Electrochemiluminescence immunoassay kit as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunodetection, in particular to an electrochemical luminescence immunodetection kit as well as a preparation method and application thereof. The detection kit comprises: an antibody 1-solid phase carrier conjugate; an antibody 2-biotin conjugate; and a streptavidin-ruthenium marker conjugate, which is a conjugate of the streptavidin-ruthenium marker, an antibody 1 in the antibody 1-solid-phase carrier conjugate and an antibody 2 in the antibody 2-biotin conjugate can be specifically combined with a target analyte and have different antigen recognition epitopes. According to the application, the ruthenium marker is coupled to the surface of the streptavidin molecule, so that the influence of the ruthenium marker on the stability of the antibody and the antigen recognition capability can be avoided, the ruthenium marker can be indirectly connected to the surface of the detection antibody molecule by utilizing the high affinity and high specificity of the streptavidin and the biotin, and compared with the traditional technology, the method has the advantages that the sensitivity is high; the quantity of ruthenium markers on the surface of a single antibody molecule is more, so that the electrochemical luminescence signal value is stronger, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassay, and in particular to an electrochemiluminescent immunoassay kit and a preparation method and application thereof. Background Art

[0002] Biotin-Avidin—System (BAS) is a commonly used signal amplification system in immunoassays. It is a pair of compounds with strong non-covalent interactions that can bind firmly and specifically. The application of this signal amplification system can greatly improve the sensitivity of detection, making BAS immunolabeling and related tracer analysis more sensitive. It has been widely used in qualitative and quantitative detection and positioning observation research of antigens and antibodies.

[0003] Biotin is composed of an imidazolone ring and a thiophene ring. The imidazolone ring is the main one that binds to avidin, while the carboxyl group at the end of the thiophene ring side chain can be used as a functional group for coupling antibodies and other macromolecules.

[0004] Streptavidin (SA) is a homotetrameric protein secreted by Streptomyces avidinii during its growth. Each SA molecule has four binding sites for biotin molecules. The binding of SA to biotin is similar to the binding of antibodies and antigens, with extremely high specificity. It can bind to each other in a complex solution environment, and its binding constant is 10 15 mol / L, approximately the Ka(10 5 ~10 11 mol / L). Acids, alkalis, denaturants, proteases and organic solvents have little effect on the binding of avidin to biotin, and the structural integrity can be maintained even in urea or guanidine hydrochloride solutions with a concentration of up to 8M. Compared with avidin, streptavidin does not carry any sugar groups and has lower non-specific binding than avidin in detection applications. BAS can be combined with a variety of labeling systems such as enzymes, fluorescein, ferritin, lectin, Staphylococcus a protein, radionuclides, etc., and can be used in a variety of biological reaction systems such as antigens, antibodies, proteins, hormones, receptors, nucleic acid systems, etc. It can also be used as an affinity medium for the separation and purification of reactants in the above-mentioned reaction systems.

[0005] Electrochemiluminescence immunoassay (ECLI) is a combination of electrochemiluminescence (ECL) and immunoassay. It is a new generation of labeled immunoassay technology after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and chemiluminescence immunoassay. The marker used in electrochemiluminescence immunoassay is ruthenium trispyridine, which can bind to antibodies or antigen molecules through N-hydroxysuccinamide (NHS) ester to produce labeled antibodies or antigens. ECLI is a specific chemiluminescence reaction triggered by electrochemistry on the electrode surface. It actually includes the following two processes of electrochemistry and chemiluminescence and the cycle of these two processes:

[0006] (1) Electrochemical reaction process: When a certain voltage energy is applied to the working electrode (anode), divalent ruthenium trichlorobipyridyl [Ru(bpy)3] 2+ It releases electrons and undergoes oxidation reaction to become trivalent ruthenium trichlorobipyridyl [Ru(bpy)3] 3+ At the same time, tripropylamine (TPA) on the electrode surface also releases electrons to undergo oxidation reaction and become cationic free radical TPA + , and rapidly and spontaneously removes a proton to form tripropylamine free radical TPA · Thus, there is a strong oxidizing trivalent ruthenium trichlorobipyridyl [Ru(bpy) 3] 3+ and tripropylamine free radical TPA with strong reducing properties · .

[0007] (2) Chemiluminescence process: The trivalent ruthenium trichlorobipyridyl [Ru(bpy)3 ]3+ and tripropylamine free radical TPA with strong reducing properties · The redox reaction occurs, resulting in the trivalent ruthenium trichlorobipyridyl [Ru(bpy)3] 3+ Reduced to an excited state of divalent trichlorobipyridyl [Ru(bpy)3] 2+ , whose energy comes from trivalent ruthenium trichlorobipyridyl [Ru(bpy)3] 3+ Tripropylamine free radical TPA · The potential difference between the excited state [Ru(bpy)3] 2+ It decays by fluorescence mechanism and releases energy by releasing a photon with a wavelength of 620nm, becoming the ground state [Ru(bpy)3] 2+ .

[0008] (3) Cyclic process: After the above chemiluminescence process, divalent ruthenium trichlorobipyridyl [Ru(bpy)3] still exists in the reaction system 2+and tripropylamine (TPA), so that the electrochemical reaction and chemiluminescence process on the electrode surface can continue, so that the whole reaction process can be cyclic.

[0009] Through the above-mentioned cycle process, the measurement signal is continuously amplified, thereby greatly improving the detection sensitivity, so ECLI measurement has the characteristics of high sensitivity. [Ru(bpy)3] 2+ The molecular ratio of the derivative to the immunoglobulin is more than 20, which will not affect the solubility and immune activity of the antibody; [Ru(bpy)3] 2+ The small molecular weight and small steric hindrance can label even small nucleic acids, greatly enriching the detection menu. More importantly, it provides broad space for the development of its detection menu.

[0010] The main advantages of ECLI include: ① The marker can be recycled, making the luminescence time longer, the intensity higher, and easy to measure; ② High sensitivity, up to pg / mL or pM level; ③ Wide linear range, up to >10 4 ④Short reaction time, the assay can be completed within 20 minutes; ⑤Good reagent stability, it can be kept at 2-5°C for more than 1 year. Currently, ECLI can be applied not only to all immunoassays, but also to the detection of DNA / RNA probes.

[0011] Magnetic particle electrochemiluminescence immunoassay is an analytical method that combines magnetic separation technology, electrochemiluminescence technology, and immunoassay technology. This technology makes full use of the rapid and easy automation of magnetic separation technology, the high sensitivity of electrochemiluminescence technology, and the specificity of immunoassay, and plays an irreplaceable role in the field of bioanalysis. The most widely used method is to coat SA on the surface of magnetic beads, biotin-label the capture antibody, couple the signal marker to the detection antibody, and detect the antigen concentration in the sample by the double antibody sandwich method.

[0012] In clinical testing, some projects have very high requirements for the detection sensitivity of reagents, which need to reach fg / mL or even lower. The conventional methods mentioned above are not suitable for projects with high sensitivity requirements due to solid phase steric hindrance and poor signal amplification effect, and cannot meet clinical requirements. Low-sensitivity detection technology often leads to inaccurate quantification or even false negative results. Summary of the invention

[0013] Based on this, one or more embodiments of the present application provide an electrochemiluminescent immunoassay kit and a preparation method and application thereof, including the following technical solutions:

[0014] One or more embodiments of the present application provide an electrochemiluminescent immunoassay kit, the assay kit comprising:

[0015] Antibody 1-solid phase carrier conjugate;

[0016] Antibody 2-biotin conjugate; and,

[0017] Streptavidin-ruthenium label conjugate;

[0018] The antibody 1 in the antibody 1-solid phase carrier conjugate and the antibody 2 in the antibody 2-biotin conjugate can both specifically bind to the target analyte and have different antigen recognition epitopes.

[0019] In some embodiments of the present application, the detection kit satisfies one or more of the following conditions:

[0020] (1) The solid phase carrier in the antibody 1-solid phase carrier conjugate comprises magnetic beads; optionally, the functional groups modified on the surface of the magnetic beads comprise one or more of carboxyl, amino, hydroxyl, epoxy and p-toluenesulfonyl groups; and,

[0021] (2) 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;

[0022] Optionally, the diluent 1 comprises NaCl, a protein protective agent 1, a preservative 1, a surfactant 1 and a neutral buffer 1;

[0023] Optionally, the protein protective agent 1 includes a carbohydrate protective agent 1 and a protein protective agent 1; optionally, the carbohydrate protective agent 1 includes sucrose, trehalose, dextran, etc.; optionally, the protein protective agent 1 includes bovine serum albumin;

[0024] Optionally, the preservative 1 includes PC300;

[0025] Optionally, the surfactant 1 includes Tween-20;

[0026] Optionally, the neutral buffer 1 includes one or more of HEPES buffer, PBS buffer and Tris buffer;

[0027] Optionally, the diluent 1 comprises 80mM to 120mM HEPES buffer and 125mM to 175mM NaCl, 45g / L to 55g / L sucrose, 0.1% (w / v) to 0.6% (w / v) Tween-20, 0.1wt% to 0.6wt% bovine serum albumin, 0.004% (v / v) to 0.006% (v / v), pH 7 to 7.6;

[0028] Optionally, the concentration of the antibody 1-solid phase carrier conjugate in the detection reagent 1 is 0.3 mg / mL to 2 mg / mL.

[0029] In some embodiments of the present application, the detection kit includes detection reagent 2, and the detection reagent 2 includes the antibody 2-biotin conjugate and diluent 2;

[0030] Optionally, the diluent 2 comprises a protein protectant 2, a preservative 2, a surfactant 2, a blocking agent 1 and a neutral buffer 2;

[0031] Optionally, the protein protective agent 2 includes a carbohydrate protective agent 2; Optionally, the carbohydrate protective agent 2 includes sucrose;

[0032] Optionally, the preservative 2 includes PC300;

[0033] Optionally, the surfactant 2 includes one or more of Tween-20 and Triton X-100;

[0034] Optionally, the blocking agent 1 comprises mouse IgG;

[0035] Optionally, the neutral buffer 2 includes one or more of PBS buffer and HEPES buffer;

[0036] Optionally, the diluent 2 comprises 80 mM to 120 mM PBS buffer and 45 g / L to 55 g / L sucrose, 0.04% (w / v) to 0.06% (w / v) Tween-20, 0.04% (v / v) to 0.06% (v / v) Triton X-100, 0.04% (v / v) to 0.06% (v / v) PC300 and 0.06M to 0.1M mouse IgG, pH 7 to 7.6;

[0037] Optionally, the concentration of the antibody 2-biotin conjugate in the detection reagent 2 is 0.5 μg / mL to 5 μg / mL.

[0038] In some embodiments of the present application, the detection kit includes detection reagent 3, and the detection reagent 3 includes the streptavidin-ruthenium marker conjugate and diluent 3;

[0039] Optionally, the diluent 3 comprises a protein protective agent 3, a preservative 3, a surfactant 3, a blocking agent 2 and a neutral buffer 3;

[0040] Optionally, the protein protective agent 3 includes a carbohydrate protective agent 3; Optionally, the carbohydrate protective agent 3 includes sucrose; Optionally, the preservative 3 includes PC300;

[0041] Optionally, the surfactant 3 includes one or more of Tween-20 and Triton X-100;

[0042] Optionally, the blocking agent 2 comprises mouse IgG;

[0043] Optionally, the neutral buffer 3 includes one or more of PBS buffer and HEPES buffer;

[0044] Optionally, the diluent 3 comprises 80 mM to 120 mM PBS buffer and 45 g / L to 55 g / L sucrose, 0.04% (w / v) to 0.06% (w / v) Tween-20, 0.04% (v / v) to 0.06% (v / v) Triton X-100, 0.04% (v / v) to 0.06% (v / v) PC300 and 0.06M to 0.1M mouse IgG, pH 7 to 7.6;

[0045] Optionally, the amount of the streptavidin-ruthenium marker conjugate in the detection reagent 3 is 1 to 10 times that of the antibody 2-biotin conjugate.

