The conditions of the first coupling reaction include: the temperature of the blocking agent 1 and the blocking agent 2 is 36° C. to 38° C., and the time is 20 h to 28 h; The conditions of the second coupling reaction include: the mass ratio of the antibody 2 to the chemiluminescent marker is (28-32):1, the reaction conditions are 36° C.-38° C., and the reaction time is 1 h-3 h; The conditions of the third coupling reaction include: the blocking temperature of the blocking agent 1 and the blocking agent 2 is 36° C. to 38° C., and the time is 20 h to 28 h.

10. A method for detecting triiodothyronine, characterized in that: The detection method comprises the steps of using the triiodothyronine chemiluminescence immunoassay kit according to any one of claims 1 to 5 to detect triiodothyronine in a sample to be tested; Optionally, the sample to be tested includes one or both of serum and plasma.

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