25-hydroxyvitamin D detection kit and application thereof

Abstract

The invention relates to a 25-hydroxyvitamin D detection kit and application thereof. The detection kit comprises a working reagent 1 and a working reagent 2, the working reagent 1 comprises a reagent R1 and a reagent R2, or comprises a reagent R3; the reagent R1 comprises a binding molecule 1-solid phase conjugate, the reagent R2 comprises an antibody marker 1, the antibody marker 1 comprises a binding molecule 2 and an antibody 1 specifically binding 25-hydroxyvitamin D, and the binding molecule 1 and the binding molecule 2 form a molecule binding pair; the R3 reagent comprises an antibody-solid phase conjugate, and an antibody in the antibody-solid phase conjugate is an antibody 1 specifically combined with 25-hydroxyvitamin D; the working reagent 2 comprises an antibody marker 2, and the antibody marker 2 comprises a tracing marker and an antibody 2 specifically combined with 25-hydroxyvitamin D. The 25-hydroxyvitamin D detection kit is high in sensitivity, large in linear range, good in repeatability, good in correlation with mass spectrum results and short in consumed time when being used for detecting 25-hydroxyvitamin D.

Description

Technical Field

[0001] The present application relates to the field of in vitro diagnostic technology, and in particular to a 25-hydroxyvitamin D detection kit and its application. Background Art

[0002] Vitamin D is a fat-soluble vitamin that is essential to the human body and is mainly produced by the skin after exposure to sunlight. It can promote intestinal absorption of calcium and regulate calcium balance. Vitamin D itself is inactive and can be converted into biologically active 1,25-dihydroxyvitamin D through a two-step hydroxylation process in the liver and kidneys. The two most important forms of vitamin D are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Unlike vitamin D3, the human body cannot synthesize vitamin D2 and can only obtain it from food or food supplements.

[0003] Vitamin D is a major element for forming bones and keeping them strong and healthy. Severe vitamin D deficiency in childhood will lead to bone deformity, namely rickets, while mild vitamin D deficiency will lead to decreased utilization efficiency of calcium in food. Vitamin D deficiency can also lead to muscle weakness, which can increase the risk of falling in middle-aged and elderly people. Vitamin D deficiency is also a common cause of secondary hyperparathyroidism. It has been reported that vitamin D has been found to affect the expression of more than 200 genes. Vitamin D deficiency is associated with diabetes, different types of cancer, cardiovascular disease, autoimmune diseases, respiratory diseases and other diseases.

[0004] It is generally believed that 25-hydroxyvitamin D is the metabolite that determines the overall vitamin D status because it is the main storage form of vitamin D in the body. Low 25-hydroxyvitamin D levels are also associated with low bone mineral density. Combined with other clinical data, the results can be used as an auxiliary means to determine bone metabolism.

[0005] Currently, the methods for detecting 25-hydroxyvitamin D mainly include:

[0006] 1. Competitive protein binding assay (CPBA): 3 H-labeled 25-hydroxyvitamin D3 competes with 25-hydroxyvitamin D in the sample for binding to vitamin D binding protein (DBP). 3 The concentration of 25-hydroxyvitamin D was calculated from the H signal intensity.

[0007] 2. Immunoassay (manual): mainly includes radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELIAS). Principle of radioimmunoassay (RIA): Compared with CPBA, RIA adopts 125 I-25-Hydroxyvitamin D3 Replacement 3H-25-hydroxyvitamin D3 is used as a tracer. This method does not require complicated sample pretreatment. Principle of enzyme-linked immunosorbent assay (ELIAS): Biotin-labeled 25-hydroxyvitamin D and 25-hydroxyvitamin D in the sample are used to competitively bind to 25-hydroxyvitamin D antibodies, and then biotin-binding protein labeled with horseradish peroxidase is used to recognize biotin. The concentration of 25-hydroxyvitamin D is calculated through the display reaction of horseradish peroxidase.

[0008] 3. LC-MS / MS principle: The samples are separated by liquid chromatography and detected by tandem mass spectrometer to achieve double mass selection of molecular ions and fragment ions, and 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 can be detected simultaneously.

[0009] 4. Chemiluminescent immunoassay (automatic): mainly includes chemiluminescent immunoassay (CLIA) and electrochemiluminescent immunoassay (eCLIA). Principle of chemiluminescent immunoassay (CLIA): a type of immunoassay that directly labels antibodies with luminescent agents; Principle of electrochemiluminescent immunoassay (eCLIA): a specific chemiluminescent reaction is induced by electrochemistry on the electrode surface, including two processes: electrochemistry and chemiluminescence.