[0046] In some embodiments of the present application, the detection kit satisfies one or more of the following conditions:

[0047] 1) Antibody 1 in the antibody 1-solid phase carrier conjugate and / or Antibody 2 in the antibody 2-biotin conjugate are monoclonal antibodies; and,

[0048] 2) The target analyte is thyroid stimulating hormone, IL-6 or cTNT.

[0049] One or more embodiments of the present application also provide a method for preparing the electrochemiluminescent immunoassay kit, the method comprising the following steps:

[0050] The antibody 1-solid phase carrier conjugate, the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate are provided respectively to prepare a detection kit.

[0051] In some embodiments of the present application, the steps of preparing the antibody 1-solid phase carrier conjugate include:

[0052] The antibody 1 and the solid phase carrier undergo a first coupling reaction, and a blocking agent 1 is added to the obtained first coupling reaction product for blocking to prepare the antibody 1-solid phase carrier conjugate;

[0053] Optionally, the blocking agent 1 is used for blocking, and one or more of the conditions (I) to (III) are met:

[0054] (I) the blocking agent 1 comprises a non-specific protein and a neutral buffer 4; optionally, the non-specific protein comprises one or more of casein, bovine serum albumin and human serum albumin; optionally, the neutral buffer 4 comprises a Tris-HCl buffer; optionally, the blocking agent 1 comprises 30 mM to 100 mM Tris-HCl and 1% (w / v) to 10% (w / v) bovine serum albumin, pH 7 to 8;

[0055] Optionally, the blocking agent 1 further comprises a blocking enhancer; optionally, the blocking enhancer comprises one or more of a surfactant, a streptavidin-biotin conjugate, a cyclodextrin and an amino acid; optionally, the small molecule amino acid is not glycine; optionally, the surfactant comprises one or more of Tween-20, Triton X-405 and Triton X-100; optionally, the small molecule amino acid comprises one or more of lysine and arginine; optionally, the concentration of Tween-20 in the blocking agent 1 is 0.1% (v / v) to 0.5% (v / v); optionally, the concentration of the streptavidin-biotin conjugate in the blocking agent 1 is 0.02 g / L to 2 g / L; optionally, the concentration of the cyclodextrin in the blocking agent 1 is 0.1% (w / v) to 2% (w / v); optionally, the concentration of the lysine in the blocking agent 1 is 1% (w / v) to 10% (w / v);

[0056] (II) the amount of the blocking agent 1 corresponding to each 5 mg of the solid phase carrier is 100 μL to 1000 μL; and,

[0057] (III) Constant temperature, the sealing temperature is 25°C to 40°C, the sealing time is 8h to 72h, and optionally 12h to 36h;

[0058] Optionally, the first coupling reaction satisfies one or more of the conditions shown in I) to IV):

[0059] Ⅰ) under alkaline conditions without primary amino groups; optionally, in one or more of a sodium bicarbonate solution and a boric acid buffer; optionally, the pH value of the boric acid solution is 9.3 to 9.6, and the concentration of boric acid is 0.08 M to 0.12 M;

[0060] II) using ammonium sulfate as a catalyst; optionally, the concentration of the ammonium sulfate in the initial reaction system is 0.5M to 10M, optionally 1M to 5M;

[0061] III) constant temperature, the reaction temperature is 25°C to 40°C, the reaction time is 18h to 72h, optionally 18h to 36h; and,

[0062] IV) The amount of the antibody 1 used per 5 mg of the solid phase carrier is 25 μg to 100 μg.

[0063] In some embodiments of the present application, the steps of preparing the antibody 2-biotin conjugate include:

[0064] The antibody 2 and the biotin modified with the active group undergo a second coupling reaction, and the blocking agent 2 is added to the obtained second coupling reaction product for blocking to prepare the antibody 2-biotin conjugate;

[0065] Optionally, the second coupling reaction satisfies one or more of the conditions (i) to (iv):

[0066] (i) the modified active groups on the biotin include one or more of NHS ester group, amino group, maleimide group and hydrazide group;

[0067] (ii) under neutral or alkaline conditions without primary amino groups; optionally, in one or more of sodium bicarbonate solution, PBS buffer, HEPES buffer and MOPS buffer; optionally, in a buffer with a pH value of 7 to 9; optionally, in a PBS buffer with a pH value of 7.1 to 7.6 and a concentration of 0.008 M to 0.012 M;

[0068] (iii) the molar concentration ratio of the antibody 2 to the biotin modified with the active group is 1:(5-80), optionally 1:(10-40);

[0069] (iv) the reaction temperature is 20°C to 40°C, and the reaction time is 10 minutes to overnight;

[0070] Optionally, the sealing agent 2 is used for sealing, and one or more of the following conditions i) to iii) are met:

[0071] i) the blocking agent 2 comprises a small molecule with a primary amino group; optionally, the small molecule with a primary amino group comprises one or more of glycine, Tris and ethanolamine; optionally, the amount of the blocking agent 2 corresponding to the blocking system containing 500 μg to 2 mg of the antibody 2 is 8 μL to 12 μL, and the concentration of glycine is 1.8M to 2.2M;

[0072] ii) The sealing temperature is 20°C to 40°C, and the sealing time is 5min to 15min;

[0073] iii) the blocked product is purified to prepare the antibody 2-biotin conjugate; optionally, the purification step is dialysis, desalting or ultrafiltration; optionally, the ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of 10kDa to 50kDa, optionally 10kDa to 30kDa.

[0074] In some embodiments of the present application, the steps of preparing the streptavidin-ruthenium label conjugate include:

[0075] Performing a third coupling reaction between streptavidin and the ruthenium marker modified with an active group, adding a blocking agent 3 to the obtained reaction product for blocking, and preparing the streptavidin-ruthenium marker conjugate;

[0076] Optionally, the third coupling reaction satisfies one or more of the following conditions (A) to (D):

[0077] (A) under neutral or alkaline conditions without primary amino groups; optionally, in one or more of sodium bicarbonate solution, PBS buffer, HEPES buffer and MOPS buffer; optionally, in a buffer with a pH value of 7 to 9; optionally, in a PBS buffer with a pH value of 7.1 to 7.6 and a concentration of 0.008 M to 0.012 M;

[0078] (B) the molar concentration ratio of the streptavidin to the active group-modified ruthenium label is 1:(5-80), optionally 1:(10-40);

[0079] (C) the modified active group on the active group-modified ruthenium label comprises an NHS ester group;

[0080] (D) The reaction temperature is 20°C to 40°C, and the reaction time is 10 minutes to overnight;

[0081] Optionally, the sealing agent 3 is used for sealing, and one or more of the following conditions are met:

[0082] A) the blocking agent 3 comprises a small molecule with a primary amino group; optionally, the small molecule with a primary amino group comprises one or more of glycine, Tris and ethanolamine; optionally, the amount of the blocking agent 3 corresponding to the blocking system containing 500 μg to 2 mg of the streptavidin is 18 μL to 22 μL, and the concentration of glycine is 1.8M to 2.2M;

[0083] B) The sealing temperature is 20℃~37℃, and the sealing time is 5min~15min;

[0084] C) The blocked product is purified to prepare the antibody 2-biotin conjugate; optionally, the purification step is dialysis, desalting or ultrafiltration; optionally, the ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of 10kDa to 50kDa, optionally 10kDa to 30kDa.

[0085] One or more embodiments of the present application further provide a method for detecting a target analyte, which comprises the step of using the electrochemiluminescence immunoassay kit to detect the target analyte in the sample to be tested.

[0086] In some embodiments of the present application, the detection method comprises the following steps:

[0087] The sample to be tested, the antibody 1-solid phase carrier conjugate and the antibody 2-biotin conjugate are mixed and incubated in the first stage to prepare a first incubation product;

[0088] The first incubation product and the streptavidin-ruthenium label conjugate are mixed and incubated in the second stage to prepare a second incubation product; and,

[0089] mixing the second incubation product and a co-reactant corresponding to the ruthenium label, and performing luminescence detection;

[0090] Optionally, the concentration ratio of the antibody 2-biotin conjugate to the streptavidin-ruthenium marker conjugate is 1:(1-10), optionally 1:(2-4).

[0091] In some embodiments of the present application, the sample to be tested is blood, serum, plasma, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid, urine, amniotic fluid or aqueous humor.

[0092] 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

[0093] 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.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0095] the term

[0096] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0097] 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").

[0098] 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.

[0099] 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.

[0100] 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.

[0101] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0102] 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.

[0103] 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.

[0104] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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 application 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.

[0110] The present application utilizes the high affinity and high specific recognition ability between streptavidin and biotin to realize the indirect amplification principle of immunoassay: a detection antibody molecule is connected to multiple biotins on its surface, and a streptavidin molecule is connected to multiple terpyridine rutheniums on its surface. One biotin can bind to a streptavidin connected to multiple terpyridine rutheniums, resulting in more terpyridine ruthenium molecules on the surface of the antibody molecule than terpyridine ruthenium directly coupled to the surface of the antibody molecule using amino groups. The generated electrochemiluminescence signal will be stronger, thereby realizing detection amplification.

[0111] In a first aspect of an embodiment of the present application, an electrochemiluminescent immunoassay kit is provided, the assay kit comprising: an antibody 1-solid phase carrier conjugate; an antibody 2-biotin conjugate; and a streptavidin-ruthenium marker conjugate;

[0112] The antibody 1 in the antibody 1-solid phase carrier conjugate and the antibody 2 in the antibody 2-biotin conjugate can both specifically bind to the target analyte and have different antigen recognition epitopes.

[0113] Antibody 1 in this application is also the capture antibody, and antibody 2 is also the detection antibody.

[0114] In the embodiment of the present application, streptavidin is coupled to a ruthenium marker, instead of the detection antibody directly coupled to the ruthenium marker, and the high affinity and high specificity between streptavidin and biotin are used to indirectly connect the ruthenium marker to the detection antibody. One streptavidin molecule can be coupled to multiple ruthenium markers, and one detection antibody molecule can be coupled to multiple biotins. The cascade reaction between the biotinylated antibody and streptavidin can significantly increase the number of ruthenium markers in the detection antibody molecule, so as to amplify the signal, improve the detection sensitivity, and meet clinical needs.

[0115] The detection kit of the present application is used for detection. The solid phase carrier (magnetic beads) coupled with the capture antibody captures the target analyte in the sample to be tested, and a double antibody sandwich structure is constructed with the detection antibody coupled with biotin. The streptavidin coupled with the ruthenium marker indirectly connects the ruthenium marker to the antibody surface through specific recognition with biotin. Through magnetic attraction, the double antibody sandwich structure with the ruthenium marker is adsorbed near the electrode detection pool, and a redox reaction occurs with the co-reactant tripropylamine to generate an electrochemical luminescence signal value.

[0116] In the present application, antibody 1 and antibody 2 can form a double antibody sandwich structure with the antigen.

[0117] In some embodiments, the detection kit satisfies one or more of the following conditions:

[0118] (1) The solid phase carrier in the antibody 1-solid phase carrier conjugate comprises magnetic beads; optionally, the functional groups modified on the surface of the magnetic beads comprise one or more of carboxyl, amino, hydroxyl, epoxy and p-toluenesulfonyl groups; and,

[0119] (2) 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; optionally, the diluent 1 comprises NaCl, a protein protective agent 1, a preservative 1, a surfactant 1 and a neutral buffer 1; optionally, the protein protective agent 1 comprises a carbohydrate protective agent 1 and a protein protective agent 1; optionally, the carbohydrate protective agent 1 comprises sucrose, trehalose, dextran, etc.; optionally, the protein protective agent 1 comprises bovine serum albumin; optionally, the preservative 1 comprises PC300; optionally, the surfactant 1 comprises Twe en-20; optionally, the neutral buffer 1 includes one or more of a HEPES buffer, a PBS buffer and a Tris buffer; optionally, the diluent 1 includes 80mM to 120mM (for example, 80, 85, 90, 95, 100, 105, 110, 115, 120mM) HEPES buffer and 125mM to 175mM (for example, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175mM) NaCl, 45g / L to 55g / L (for example, 45 , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 g / L) sucrose, 0.1% to 0.6% (w / v) (e.g. 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%) Tween-20, 0.1wt% to 0.6wt% (e.g. 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%) bovine serum Albumin, 0.004% (v / v) ~ 0.006% (v / v) (for example, 0.004%, 0.0045%, 0.005%, 0.0055%, 0.006%) PC300, pH 7 ~ 7.6 (for example, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6); Optionally, the concentration of the antibody 1-solid phase carrier conjugate in the detection reagent 1 is 0.3 mg / mL ~ 2 mg / mL (for example, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2 mg / mL).

[0120] In some embodiments, the detection kit includes a detection reagent 2, the detection reagent 2 includes the antibody 2-biotin conjugate and a diluent 2; optionally, the diluent 2 includes a protein protectant 2, a preservative 2, a surfactant 2, a blocking agent 1 and a neutral buffer 2; optionally, optionally, the protein protectant 2 includes a carbohydrate protectant 2; optionally, the carbohydrate protectant 2 includes sucrose; optionally, the preservative 2 includes PC300; optionally, the surfactant 2 includes one or more of Tween-20 and Triton X-100; optionally, the blocking agent 1 includes mouse IgG; optionally, the neutral buffer 2 includes one or more of PBS buffer and HEPES buffer; optionally, the diluent 2 includes 80mM to 120mM (for example, 80, 85, 90, 95, 100, 105, 110, 115, 120mM) PBS buffer and 45g / L to 55g / L (45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 g / L) sucrose, 0.04% (w / v) to 0.06% (w / v) (e.g., 0.04%, 0.045%, 0.05%, 0.055%, 0.06%) Tween-20, 0.04% (v / v) to 0.06% (v / v) (e.g., 0.04%, 0.045%, 0.05%, 0.055%, 0.06%) Triton X-100, 0.04% (v / v) to 0.06% (v / v) (e.g., 0.04%, 0.045%, 0.05%, 0.055%, 0.06%) PC300 and 0.06M to 0.1M (e.g., 0.06, 0.07, 0.08, 0.09, 0.1M) mouse IgG, pH 7 to 7.6 (e.g., 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6); Optionally, the concentration of the antibody 2-biotin conjugate in the detection reagent 2 is 0.5 μg / mL to 5 μg / mL (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μg / mL). The diluent of the antibody 2-biotin conjugate should allow the antibody to be stable for a long time without affecting its activity and structure. Generally, a neutral buffer is selected, and protective proteins and preservatives are added to the buffer to facilitate the long-term preservation of antibodies.

[0121] In some embodiments, the detection kit includes a detection reagent 3, the detection reagent 3 includes the streptavidin-ruthenium marker conjugate and a diluent 3; optionally, the diluent 3 includes a protein protectant 3, a preservative 3, a surfactant 3, a blocking agent 2 and a neutral buffer 3; optionally, the protein protectant 3 includes a carbohydrate protectant 3; optionally, the carbohydrate protectant 3 includes sucrose; optionally, the preservative 3 includes PC300; optionally, the surfactant 3 includes one or more of Tween-20 and Triton X-100; optionally, the blocking agent 2 includes mouse IgG; optionally, the neutral buffer 3 includes one or more of PBS buffer and HEPES buffer; optionally, the diluent 3 includes 80mM to 120mM (for example, 80, 85, 90, 95, 100, 105, 110, 115, 120mM) PBS buffer and 45g / L to 55g / L (45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 g / L) sucrose, 0.04% (w / v) to 0.06% (w / v) (e.g., 0.04%, 0.045%, 0.05%, 0.055%, 0.06%) Tween-20, 0.04% (v / v) to 0.06% (v / v) (e.g., 0.04%, 0.045%, 0.05%, 0.055%, 0.06%) Triton X-100, 0.04% (v / v) to 0.06% (v / v) (for example, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%) PC300 and 0.06M to 0.1M (for example, 0.06, 0.07, 0.08, 0.09, 0.1M) mouse IgG, pH 7 to 7.6 (for example, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6); Optionally, the amount of the streptavidin-ruthenium marker conjugate in the detection reagent 3 is 1 to 10 times (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times) of the antibody 2-biotin conjugate. The storage buffer of the SA-Ru conjugate can keep the protein stable for a long time without affecting its activity and structure. Generally, a neutral buffer is selected, and protective proteins and preservatives are added to the buffer to facilitate long-term storage of antibodies. The Ru complex should be stored away from light.

[0122] In some embodiments, the detection kit satisfies one or more of the following conditions:

[0123] 1) Antibody 1 in the antibody 1-solid phase carrier conjugate and / or Antibody 2 in the antibody 2-biotin conjugate are monoclonal antibodies; and,

[0124] 2) The target analyte is thyroid stimulating hormone, IL-6 or cTNT.

[0125] The diluents in this application (e.g., diluent 1 and diluent 2) not only protect the activity and stability of the antibody from loss, but also ensure that there is no interference from clinical samples during the test. It is understandable that different target analytes have different corresponding antibodies, and based on this, the formula of the diluent can also be adjusted accordingly.

[0126] In a second aspect of the embodiment of the present application, a method for preparing the electrochemiluminescent immunoassay kit is provided, the method comprising the following steps:

[0127] The antibody 1-solid phase carrier conjugate, the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate are provided respectively to prepare a detection kit.

[0128] In some embodiments, the steps of preparing the antibody 1-solid phase carrier conjugate include:

[0129] The antibody 1 and the solid phase carrier undergo a first coupling reaction, and a blocking agent 1 is added to the obtained first coupling reaction product for blocking to prepare the antibody 1-solid phase carrier conjugate; the first coupling reaction is described as follows using p-toluenesulfonyl magnetic beads as an example: by physical adsorption, the antibody 1 gradually approaches the p-toluenesulfonyl group on the surface of the magnetic beads, and after reaching a suitable distance, the amino group carried by the antibody 1 and the p-toluenesulfonyl group undergo a covalent reaction, thereby connecting the antibody 1 to the surface of the magnetic beads.

[0130] Optionally, the blocking agent 1 is used for blocking, and one or more of the conditions (I) to (III) are met:

[0131] (I) the blocking agent 1 comprises a non-specific protein and a neutral buffer 4; optionally, the non-specific protein comprises one or more of casein, bovine serum albumin and human serum albumin; optionally, the neutral buffer 4 comprises a Tris-HCl buffer; optionally, the blocking agent 1 comprises 30 mM to 100 mM (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM) Tris-HCl and 1% (w / v) to 10% (w / v) (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) bovine serum albumin, pH 7 to 8 (e.g., 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8);

[0132] Optionally, the blocking agent 1 further includes a blocking enhancer; optionally, the blocking enhancer includes one or more of a surfactant, a streptavidin-biotin conjugate, a cyclodextrin and an amino acid, and optionally, the small molecule amino acid is not glycine; optionally, the surfactant includes one or more of Tween-20, Triton X-405 and Triton X-100; optionally, the amino acid includes one or more of lysine and arginine; optionally, the concentration of Tween-20 in the blocking agent 1 is 0.1% (v / v) to 0.5% (v / v) (for example, 0.1, 0.2, 0.3, 0.4, 0.5%); optionally, the concentration of the streptavidin-biotin conjugate in the blocking agent 1 is 0.02 g / L to 2 g / L (for example, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2 g / L); optionally, the concentration of the cyclodextrin in the blocking agent 1 is 0.1% (w / v) to 2% (w / v) (for example, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%); optionally, the concentration of the lysine in the blocking agent 1 is 1% (w / v) to 10% (w / v) (for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%);

[0133] In some embodiments, the blocking agent 1 includes 30mM to 100mM (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM) Tris-HCl and 1% (w / v) to 10% (w / v) (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) bovine serum albumin and 0.1% (v / v) to 0.5% (v / v) (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%) Tween-20, pH 7 to 8 (e.g., 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8);

[0134] In some embodiments, the blocking agent 1 includes 30mM to 100mM (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM) Tris-HCl and 1% (w / v) to 10% (w / v) (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%). ) bovine serum albumin and 0.02 g / L to 2 g / L (e.g., 0.02, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2 g / L) streptavidin-biotin conjugate, pH 7 to 8 (e.g., 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8);

[0135] In some embodiments, the blocking agent 1 includes 30mM to 100mM (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM) Tris-HCl and 1% (w / v) to 10% (w / v) (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) bovine serum albumin and 0.1% (w / v) to 2% (w / v) (e.g., 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%) cyclodextrin, pH 7 to 8 (e.g., 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8);

[0136] In some embodiments, the blocking agent 1 includes 30mM to 100mM (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM) Tris-HCl and 1% (w / v) to 10% (w / v) (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) bovine serum albumin and 1% (w / v) to 10% (w / v) (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) lysine, pH 7 to 8 (e.g., 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8);

[0137] (II) the amount of the blocking agent 1 per 5 mg of the solid phase carrier is 100 μL to 1000 μL (for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μL); and,

[0138] (III) constant temperature, the sealing temperature is 25°C to 40°C (for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C), the sealing time is 8h to 72h (for example, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72h), optionally 12h to 36h (for example, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36h);

[0139] Optionally, the first coupling reaction satisfies one or more of the conditions shown in I) to IV):

[0140] Ⅰ) under alkaline conditions without primary amino groups; optionally, in one or more of a sodium bicarbonate solution and a boric acid buffer; optionally, the pH value of the boric acid solution is 9.3 to 9.6 (e.g., 9.3, 9.4, 9.5, 9.6), and the concentration of boric acid is 0.08M to 0.12M (e.g., 0.08, 0.09, 0.1, 0.11, 0.12M);

[0141] II) using ammonium sulfate as a catalyst; optionally, the concentration of ammonium sulfate in the initial reaction system is 0.5M to 10M (for example, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10M), optionally 1M to 5M; in the process of coupling the antibody to toluenesulfonyl magnetic beads, ammonium sulfate can be used as a catalyst to improve the reaction efficiency;

[0142] III) constant temperature, the reaction temperature is 25°C to 40°C (for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C), the reaction time is 18h to 72h (for example, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72h), optionally 18h to 36h; and,

[0143] IV) The amount of the antibody 1 used per 5 mg of the solid phase carrier is 25 μg to 100 μg (for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg).

[0144] The reaction temperature of antibody-coupled magnetic beads can be selected according to the temperature tolerance of the antibody. Generally, the reaction speed at 37°C is faster, which can accelerate the coupling efficiency of the antibody on the surface of the magnetic beads. However, some antibodies are sensitive to temperature, and long-term reaction at 37°C may lead to reduced antibody activity. Therefore, a lower temperature can be selected for coupling, for example: 25°C. Extending the coupling time can also achieve a coupling effect equivalent to 37°C. The coupling process of p-toluenesulfonyl magnetic beads with antibodies is slow, with a reaction time of 18h to 72h, preferably 18h to 36h, and more preferably 24h. P-toluenesulfonyl magnetic beads are hydrophobic in themselves, and non-specific adsorption is easily produced during the reaction with the sample. Therefore, after the magnetic beads are coupled to the antibody, the surface of the magnetic beads can be further blocked to block the binding sites on the surface of the magnetic beads. The blocking solution generally selects non-specific proteins, surfactants, small molecule amino acids, etc.

[0145] In some embodiments, the steps of preparing the antibody 2-biotin conjugate include:

[0146] The antibody 2 and the biotin modified with the active group undergo a second coupling reaction, and the blocking agent 2 is added to the obtained second coupling reaction product for blocking to prepare the antibody 2-biotin conjugate;

[0147] Optionally, the second coupling reaction satisfies one or more of the conditions (i) to (iv):

[0148] (i) the modified active groups on the biotin include one or more of NHS ester group, amino group, maleimide group and hydrazide group;

[0149] (ii) under neutral or alkaline conditions without primary amino groups; optionally, in one or more of sodium bicarbonate solution, PBS buffer, HEPES buffer and MOPS buffer; optionally, in a buffer with a pH value of 7 to 9; optionally, in a PBS buffer with a pH value of 0.008M to 0.012M (e.g., 0.008, 0.009, 0.01, 0.011, 0.012M) and a pH value of 7.1 to 7.6 (e.g., 7.1, 7.2, 7.3, 7.4, 7.5, 76);

[0150] (iii) the molar concentration ratio of the antibody 2 to the active group-modified biotin is 1:(5-80) (e.g., 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80), optionally 1:(10-40);

[0151] (iv) the reaction temperature is 20° C. to 40° C. (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40° C.), and the reaction time is 10 min to overnight (e.g., 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 min);

[0152] Optionally, the sealing agent 2 is used for sealing, and one or more of the following conditions i) to iii) are met:

[0153] i) the blocking agent 2 comprises a small molecule with a primary amino group; optionally, the small molecule with a primary amino group comprises one or more of glycine, Tris and ethanolamine; optionally, the amount of the blocking agent 2 corresponding to the blocking system containing 500 μg to 2 mg (e.g., 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 μg) of the antibody 2 is 8 μL to 12 μL (e.g., 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 μL), and the concentration of glycine is 1.8M to 2.2M (e.g., 1.8, 1.9, 2.0, 2.1, 2.2M);

[0154] ii) the sealing temperature is 20°C to 40°C (for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C), and the sealing time is 5min to 15min (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15min);

[0155] iii) the blocked product is purified to prepare the antibody 2-biotin conjugate; optionally, the purification step is dialysis, desalting or ultrafiltration; optionally, the ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of 10 kDa to 50 kDa (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 kDa), optionally 10 kDa to 30 kDa.

[0156] Amino, carboxyl, sulfhydryl, sugar and other groups in the detection antibody structure can be used for covalent coupling with biotin modified with NHS ester, amino, maleimide or hydrazide. Preferably, the amino group in the antibody structure is used for covalent coupling with biotin modified with NHS ester.

[0157] In some embodiments, the steps of preparing the streptavidin-ruthenium label conjugate include:

[0158] Performing a third coupling reaction between streptavidin and the ruthenium marker modified with an active group, adding a blocking agent 3 to the obtained reaction product for blocking, and preparing the streptavidin-ruthenium marker conjugate;

[0159] Optionally, the third coupling reaction satisfies one or more of the following conditions (A) to (D):

[0160] (A) is carried out under neutral or alkaline conditions without primary amino groups; optionally, in one or more of sodium bicarbonate solution, PBS buffer, HEPES buffer and MOPS buffer; optionally, in a buffer with a pH value of 7 to 9 (e.g., 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, 9); optionally, in a PBS buffer with a pH value of 0.008M to 0.012M (e.g., 0.008, 0.009, 0.01, 0.011, 0.012M) and a pH value of 7.1 to 7.6 (e.g., 7.1, 7.2, 7.3, 7.4, 7.5, 7.6);

[0161] (B) the molar concentration ratio of the streptavidin to the active group-modified ruthenium label is 1:(5-80) (for example, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80), optionally 1:(10-40);

[0162] (C) the modified active group on the active group-modified ruthenium label comprises an NHS ester group;

[0163] (D) the reaction temperature is 20° C. to 40° C. (e.g., 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40° C.), and the reaction time is 10 min to overnight;

[0164] Optionally, the sealing agent 3 is used for sealing, and one or more of the following conditions are met:

[0165] A) the blocking agent 2 comprises a small molecule with a primary amino group; optionally, the small molecule with a primary amino group comprises one or more of glycine, Tris and ethanolamine; optionally, the amount of the blocking agent 3 corresponding to the blocking system containing 500 μg to 2 mg (e.g., 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 μg) of streptavidin is 18 μL to 22 μL (e.g., 18, 19, 20, 21, 22 μL), and the concentration of glycine is 1.8M to 2.2M (e.g., 1.8, 1.9, 2, 2.1, 2.2M);

[0166] B) the sealing temperature is 20°C to 40°C (e.g., 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40°C), and the sealing time is 5 min to 15 min (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min);

[0167] C) the blocked product is purified to prepare the antibody 2-biotin conjugate; optionally, the purification step is dialysis, desalting or ultrafiltration; optionally, the ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of 10 kDa to 50 kDa (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50 kDa), optionally 10 kDa to 30 kDa.

[0168] In a third aspect of the embodiments of the present application, a method for detecting a target analyte is provided, the method comprising the step of using the electrochemiluminescence immunoassay kit to detect the target analyte in a sample to be tested.

[0169] In some embodiments, the detection method comprises the following steps:

[0170] The sample to be tested, the antibody 1-solid phase carrier conjugate and the antibody 2-biotin conjugate are mixed and incubated in the first stage to prepare a first incubation product;

[0171] The first incubation product and the streptavidin-ruthenium label conjugate are mixed and incubated in the second stage to prepare a second incubation product; and,

[0172] The second incubation product and the co-reactant corresponding to the ruthenium label are mixed and luminescence detection is performed.

[0173] One streptavidin molecule can bind to 4 biotins, and each antibody molecule surface is coupled with multiple biotin molecules. Therefore, streptavidin and antibody biotin will cause cross-linking to form a macromolecular polymer, which will not only affect the stability of the reagent and cause the reagent to precipitate, but also its large steric hindrance will affect the antigen recognition ability of the antibody, making it difficult to achieve the effect of improving the detection sensitivity. In order to solve this problem, the present application first mixes and incubates the antibody-solid phase carrier conjugate, the sample, and the antibody-biotin conjugate to construct a specific double antibody sandwich complex, and then adds SA-Ru for incubation, and the molar ratio of the added SA-Ru to the biotinylated antibody needs to be strictly controlled, so as to avoid the large steric hindrance caused by excessive cross-linking between SA-Ru and the antibody-biotin complex, which affects the antigen recognition of the antibody, and also avoids the precipitation of the final immune complex due to excessive reaction.

[0174] In some embodiments, the concentration ratio of the antibody 2-biotin conjugate and the streptavidin-ruthenium label conjugate is 1:(1-10) (for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10), optionally 1:(2-4).

[0175] In some embodiments, the sample to be tested is blood, serum, plasma, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid, urine, amniotic fluid or aqueous humor.

[0176] 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.

[0177] 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.

[0178] In the following specific examples, the kit of the present application is referred to as Kit B (eg, Kit B1, Kit B2, etc.), and the kit used for comparison is referred to as Kit D (eg, Kit D1, Kit D2, etc.).

[0179] Example 1

[0180] The present invention provides an electrochemiluminescent immunoassay kit and a preparation method and application thereof, which involve the following contents:

[0181] 1. Preparation of Antibody 1-Magnetic Bead Conjugate

[0182] 1.1 After mixing the magnetic beads in the magnetic bead suspension evenly, take 5 mg of p-toluenesulfonyl magnetic beads, place them on a magnetic stand for 2 minutes, remove the original buffer, and wash them repeatedly with 500 μL of boric acid buffer (0.1 M, pH 9.5) for 3 times. After washing, place them on a magnetic stand for 2 minutes and remove the buffer.

[0183] 1.2 Add 300 μL of 3M ammonium sulfate solution (the final concentration of ammonium sulfate is 1.8 M) and 50 μg of antibody 1 to the washed p-toluenesulfonyl magnetic beads in sequence, make up the reaction volume to 500 μL with boric acid buffer (0.1 M, pH 9.5), vortex mix, and place in a 25°C constant temperature incubator on a mixer for coupling reaction (i.e., the first coupling reaction) for 24 hours.

[0184] 1.3 After the coupling reaction is completed, the antibody 1-magnetic bead conjugate is placed on a magnetic stand and allowed to stand for 2 minutes, then the buffer is removed. 500 μL of blocking agent 1 (50 mM Tris-HCl + 5% (w / v) bovine serum albumin (BSA) + 0.5% (v / v) Tween-20, 0.1 g / L SA-Bio / 0.5% (w / v) cyclodextrin / 5% (w / v) Lys / 5% (w / v) glycine, pH = 7.4) is added, vortexed and placed on a mixer in a 25°C constant temperature incubator for blocking overnight.

[0185] 1.4 Place the blocked antibody 1-magnetic bead conjugate on a magnetic stand for 2 minutes, remove the supernatant, add 500 μL preservation solution 1 (50 mM Tris-HCl, 150 mM NaCl, 50 g / L sucrose, 0.5% (v / v) Tween-20, 0.5% (w / v) BSA, 0.05% (v / v) PC300, pH 7.4), wash repeatedly 3 times, adjust the concentration of antibody 1-magnetic bead conjugate to 10 mg / mL with protection solution 1, and place in a refrigerator at 2-8°C and seal for storage.

[0186] 2. Preparation of Antibody 2-Biotin Conjugate

[0187] 2.1 Take 500 μg of antibody 2 solution and ultrafilter the storage buffer of the antibody into PBS buffer (pH 7.4, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0188] 2.2 The concentration of antibody 2 after ultrafiltration was measured by UV spectrophotometer, and antibody 2 was diluted to 2 g / L with PBS buffer (pH 7.4, 0.01 M).

[0189] 2.3 Add 30.27 μg of NHS ester-modified biotin to the solution of antibody 2 (the molar ratio of antibody 2 to NHS ester-modified biotin is 1:20), vortex mix, and incubate with rotation at room temperature for 60 min (second coupling reaction).

[0190] 2.4 After the incubation, add 10 μL of 2M glycine solution (i.e., blocking agent 2) to the reaction solution for blocking, and continue to incubate with rotation at room temperature for 10 min.

[0191] 2.5 After blocking, use a 30 kDa ultrafiltration tube to remove excess biotin, glycine and other small molecules in the reaction solution, and redissolve the resulting antibody 2-biotin conjugate in pH 7.4, 0.01 M PBS buffer-NaN3 (0.1%, w / v) (referred to as redissolution solvent 1). Ultrafiltration conditions: 10000 rpm, 8 min, and ultrafiltration for 3 consecutive times.

[0192] 2.6 The antibody 2-biotin conjugate obtained by coupling was quantified by UV spectrophotometer, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and the solution was sealed and stored in a refrigerator at 2°C to 8°C for future use. That is, the protective solution 2 in this step is pH 7.4, 0.01M PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0193] 3. Preparation of Streptavidin-Ruthenium Terpyridine Conjugate

[0194] 3.1 Take 1 mg of streptavidin solution and ultrafilter the streptavidin storage buffer into PBS buffer (pH 7.4, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0195] 3.2 The concentration of streptavidin after ultrafiltration was measured by UV spectrophotometer and diluted to 2 g / L with PBS buffer (pH 7.4, 0.01 M).

[0196] 3.3 Add 301 μg of NHS ester-modified terpyridine ruthenium (the molar concentration ratio of streptavidin to NHS ester-modified terpyridine ruthenium is 1:15) to the streptavidin solution, vortex mix, and incubate with rotation at room temperature for 60 min (third coupling reaction).

[0197] 3.4 After the incubation, add 20 μL of 2M glycine solution (blocking agent 3) to the reaction solution for blocking, and continue to incubate at room temperature with rotation for 10 min.

[0198] 3.5 After the blocking is completed, the excess terpyridine ruthenium, glycine and other small molecules in the reaction solution are removed by a 30 kDa ultrafiltration tube, and the obtained streptavidin-terpyridine ruthenium conjugate is redissolved in 0.01 M, pH 7.4 PBS buffer-NaN3 (0.1%, w / v) (i.e., redissolution solvent 1), and the ultrafiltration conditions are: 10000 rpm, 8 min, and continuous ultrafiltration is repeated 3 times.

[0199] 3.6 BCA protein quantification method The streptavidin-terpyridine ruthenium conjugate obtained by coupling was quantified, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and placed in a refrigerator at 2°C-8°C for sealed storage. That is, the protective solution 3 in this step is 0.01M, pH 7.4 PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0200] 4 Assembly of the kit

[0201] 4.1 Assembly of the kit of this application

[0202] The antibody 1-magnetic bead conjugate prepared under item 1, the antibody 2-biotin conjugate prepared under item 2, and the streptavidin-terpyridine ruthenium conjugate prepared under item 3 were respectively diluted with antibody 1-magnetic bead conjugate diluent 1, antibody 2-biotin conjugate diluent 2, and streptavidin-terpyridine ruthenium conjugate diluent 3, respectively, to prepare detection reagent 1, detection reagent 2, and detection reagent 3, respectively, wherein detection reagent 1 includes antibody 1-solid phase carrier conjugate and diluent 1, detection reagent 2 includes antibody 2-biotin conjugate and diluent 2, and detection reagent 3 includes streptavidin-ruthenium marker conjugate and diluent 3. Finally, the working solution tank is placed in the test kit and stored at 2°C to 8°C for future use. The optimal working solution concentration is adjusted depending on the project.

[0203] In the detection reagent 1, the formula of diluent 1 is 0.1M HEPES, 0.15M NaCl, 0.15M (51.345g / L) sucrose, 0.1% (w / v) Tween-20, 0.1% (w / w) BSA, 0.005% (v / v) PC300, the pH value is 7.4, and the concentration of antibody 1-magnetic bead conjugate is 0.75mg / mL.

[0204] In detection reagent 2, the formula of diluent 2 is 0.1M PBS, 0.15M (51.345g / L) sucrose, 0.05% (w / v) Tween-20, 0.05% (v / v) Triton X-100, 0.05% (v / v) PC300, 0.08M mouse IgG, pH value is 7.4, and the concentration of antibody 2-biotin coupling is 1.5μg / mL.

[0205] In detection reagent 3, the formula of diluent 3 is 0.1M PBS, 0.15M sucrose, 0.05% (w / v) Tween-20, 0.05% (v / v) Triton X-100, 0.05% (v / v) PC300, 0.08M mouse IgG, pH value is 7.4, and the concentration of streptavidin-terpyridine ruthenium conjugate is 2.2 times the concentration of antibody 2-biotin conjugate.

[0206] In the section "1. Preparation of Antibody 1-Magnetic Bead Conjugate", in order to achieve the best detection sensitivity, the magnetic bead blocker 1 is optimized to more completely block the immunomagnetic beads, reduce non-specific adsorption, achieve a better blocking effect, further reduce the detection background value, and further improve the detection sensitivity. The optimization of the magnetic bead blocker 1 is as follows: The new process indirectly connects the terpyridine ruthenium to the surface of the detection antibody molecule through the high affinity and high specificity of streptavidin and biotin, thereby improving the detection sensitivity.

[0207] The conventional blocking agent 1 component is a Tris-BSA system. Referring to the operation under item 1, the present application adds a surfactant, a streptavidin-biotin (SA-Bio) conjugate, a small molecule compound cyclodextrin and a small molecule amino acid to the closed system to further block the active sites on the surface of the magnetic beads, and tests the corresponding blocking effects and compares the signal-to-noise ratio. The SA-Bio complex is a complex obtained by blocking SA with a 30-fold excess of Biotin in advance and then removing excess Biotin by ultrafiltration. Specifically, the preparation method of the SA-Bio conjugate comprises the following steps:

[0208] (1) Take a streptavidin solution containing 1 mg of streptavidin, and ultrafilter the streptavidin storage buffer into PBS buffer (pH 7.4, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10,000 rpm, 8 min, and ultrafiltration is repeated twice.

[0209] (2) The concentration of streptavidin after ultrafiltration was measured by UV spectrophotometer and diluted to 2 g / L with PBS buffer (pH 7.4, 0.01 M).

[0210] (3) Add 204.3 μg of biotin to the streptavidin solution (the molar concentration ratio of streptavidin to biotin is 1:30, and the biotin is unmodified biotin), vortex mix, and incubate at room temperature for 60 min.

[0211] (4) After blocking, excess biotin in the reaction solution was removed using a 30 kDa ultrafiltration tube. The operation was repeated three times. The resulting SA-Bio conjugate was redissolved in 0.01 M, pH 7.4 PBS buffer-NaN3 (0.1%, w / v) (redissolution solvent 1). The ultrafiltration conditions were: 10,000 rpm, 8 min, and the ultrafiltration was repeated three times.

[0212] (5) The SA-Bio conjugate obtained by coupling was quantified by ultraviolet spectrophotometry, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and the solution was sealed and stored in a refrigerator at 2-8°C for later use. That is, the protective solution 3 in this step is 0.01M, pH 7.4 PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0213] The results are as follows (taking TSH project as an example):

[0214] Table 1. Detection effects of the kits assembled with various formulations of sealant 1

[0215]

[0216] Formulation 1 contained 50 mM Tris-HCl and 5% (w / v) bovine serum albumin (BSA), pH = 7.4;

[0217] Formulation 2 contained 50 mM Tris-HCl, 5% (w / v) bovine serum albumin (BSA), and 0.5% (v / v) Tween-20, pH = 7.4;

[0218] Formulation 3 contained 50 mM Tris-HCl, 5% (w / v) bovine serum albumin (BSA), 0.5% (v / v) Tween-20, and 0.1 g / L SA-Bio, pH = 7.4;

[0219] Formulation 4 contained 50 mM Tris-HCl, 5% (w / v) bovine serum albumin (BSA), 0.5% (v / v) Tween-20, and 0.5% (w / v) / cyclodextrin, pH = 7.4;

[0220] Formulation 5 contained 50 mM Tris-HCl, 5% (w / v) bovine serum albumin (BSA), 0.5% (v / v) Tween-20, and 5% (w / v) lysine, pH = 7.4;

[0221] Formulation 6 contained 50 mM Tris-HCl, 5% (w / v) bovine serum albumin (BSA), 0.5% (v / v) Tween-20 and 5% (w / v) glycine, pH=7.4.

[0222] The kits corresponding to Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, and Formula 6 are respectively recorded as Kit B1, Kit B2, Kit B3, Kit B4, Kit B5, and Kit B6.

[0223] Result analysis: From the above results, it can be seen that Tween-20, SA-Bio complex, cyclodextrin and lysine can all block the active sites on the surface of antibody 1-magnetic bead conjugate, among which lysine has a better blocking effect and the highest detection sensitivity. Compared with conventional blocking buffer, its sensitivity is increased by 2.37 to 2.72 times.

[0224] The method for detecting using the kit B of the present application comprises the following steps:

[0225] (1) Add antibody 1-magnetic bead conjugate, the sample to be tested, and antibody 2-biotin conjugate to the incubation cup, mix, and incubate for 9 minutes;

[0226] (2) adding a suitable ratio of streptavidin-ruthenium label conjugate to the reaction solution of step (1), mixing for 9 minutes, and then incubating and washing in sequence;

[0227] (3) Add the mixture of step (2) into the measuring cell, the reactants reach the electrode surface through magnetic attraction, add the co-reactant, and under the action of continuous voltage, the ruthenium marker and the co-reactant undergo redox reaction to generate a luminescent signal value.

[0228] Wherein, the concentration ratio of the antibody 2-biotin conjugate to the streptavidin-ruthenium marker conjugate is 1:2.2.

[0229] In the subsequent performance testing process, if there are special instructions, this method will be used for testing.

[0230] 4.2 Assembly of Comparative Kit D1

[0231] The traditional kit D1 is recorded as, including: SA-magnetic bead conjugate, biotin-antibody 1 (first antibody for capture) conjugate, terpyridine ruthenium-antibody 2 (second antibody for detection) conjugate. The reaction sequence is as follows:

[0232] (1) Add biotin-antibody 1 conjugate, the sample to be tested, and terpyridine ruthenium-antibody 2 conjugate to the incubation cup, mix, and incubate for 9 minutes;

[0233] (2) adding SA-magnetic bead conjugate to the reaction product of step (1), mixing, incubating for 9 min, and then washing;

[0234] (3) Add the mixture of step (2) into the measuring cell, the reactants reach the electrode surface through magnetic attraction, add the co-reactant, and under the action of continuous voltage, the ruthenium marker and the co-reactant undergo redox reaction to generate a luminescent signal value.

[0235] 5. Performance Testing

[0236] 5.1TSH (Thyroid Stimulating Hormone) Kit Performance Test

[0237] The assembled TSH kit B5 was used to test samples containing different concentrations of TSH on an electrochemiluminescence immunoassay, with the TSH kit D1 under item 4 as a control. Antibody 1 is specifically an anti-TSH capture mouse monoclonal antibody, and antibody 2 is specifically an anti-TSH detection mouse monoclonal antibody. The detection steps are described in item 4.

[0238] The signal value of the sample test with a concentration lower than 0.005 μIU / mL was used as the background value, and the ratio of the signal value to the background value of the sample test with other concentrations was used as the signal-to-noise ratio. The sample concentration lower than 0.005 μIU / mL was determined by other reference test methods. The results are shown in the following table. In addition, a TSH blank sample matrix was prepared, and the test was repeated 20 times a day for 3 consecutive days for the Limit of Blank (LoB) test. Five low-concentration samples (within the concentration range of LoB to 5 times LoB) were prepared with the blank sample matrix, and the test was repeated 3 times a day for 4 consecutive days for the Limit of Detection (LoD) test of TSH.

[0239] Table 2. Test results corresponding to TSH test kit

[0240]

[0241] Result analysis: From the results of the signal-to-noise ratio, the signal value and signal-to-noise ratio of the kit of this application are significantly higher than those of kit D1, and this application effectively improves the sensitivity of the detection kit. From the results of the blank limit and detection limit, the blank limit LoB of kit D1 is 0.023μIU / mL, and the detection limit LoD is 0.069μIU / mL; the LoB of the kit of this application is 0.002μIU / mL, and the LoD is 0.0015μIU / mL. Compared with the traditional kit, the sensitivity of this application is increased by 3.33 times after amplification by the streptavidin-biotin system, which can better meet the needs of clinical testing.

[0242] 5.2IL-6 kit performance test

[0243] The assembled IL-6 kit B5 was used to test samples containing different concentrations of IL-6 on an electrochemiluminescence immunoassay, with the kit D1 under item 4 as a control. In the IL-6 kit B5, antibody 1 is specifically an anti-IL-6 capture mouse monoclonal antibody, and antibody 2 is specifically an anti-IL-6 detection mouse monoclonal antibody, and the rest is the same as the TSH kit B5. The detection steps are described in item 4. Similarly, the difference between the IL-6 kit D1 and the TSH kit D1 is only the type of antibody 1 and antibody 2.

[0244] The signal value of the sample test with a concentration lower than 1.5pg / mL was used as the background value, and the ratio of the signal value to the background value of the sample test with other concentrations was used as the signal-to-noise ratio. The sample concentration lower than 1.5pg / mL was determined by other reference test methods. The results are shown in the following table. In addition, an IL-6 blank sample matrix was prepared, and the test was repeated 20 times a day for 3 consecutive days for the Limit of Blank (LoB) test. Five low-concentration samples (within the concentration range of LoB to 5 times LoB) were prepared with the blank sample matrix, and the test was repeated 3 times a day for 4 consecutive days for the Limit of Detection (LoD) test of IL-6.

[0245] Table 3. Detection results of IL-6 kit

[0246]

[0247] Result analysis: From the results of the signal-to-noise ratio, the signal value and signal-to-noise ratio of the kit of this application are significantly higher than those of kit D1, and this application effectively improves the sensitivity of the detection kit. From the results of the blank limit and detection limit, the blank limit LoB of kit D1 is 1.0pg / mL, and the detection limit LoD is 1.5pg / mL; the LoB of the kit of this application is 0.1pg / mL, and the LoD is 0.2pg / mL. Compared with the traditional kit, the sensitivity of this application is increased by 5 times after amplification by the streptavidin-biotin system, which can better meet the needs of clinical testing.

[0248] 5.3cTNT kit performance test

[0249] The assembled cTNT kit B5 was used to test samples containing different concentrations of cTNT on an electrochemiluminescence immunoassay, with the kit D1 under item 4 as a control. In the cTNT kit B5, antibody 1 is specifically a mouse monoclonal antibody for anti-cTNT capture, and antibody 2 is specifically a mouse monoclonal antibody for anti-cTNT detection, and the rest is the same as the TSH kit B5. The detection steps are described in item 4. Similarly, the difference between the cTNT kit D1 and the TSH kit D1 is only the type of antibody 1 and antibody 2.

[0250] The signal value of the sample with a concentration lower than 3pg / mL was used as the background value, and the ratio of the signal value to the background value of the sample with other concentrations was used as the signal-to-noise ratio. The sample with a concentration lower than 3pg / mL was determined by other reference test methods. The results are shown in the following table. In addition, a cTNT blank sample matrix was prepared, and the test was repeated 20 times a day for 3 consecutive days for the Limit of Blank (LoB) test. Five low-concentration samples (within the concentration range of LoB to 5 times LoB) were prepared with the blank sample matrix, and the test was repeated 3 times a day for 4 consecutive days for the Limit of Detection (LoD) test of cTNT.

[0251] Table 4. Detection results corresponding to cTNT detection kit

[0252]

[0253] Result analysis: From the results of the signal-to-noise ratio, the signal value and signal-to-noise ratio of the kit of this application are significantly higher than those of kit D1, and the new process effectively improves the sensitivity of the detection kit. From the results of the blank limit and detection limit, the blank limit LoB of kit D1 is 1.3pg / mL, and the detection limit LoD is 5pg / mL; the LoB of the kit of this application is 0.4pg / mL, and the LoD is 0.6pg / mL. Compared with the traditional kit, after amplification by the streptavidin-biotin system, the sensitivity is increased by 5 times, which can better meet the needs of clinical testing.

[0254] On the whole, the embodiment of the present application couples terpyridine ruthenium to the surface of streptavidin molecules instead of directly coupling it to the surface of detection antibody molecules, which can avoid the influence of terpyridine ruthenium label on the stability of antibody molecules and antigen recognition ability; the high affinity and high specificity of streptavidin and biotin are used to indirectly connect terpyridine ruthenium to the surface of detection antibody molecules. Compared with directly coupling terpyridine ruthenium to the surface of antibody molecules, the number of terpyridine ruthenium on the surface of a single antibody molecule is greater, resulting in a stronger electrochemiluminescence signal value, thereby improving the detection sensitivity.

[0255] Example 2

[0256] This example uses the TSH kit B5 in Example 1 to detect TSH, involving the following detection scheme:

[0257] Detection scheme 1, relative to the detection method in Example 1, is changed only in that the concentration ratio of the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate is 1:1.

[0258] Detection Scheme 2, relative to the detection method in Example 1, is changed only in that the concentration ratio of the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate is 1:2.

[0259] Detection scheme 3, relative to the detection method in Example 1, is changed only in that the concentration ratio of the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate is 1:4.

[0260] Detection scheme 4, relative to the detection method in Example 1, is changed only in that the concentration ratio of the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate is 1:8.

[0261] Detection scheme 5, relative to the detection method in Example 1, is changed only in that the concentration ratio of antibody 2-biotin conjugate to streptavidin-ruthenium marker conjugate is 1:2.2.

[0262] Detection Scheme 6, relative to the detection method in Example 1, is changed only in that the concentration ratio of the antibody 2-biotin conjugate to the streptavidin-ruthenium marker conjugate is 1:2.5.

[0263] Detection scheme 7, relative to the detection method in Example 1, is changed only in that the concentration ratio of the antibody 2-biotin conjugate and the streptavidin-ruthenium marker conjugate is 1:3.

[0264] Detection scheme 8, relative to the detection method in Example 1, is changed only in that the concentration ratio of antibody 2-biotin conjugate to streptavidin-ruthenium marker conjugate is 1:3.5.

[0265] Table 5. Optimization of concentrations of antibody 2-biotin conjugate and streptavidin-ruthenium label conjugate in kit B5 TSH assay

[0266]

[0267] Analysis of results: The optimization of the concentration ratio of antibody 2-biotin conjugate and streptavidin-ruthenium marker conjugate was evaluated by the signal-to-noise ratio. When the concentration ratio increased from 1:2 to 1:4, with the increase of the concentration of streptavidin-ruthenium marker conjugate, both the background signal value and the detection signal value increased. However, when the concentration ratio increased from 1:4 to 1:8, with the further increase of the concentration of streptavidin-ruthenium marker conjugate, its background signal value increased significantly, but the increase rate of its detection signal value was slow, indicating that the concentration of streptavidin-ruthenium marker conjugate had reached saturation at 4 times the concentration of antibody 2-biotin conjugate, and it was no longer appropriate to further increase the concentration of SA-Ru complex. The concentration ratio of antibody 2-biotin conjugate and streptavidin-ruthenium marker conjugate was further optimized between 1:2 and 1:4, and it was found that when the concentration ratio of antibody 2-biotin conjugate and streptavidin-ruthenium marker conjugate was 1:2.2, the signal-to-noise ratio was the highest. Therefore, the concentration ratio of antibody 2-biotin conjugate and streptavidin-ruthenium marker conjugate in this kit is 1:2.2.

[0268] Example 3

[0269] This example prepares TSH kit B with reference to the preparation method of TSH kit B5 in Example 1 and uses it for TSH detection, involving the following preparation scheme:

[0270] TSH kit B5-1, compared with the preparation method in Example 1, is changed only in that the concentration of the antibody in the antibody 1-magnetic bead conjugate coupling process is 25 μg of antibody 1 added to 5 mg of magnetic beads.

[0271] TSH kit B5-2, compared with the preparation method in Example 1, is changed only in that the concentration of the antibody in the antibody 1-magnetic bead conjugate coupling process is 37.5 μg of antibody 1 added to 5 mg of magnetic beads.

[0272] TSH kit B5-3, compared with the preparation method in Example 1, is changed only in that the concentration of the antibody in the antibody 1-magnetic bead conjugate coupling process is 100 μg of antibody 1 added to 5 mg of magnetic beads.

[0273] Table 6. Optimization of Antibody 1 Concentration in Antibody 1-Magnetic Bead Conjugate in Kit B5 TSH Detection

[0274]

[0275] Results analysis: Antibody 1 was coupled to the surface of magnetic beads to prepare the conjugate, and the amount of antibody 1 added to the magnetic beads was optimized. It was found that when the amount of antibody 1 was increased from 25μg to 50μg (the amount of magnetic beads remained unchanged), the detection signal value was significantly improved, and the background signal value did not increase significantly, and the signal-to-noise ratio was significantly improved. However, when the amount of antibody 1 was further increased from 50μg to 100μg, the background signal value and the detection signal value did not increase significantly, indicating that the amount of antibody 1 had reached saturation at 50μg and was not suitable for further increase. Therefore, the amount of antibody 1 added to 5mg magnetic beads was 50μg, and the detection effect was optimal.

[0276] Example 4

[0277] This example prepares a TSH kit by referring to the preparation method of the TSH kit B5 in Example 1 and uses it to detect TSH, involving the following preparation scheme:

[0278] TSH kit B5-A, compared with the preparation method in Example 1, is changed only in that the final concentration of ammonium sulfate during the coupling process of antibody 1-magnetic bead conjugate is 1.0M.

[0279] TSH kit B5-B, compared with the preparation method in Example 1, is changed only in that the final concentration of ammonium sulfate during the antibody 1-magnetic bead conjugate coupling process is 1.2M.

[0280] TSH kit B5-C, compared with the preparation method in Example 1, is changed only in that the final concentration of ammonium sulfate during the antibody 1-magnetic bead conjugate coupling process is 1.5M.

[0281] TSH kit B5-D, compared with the preparation method in Example 1, is changed only in that the final concentration of ammonium sulfate during the antibody 1-magnetic bead conjugate coupling process is 1.8M.

[0282] TSH kit B5-E, compared with the preparation method in Example 1, is changed only in that the final concentration of ammonium sulfate during the antibody 1-magnetic bead conjugate coupling process is 2.0M.

[0283] Table 7. Optimization of the final concentration of ammonium sulfate in the antibody 1-magnetic bead conjugate in the TSH test kit B5

[0284]

[0285] Results analysis: Antibody 1 was coupled to the surface of magnetic beads to prepare the conjugate, and the concentration of ammonium sulfate added to the coupling system was optimized. It was found that when the concentration of ammonium sulfate was increased from 1.0M to 1.8M, the detection signal value was significantly improved, and the signal-to-noise ratio was significantly improved. When the concentration of ammonium sulfate was lower than 1.5M, its detection signal value was very low, indicating that ammonium sulfate in the reaction system can indeed improve the reaction rate, so that more antibody 1 can reach the surface of magnetic beads through hydrophobic forces to achieve coupling. However, when the amount of ammonium sulfate was further increased from 1.8M to 2.0M, its detection signal value did not increase further, but showed a downward trend, indicating that excessive ammonium sulfate in the reaction system may lead to a decrease in the activity of antibody 1, resulting in a decrease in the detection signal value. Therefore, the optimized final concentration of ammonium sulfate in the antibody 1-magnetic bead conjugate coupling process is 1.8M.

[0286] Example 5

[0287] This example provides electrochemiluminescent immunoassay kits B7 to B14, which are different from the TSH kit B5 in Example 1 in that the blocking agent 1 shown in Formulas 7 to 14 is used under item 1, respectively.

[0288] Table 8

[0289]

[0290] Taking the TSH project as an example, the effects of TSH kits B7 to B14 were verified, and the results are as follows:

[0291] Table 9. TSH detection effect

[0292]

[0293] Example 6

[0294] This embodiment is a variation of embodiment 1, and the only difference compared with embodiment 1 is the preparation method of TSH kit B, and the contents involved are as follows:

[0295] 1. Preparation of Antibody 1-Magnetic Bead Conjugate

[0296] 1.1 After mixing the magnetic beads in the magnetic bead suspension evenly, take 5 mg of carboxyl magnetic beads, place them on a magnetic stand for 2 minutes, remove the original buffer, and wash them repeatedly with 500 μL of MES buffer (0.1 M, pH 5.0) for 3 times. After washing, place them on a magnetic stand for 2 minutes and remove the buffer.

[0297] 1.2 Add 100 μL of 10 g / L EDC aqueous solution to the washed carboxyl magnetic beads, make up the reaction volume to 500 μL with MES buffer (0.1 M, pH 5.0), vortex to mix, and incubate at room temperature for 30 min.

[0298] 1.3 After magnetic separation of the activated magnetic beads and removal of the supernatant, add 1 ml of MES buffer (0.1 M, pH 5.0) and wash repeatedly for 3 times. After washing, place on a magnetic stand for 2 minutes and then remove the buffer.

[0299] 1.4 Add 50 μg of antibody 1 and 100 μL of 10 g / L Sulfo-NHS to the washed magnetic beads, vortex to mix, and incubate with rotation at room temperature for 2 h.

[0300] 1.5 After the coupling reaction is completed, the antibody 1-magnetic bead conjugate is placed on a magnetic stand for 2 minutes and then the buffer is removed. 100 μL of blocking agent 1 (50 mM Tris-HCl + 5% (w / v) bovine serum albumin (BSA) + 0.5% (v / v) Tween-20, 5% (w / v) Lys, pH = 7.4) is added, vortexed and placed on a mixer in a 37°C constant temperature incubator for blocking for 3 hours.

[0301] 1.6 Place the blocked antibody 1-magnetic bead conjugate on a magnetic stand for 2 minutes, remove the supernatant, add 500 μL preservation solution 1 (50 mM Tris-HCl, 150 mM NaCl, 50 g / L sucrose, 0.5% (v / v) Tween-20, 0.5% (w / v) BSA, 0.05% (v / v) PC300, pH 7.4), wash repeatedly 3 times, adjust the concentration of antibody 1-magnetic bead conjugate to 10 mg / mL with protection solution 1, and place in a 2-8°C refrigerator for sealed storage.

[0302] Dilution solution 1 includes 80 mM HEPES buffer, 125 mM NaCl, 45 g / L sucrose, 0.4% (w / v) Tween-20, 0.1 wt% bovine serum albumin and 0.004% (v / v) PC300, with a pH value of 7-7.6.

[0303] The concentration of the antibody 1-magnetic bead conjugate in the detection reagent 1 is 0.3 mg / mL.

[0304] 2. Preparation of Antibody 2-Biotin Conjugate

[0305] 2.1 Take 500 μg of antibody 2 solution and ultrafilter the storage buffer of the antibody into PBS buffer (pH 7.4, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0306] 2.2 Add 5 mM TCEP to the antibody 2 solution, incubate at room temperature for 5 min, and then ultrafilter the antibody storage buffer into PBS buffer (pH 7.4, 0.01 M) using a 30 kDa ultrafiltration tube. Ultrafiltration conditions: 10,000 rpm, 8 min, and ultrafiltration twice continuously.

[0307] 2.3 Add 30.27 μg of maleimide-modified biotin to the solution of antibody 2 (the molar ratio of antibody 2 to maleimide-modified biotin is 1:5), vortex mix, and place in a 37° C. constant temperature incubator for rotation and incubation for 30 min (second coupling reaction).

[0308] 2.4 After the coupling is completed, the excess biotin and other small molecules in the reaction solution are removed by a 10 kDa ultrafiltration tube, and the obtained antibody 2-biotin conjugate is redissolved in pH 7.4, 0.01 M PBS buffer-NaN3 (0.1%, w / v) (referred to as redissolution solvent 1), and the ultrafiltration conditions are: 10000 rpm, 8 min, and continuous ultrafiltration is repeated 3 times.

[0309] 2.5 The antibody 2-biotin conjugate obtained by coupling was quantified by UV spectrophotometer, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and the solution was sealed and stored in a refrigerator at 2°C to 8°C for later use. That is, the protective solution 2 in this step is pH 7.4, 0.01M PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0310] The diluent 2 includes 120 mM PBS buffer, 55 g / L sucrose, 0.06% (w / v) Tween-20, 0.06% (v / v) Triton X-100, 0.06% (v / v) PC300 and 0.1 M mouse IgG, with a pH value of 7 to 7.6; the concentration of the antibody 2-biotin conjugate in the detection reagent 2 is 5 μg / mL.

[0311] 3. Preparation of Streptavidin-Ruthenium Terpyridine Conjugate

[0312] 3.1 Take 1 mg of streptavidin solution and ultrafilter the streptavidin storage buffer into PBS buffer (pH 7.4, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0313] 3.2 The concentration of streptavidin after ultrafiltration was measured by UV spectrophotometer and diluted to 2 g / L with PBS buffer (pH 7.4, 0.008 M).

[0314] 3.3 Add 301 μg of NHS ester-modified terpyridine ruthenium (the molar concentration ratio of streptavidin to NHS ester-modified terpyridine ruthenium is 1:5) to the streptavidin solution, vortex mix well, and place in a 37°C constant temperature incubator for rotation and incubation for 30 min (third coupling reaction).

[0315] 3.4 After the incubation, add 18 μL of 2M glycine solution (blocking agent 3) to the reaction solution for blocking, and continue to incubate at 20°C with rotation for 15 min.

[0316] 3.5 After the blocking is completed, the excess small molecules such as terpyridine ruthenium in the reaction solution are removed by a 30 kDa ultrafiltration tube, and the obtained streptavidin-terpyridine ruthenium conjugate is redissolved in 0.01 M, pH 7.4 PBS buffer-NaN3 (0.1%, w / v) (i.e., redissolution solvent 1), and the ultrafiltration conditions are: 10000 rpm, 8 min, and continuous ultrafiltration is repeated 3 times.

[0317] 3.6 BCA protein quantification method The streptavidin-terpyridine ruthenium conjugate obtained by coupling was quantified, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and placed in a refrigerator at 2°C-8°C for sealed storage. That is, the protective solution 3 in this step is 0.01M, pH 7.4 PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0318] Dilution solution 3 includes 80 mM PBS buffer and 45 g / L sucrose, 0.04% (w / v) Tween-20, 0.04% (v / v) Triton X-100, 0.04% (v / v) PC300 and 0.06 M mouse IgG, with a pH value of 7-7.6.

[0319] Example 7

[0320] This embodiment is a variation of embodiment 1, and the only difference from embodiment 1 is the preparation method of TSH kit B, involving the following contents:

[0321] 1. Preparation of Antibody 1-Magnetic Bead Conjugate

[0322] 1.1 Take 500 μg of antibody 1 solution and ultrafilter the storage buffer of the antibody into MES buffer (pH 5.0, 0.1 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0323] 1.2 Add 100 μL of 10 g / L EDC aqueous solution to the ultrafiltered antibody 1, make up the reaction volume to 500 μL with MES buffer (0.1 M, pH 5.0), vortex mix, and incubate at room temperature for 30 min.

[0324] 1.3 The activated antibody 1 was filtered through a 30 kDa ultrafiltration tube to remove excess activator. The ultrafiltration conditions were: 10,000 rpm, 8 min, and repeated ultrafiltration twice.

[0325] 1.4 After mixing the magnetic beads in the magnetic bead suspension evenly, take 5 mg of amino magnetic beads, place them on a magnetic stand and let them stand for 2 minutes, then remove the original buffer solution, and wash them repeatedly with 500 μL of MES buffer solution (0.1 M, pH 5.0) for 3 times. After washing, place them on a magnetic stand and let them stand for 2 minutes before removing the buffer solution.

[0326] 1.5 Add activated antibody 1 and 100 μL 10 g / L Sulfo-NHS to the washed magnetic beads, vortex to mix, and incubate with rotation at room temperature for 2 h.

[0327] 1.6 After the coupling reaction is completed, the antibody 1-magnetic bead conjugate is placed on a magnetic stand for 2 minutes and then the buffer is removed. 100 μL of blocking agent 1 (50 mM Tris-HCl + 5% (w / v) bovine serum albumin (BSA) + 0.5% (v / v) Tween-20, 5% (w / v) Lys, pH = 7.4) is added, vortexed and placed on a mixer in a 37°C constant temperature incubator for blocking for 3 hours.

[0328] 1.7 Place the blocked antibody 1-magnetic bead conjugate on a magnetic stand for 2 minutes, remove the supernatant, add 500 μL preservation solution 1 (50 mM Tris-HCl, 150 mM NaCl, 50 g / L sucrose, 0.5% (v / v) Tween-20, 0.5% (w / v) BSA, 0.05% (v / v) PC300, pH 7.4), wash repeatedly 3 times, adjust the concentration of antibody 1-magnetic bead conjugate to 10 mg / mL with protection solution 1, and place in a refrigerator at 2-8°C and seal for storage.

[0329] Diluent 1 includes 120 mM HEPES buffer and 175 mM NaCl, 55 g / L sucrose, 0.6% (w / v) Tween-20, 0.6 wt % bovine serum albumin, and 0.006% (v / v) PC300, with a pH value of 7 to 7.6.

[0330] The concentration of the antibody 1-magnetic bead conjugate in the detection reagent 1 is 2 mg / mL.

[0331] 2. Preparation of Antibody 2-Biotin Conjugate

[0332] 2.1 Take 500 μg of antibody 2 solution and ultrafilter the storage buffer of the antibody into NaAc-HAC buffer (pH 5.5, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0333] 2.2 The concentration of antibody 2 after ultrafiltration was measured by UV spectrophotometer, and antibody 2 was diluted to 2 g / L with NaAc-HAC buffer (pH 5.5, 0.01 M).

[0334] 2.3 Add 20 mM NaIO4 (NaAc-HAC dilution, pH 5.5, 0.01 M) to the antibody 2 solution, vortex mix, and incubate at 4°C for 30 min. Immediately add 2 M ethylene glycol and continue incubation for 10 min.

[0335] 2.4 Add 30.27 μg of hydrazide-modified biotin to the solution of antibody 2 (the molar ratio of antibody 2 to hydrazide-modified biotin is 1:20), vortex mix, and incubate at 40° C. for 4 h (second coupling reaction).

[0336] 2.5 After the incubation, add 10 μL of 2M glycine solution (i.e., blocking agent 2) to the reaction solution for blocking, and continue to incubate at 40°C with rotation for 15 min.

[0337] 2.6 After blocking, use a 50 kDa ultrafiltration tube to remove excess biotin and other small molecules in the reaction solution, and redissolve the resulting antibody 2-biotin conjugate in pH 7.4, 0.01 M PBS buffer-NaN3 (0.1%, w / v) (referred to as redissolution solvent 1). Ultrafiltration conditions: 10000 rpm, 8 min, and ultrafiltration for 3 consecutive times.

[0338] 2.7 The antibody 2-biotin conjugate obtained by coupling was quantified by UV spectrophotometer, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and the solution was sealed and stored in a refrigerator at 2°C to 8°C for later use. That is, the protective solution 2 in this step is pH 7.4, 0.01M PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0339] Diluent 2 includes 80 mM PBS buffer and 45 g / L sucrose, 0.04% (w / v) Tween-20, 0.04% (v / v) Triton X-100, 0.04% (v / v) PC300 and 0.06 M mouse IgG, with a pH of 7 to 7.6;

[0340] The concentration of the antibody 2-biotin conjugate in the detection reagent 2 is 0.5 μg / mL.

[0341] 3. Preparation of Streptavidin-Ruthenium Terpyridine Conjugate

[0342] 3.1 Take 1 mg of streptavidin solution and ultrafilter the streptavidin storage buffer into PBS buffer (pH 7.4, 0.01 M) through a 30 kDa ultrafiltration tube. The ultrafiltration conditions are: 10000 rpm, 8 min, and ultrafiltration is repeated twice.

[0343] 3.2 The concentration of streptavidin after ultrafiltration was measured by UV spectrophotometer and diluted to 2 g / L with PBS buffer (pH 7.4, 0.01 M).

[0344] 3.3 Add 301 μg of NHS ester-modified terpyridine ruthenium (the molar concentration ratio of streptavidin to NHS ester-modified terpyridine ruthenium is 1:15) to the streptavidin solution, vortex mix, and incubate with rotation at room temperature for 60 min (third coupling reaction).

[0345] 3.4 After the incubation, add 20 μL of 2M glycine solution (blocking agent 3) to the reaction solution for blocking, and continue to incubate at room temperature with rotation for 10 min.

[0346] 3.5 After the blocking is completed, the excess terpyridine ruthenium, glycine and other small molecules in the reaction solution are removed by a 30 kDa ultrafiltration tube, and the obtained streptavidin-terpyridine ruthenium conjugate is redissolved in 0.01 M, pH 7.4 PBS buffer-NaN3 (0.1%, w / v) (i.e., redissolution solvent 1), and the ultrafiltration conditions are: 10000 rpm, 8 min, and continuous ultrafiltration is repeated 3 times.

[0347] 3.6 BCA protein quantification method The streptavidin-terpyridine ruthenium conjugate obtained by coupling was quantified, and BSA was added to the solution after quantification to make the final concentration of BSA 1% (w / v), and placed in a refrigerator at 2°C-8°C for sealed storage. That is, the protective solution 3 in this step is 0.01M, pH 7.4 PBS buffer-BSA (1%, w / v)-NaN3 (0.1%, w / v) (i.e., re-dissolution solvent 2).

[0348] Dilution solution 3 includes 120 mM PBS buffer and 55 g / L sucrose, 0.06% (w / v) Tween-20, 0.06% (v / v) Triton X-100, 0.06% (v / v) PC300 and 0.1 M mouse IgG, with a pH value of 7-7.6.

[0349] Referring to the detection method of TSH kit B5, the corresponding results shown in the following table were obtained.

[0350] Table 10. TSH detection effect

[0351]

[0352] As can be seen from Table 10, the detection kits provided in Examples 6 and 7 can detect thyroid stimulating hormone with high sensitivity.

[0353] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0354] 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. An electrochemiluminescence immunoassay kit, characterized in that: The detection kit comprises: Antibody 1-solid phase carrier conjugate; Antibody 2-biotin conjugate; and, Streptavidin-ruthenium label conjugate; The antibody 1 in the antibody 1-solid phase carrier conjugate and the antibody 2 in the antibody 2-biotin conjugate can both specifically bind to the target analyte and have different antigen recognition epitopes.

2. The electrochemiluminescence immunoassay kit according to claim 1, characterized in that: The detection kit satisfies one or more of the following conditions (1) and (2): (1) The solid phase carrier in the antibody 1-solid phase carrier conjugate comprises magnetic beads; optionally, the functional groups modified on the surface of the magnetic beads comprise one or more of carboxyl, amino, hydroxyl, epoxy and p-toluenesulfonyl groups; and, (2) 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; Optionally, the diluent 1 comprises NaCl, a protein protective agent 1, a preservative 1, a surfactant 1 and a neutral buffer 1; Optionally, the protein protective agent 1 includes a carbohydrate protective agent 1 and a protein protective agent 1; optionally, the carbohydrate protective agent 1 includes one or more of sucrose, trehalose and dextran; optionally, the protein protective agent 1 includes bovine serum albumin; Optionally, the preservative 1 includes PC300; Optionally, the surfactant 1 includes Tween-20; Optionally, the neutral buffer 1 includes one or more of HEPES buffer, PBS buffer and Tris buffer; Optionally, the diluent 1 comprises 80mM to 120mM HEPES buffer and 125mM to 175mM NaCl, 45g / L to 55g / L sucrose, 0.1% (w / v) to 0.6% (w / v) Tween-20, 0.1wt% to 0.6wt% bovine serum albumin, and 0.004% (v / v) to 0.006% (v / v) PC300, with a pH value of 7 to 7.6; Optionally, the concentration of the antibody 1-solid phase carrier conjugate in the detection reagent 1 is 0.3 mg / mL to 2 mg / mL.

3. The electrochemiluminescence immunoassay kit according to claim 1, characterized in that: The detection kit comprises detection reagent 2, and the detection reagent 2 comprises the antibody 2-biotin conjugate and diluent 2; Optionally, the diluent 2 comprises a protein protectant 2, a preservative 2, a surfactant 2, a blocking agent 1 and a neutral buffer 2; Optionally, the protein protective agent 2 includes a carbohydrate protective agent 2; Optionally, the carbohydrate protective agent 2 includes sucrose; Optionally, the preservative 2 includes PC300; Optionally, the surfactant 2 includes one or more of Tween-20 and Triton X-100; Optionally, the blocking agent 1 comprises mouse IgG; Optionally, the neutral buffer 2 includes one or more of PBS buffer and HEPES buffer; Optionally, the diluent 2 comprises 80 mM to 120 mM PBS buffer and 45 g / L to 55 g / L sucrose, 0.04% (w / v) to 0.06% (w / v) Tween-20, 0.04% (v / v) to 0.06% (v / v) Triton X-100, 0.04% (v / v) to 0.06% (v / v) PC300 and 0.06M to 0.1M mouse IgG, with a pH value of 7 to 7.6; Optionally, the concentration of the antibody 2-biotin conjugate in the detection reagent 2 is 0.5 μg / mL to 5 μg / mL.

4. The electrochemiluminescence immunoassay kit according to claim 1, characterized in that: The detection kit comprises a detection reagent 3, wherein the detection reagent 3 comprises the streptavidin-ruthenium marker conjugate and a diluent 3; Optionally, the diluent 3 comprises a protein protective agent 3, a preservative 3, a surfactant 3, a blocking agent 2 and a neutral buffer 3; Optionally, the protein protective agent 3 includes a carbohydrate protective agent 3; Optionally, the carbohydrate protective agent 3 includes sucrose; Optionally, the preservative 3 includes PC300; Optionally, the surfactant 3 includes one or more of Tween-20 and Triton X-100; Optionally, the blocking agent 2 comprises mouse IgG; Optionally, the neutral buffer 3 includes one or more of PBS buffer and HEPES buffer; Optionally, the diluent 3 comprises 80 mM to 120 mM PBS buffer and 45 g / L to 55 g / L sucrose, 0.04% (w / v) to 0.06% (w / v) Tween-20, 0.04% (v / v) to 0.06% (v / v) Triton X-100, 0.04% (v / v) to 0.06% (v / v) PC300 and 0.06M to 0.1M mouse IgG, with a pH value of 7 to 7.6; Optionally, the amount of the streptavidin-ruthenium marker conjugate in the detection reagent 3 is 1 to 10 times that of the antibody 2-biotin conjugate.

5. The electrochemiluminescence immunoassay kit according to any one of claims 1 to 4, characterized in that: The antibody 1 in the antibody 1-solid phase carrier conjugate and / or the antibody 2 in the antibody 2-biotin conjugate are monoclonal antibodies.

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

7. The method for preparing the electrochemiluminescent immunoassay kit according to claim 6, characterized in that: The steps for preparing the antibody 1-solid phase carrier conjugate include: The antibody 1 and the solid phase carrier undergo a first coupling reaction, and a blocking agent 1 is added to the obtained first coupling reaction product for blocking to prepare the antibody 1-solid phase carrier conjugate; Optionally, the blocking agent 1 is used for blocking, and one or more of the conditions (I) to (III) are met: (I) the blocking agent 1 comprises a non-specific protein and a neutral buffer 4; optionally, the non-specific protein comprises one or more of casein, bovine serum albumin and human serum albumin; optionally, the neutral buffer 4 comprises a Tris-HCl buffer; optionally, the blocking agent 1 comprises 30 mM to 100 mM Tris-HCl and 1% (w / v) to 10% (w / v) bovine serum albumin, with a pH value of 7 to 8; Optionally, the blocking agent 1 further comprises a blocking enhancer; optionally, the blocking enhancer comprises one or more of a surfactant, a streptavidin-biotin conjugate, a cyclodextrin and an amino acid; optionally, the amino acid is not glycine; optionally, the surfactant comprises one or more of Tween-20, Triton X-405 and Triton X-100; optionally, the amino acid comprises one or more of lysine and arginine; optionally, the concentration of Tween-20 in the blocking agent 1 is 0.1% (v / v) to 0.5% (v / v); optionally, the concentration of the streptavidin-biotin conjugate in the blocking agent 1 is 0.02 g / L to 2 g / L; optionally, the concentration of the cyclodextrin in the blocking agent 1 is 0.1% (w / v) to 2% (w / v); optionally, the concentration of the lysine in the blocking agent 1 is 1% (w / v) to 10% (w / v); (II) the amount of the blocking agent 1 corresponding to each 5 mg of the solid phase carrier is 100 μL to 1000 μL; and, (III) Constant temperature, the sealing temperature is 25°C to 40°C, the sealing time is 8h to 72h, and optionally 12h to 36h; Optionally, the first coupling reaction satisfies one or more of the conditions shown in I) to IV): Ⅰ) under alkaline conditions without primary amino groups; optionally, in one or more of a sodium bicarbonate solution and a boric acid buffer; optionally, the pH value of the boric acid solution is 9.3 to 9.6, and the concentration of boric acid is 0.08 M to 0.12 M; II) using ammonium sulfate as a catalyst; optionally, the concentration of the ammonium sulfate in the initial reaction system is 0.5M to 10M, optionally 1M to 5M; III) constant temperature, the reaction temperature is 25°C to 40°C, the reaction time is 18h to 72h, optionally 18h to 36h; and, IV) The amount of the antibody 1 used per 5 mg of the solid phase carrier is 25 μg to 100 μg.

8. The method for preparing the electrochemiluminescence immunoassay kit according to any one of claims 6 to 7, characterized in that: The steps of preparing the antibody 2-biotin conjugate include: The antibody 2 and the biotin modified with the active group undergo a second coupling reaction, and the blocking agent 2 is added to the obtained second coupling reaction product for blocking to prepare the antibody 2-biotin conjugate; Optionally, the second coupling reaction satisfies one or more of the conditions (i) to (iv): (i) the modified active groups on the biotin include one or more of NHS ester group, amino group, maleimide group and hydrazide group; (ii) under neutral or alkaline conditions without primary amino groups; optionally, in one or more of sodium bicarbonate solution, PBS buffer, HEPES buffer and MOPS buffer; optionally, in a buffer with a pH value of 7 to 9; optionally, in a PBS buffer with a pH value of 7.1 to 7.6 and a concentration of 0.008 M to 0.012 M; (iii) the molar concentration ratio of the antibody 2 to the active group-modified biotin is 1:(5-80), optionally 1:(10-40); and, (iv) the reaction temperature is 20°C to 40°C, and the reaction time is 10 minutes to overnight; Optionally, the sealing agent 2 is used for sealing, and one or more of the following conditions i) to iii) are met: i) the blocking agent 2 comprises a small molecule with a primary amino group; optionally, the small molecule with a primary amino group comprises one or more of glycine, Tris and ethanolamine; optionally, the amount of the blocking agent 2 corresponding to the blocking system containing 500 μg to 2 mg of the antibody 2 is 8 μL to 12 μL, and the concentration of glycine is 1.8M to 2.2M; ii) the sealing temperature is 20°C to 40°C, and the sealing time is 5min to 15min; and, iii) the blocked product is purified to prepare the antibody 2-biotin conjugate; optionally, the purification step is dialysis, desalting or ultrafiltration; optionally, the ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of 10kDa to 50kDa, optionally 10kDa to 30kDa.

9. The method for preparing the electrochemiluminescent immunoassay kit according to any one of claims 6 to 7, characterized in that: The steps of preparing the streptavidin-ruthenium label conjugate include: Performing a third coupling reaction between streptavidin and the ruthenium marker modified with an active group, adding a blocking agent 3 to the obtained reaction product for blocking, and preparing the streptavidin-ruthenium marker conjugate; Optionally, the third coupling reaction satisfies one or more of the following conditions (A) to (D): (A) under neutral or alkaline conditions without primary amino groups; optionally, in one or more of sodium bicarbonate solution, PBS buffer, HEPES buffer and MOPS buffer; optionally, in a buffer with a pH value of 7 to 9; optionally, in a PBS buffer with a pH value of 7.1 to 7.6 and a concentration of 0.008 M to 0.012 M; (B) the molar concentration ratio of the streptavidin to the active group-modified ruthenium label is 1:(5-80), optionally 1:(10-40); (C) the modified active group on the active group-modified ruthenium label comprises an NHS ester group; and (D) The reaction temperature is 20°C to 40°C, and the reaction time is 10 minutes to overnight; Optionally, the sealing agent 3 is used for sealing, and one or more of the following conditions A) to C) are met: A) the blocking agent 3 comprises a small molecule with a primary amino group; optionally, the small molecule with a primary amino group comprises one or more of glycine, Tris and ethanolamine; optionally, the amount of the blocking agent 3 corresponding to the blocking system containing 500 μg to 2 mg of the streptavidin is 18 μL to 22 μL, and the concentration of glycine is 1.8M to 2.2M; B) the sealing temperature is 20°C to 40°C, and the sealing time is 5min to 15min; and, C) The blocked product is purified to prepare the antibody 2-biotin conjugate; optionally, the purification step is dialysis, desalting or ultrafiltration; optionally, the ultrafiltration tube used in ultrafiltration has a molecular weight cutoff of 10kDa to 50kDa, optionally 10kDa to 30kDa.

10. A method for detecting a target analyte, characterized in that: The detection method comprises the steps of using the electrochemiluminescence immunoassay kit according to any one of claims 1 to 5 to detect the target analyte in the sample to be detected; Optionally, the detection method comprises the following steps: The sample to be tested, the antibody 1-solid phase carrier conjugate and the antibody 2-biotin conjugate are mixed, and a first stage of incubation is performed to prepare a first incubation product; The first incubation product and the streptavidin-ruthenium label conjugate are mixed and incubated in the second stage to prepare a second incubation product; as well as, Mixing the second incubation product with the co-reactant of the ruthenium label in the streptavidin-ruthenium label conjugate, and performing luminescence detection; Optionally, the concentration ratio of the antibody 2-biotin conjugate to the streptavidin-ruthenium marker conjugate is 1:(1-10), optionally 1:(2-4); Optionally, the sample to be tested is blood, serum, plasma, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid, urine, amniotic fluid or aqueous humor.

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