[0010] Among the above methods, chemiluminescent immunoassay is a relatively advanced labeling immunoassay method. In traditional immunoassay, the detection of 25-hydroxyvitamin D usually adopts the competitive method. Generally, after the 25-hydroxyvitamin D sample is pretreated, the solid phase carrier coated with 25-hydroxyvitamin D monoclonal antibody specifically binds to the 25-hydroxyvitamin D in the sample, and the chemiluminescent marker labeled with vitamin D derivative is further added for competitive binding, and the concentration is obtained after calibration by the standard curve. In the competitive reaction of 25-hydroxyvitamin, the amount of labeled 25-hydroxyvitamin D hapten and antibody is limited. The signal intensity is read after the limited antibody, antigen and hapten bind and dissociate to reach equilibrium, which limits the linear range of the competitive method; and the antibody affinity constant of 25-hydroxyvitamin D small molecule compound is usually low, and the sensitivity is very limited by the affinity of the antibody, resulting in the low analytical sensitivity of the existing competitive method for detecting 25-hydroxyvitamin D, poor zero value distinction, and long incubation time of the competitive method reaction, and poor reproducibility at low concentrations. Therefore, based on the limitations of small molecule competition detection, there is still a need for a reliable method to detect 25-hydroxyvitamin D that is fast, highly sensitive, specific, reproducible, and has a wide linear range to meet clinical requirements for detection precision and accuracy.

[0011] In view of this, this application is hereby filed. Summary of the invention

[0012] One or more embodiments of the present application provide a 25-hydroxyvitamin D detection kit and its application, including the following technical solutions:

[0013] One or more embodiments of the present application provide a 25-hydroxyvitamin D detection kit, the detection kit comprising a working reagent 1 and a working reagent 2; wherein,

[0014] The working reagent 1 includes R1 reagent and R2 reagent, wherein the R1 reagent includes a binding molecule 1-solid phase conjugate, and the R2 reagent includes an antibody marker 1, wherein the antibody marker 1 includes a binding molecule 2 and an antibody 1 that specifically binds to 25-hydroxyvitamin D, and the binding molecule 1 and the binding molecule 2 constitute a molecular binding pair;

[0015] Alternatively, the working reagent 1 includes an R3 reagent, the R3 reagent includes an antibody-solid phase conjugate, and the antibody in the antibody-solid phase conjugate is an antibody 1 that specifically binds to 25-hydroxyvitamin D;

[0016] The working reagent 2 includes an antibody marker 2, and the antibody marker 2 includes a tracer marker and an antibody 2 that specifically binds to 25-hydroxyvitamin D.

[0017] In some embodiments of the present application, the solvent of the working reagent 2 includes a buffer and 0.01wt%-5wt% of a protective agent, 0.02wt%-1wt% of a surfactant, 0.05wt%-0.5wt% of a blocking agent, and 0.01wt%-5wt% of a preservative; wherein the buffer includes one or more of a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer, and a TES buffer;

[0018] Optionally, in the solvent of the working reagent 2, the blocking agent includes anti-biotin antibody, MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, sheep IgG, goat IgG, HBR-5, HBR-7, TRU Block TM , HBR-1, HBR-X and P-heterophilic antibodies, and further optionally, the blocking agent includes anti-biotin antibody, HBR-5, HBR-7, TRU Block TM , at least one of HBR-1, HBR-X and MAK33 Poly, and one or more of P-heterophilic antibody, rabbit IgG, mouse IgG, bovine IgG, sheep IgG and goat IgG;

[0019] Optionally, in the solvent of the working reagent 2, the protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, sodium chloride, sucrose, trehalose, lactose and mannitol;

[0020] Optionally, in the solvent of the working reagent 2, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, Pluronic F-68, Pluronic F-127, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan;

[0021] Optionally, in the solvent of the working reagent 2, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives and ProClin series preservatives;

[0022] Optionally, the concentration of the antibody marker 2 in the working reagent 2 is 0.5 μg / mL-2.0 μg / mL.

[0023] In some embodiments of the present application, the detection kit further comprises a sample dissociation solution;

[0024] The sample dissociation solution includes a buffer solution and 0.02wt%-1wt% of a surfactant, 1wt%-20wt% of a dissociation agent, and 0.01wt%-5wt% of a preservative; wherein the buffer solution includes one or more of a HEPES buffer solution having a pH value of 3.0-7.5 and a concentration of 0.01mol / L-0.2mol / L, a citric acid buffer solution, an acetate buffer solution, a phosphate buffer solution, a Bis-Tris buffer solution, a MOPSO buffer solution, a PIPES buffer solution, and a TES buffer solution;

[0025] Optionally, in the sample dissociation solution, the dissociation agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, mesna, tris(2-carbonylethyl)phosphine hydrochloride, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, perfluorobutyric acid, perfluorononanoic acid and perfluorooctanoic acid. Further optionally, the dissociation agent includes at least one of 8-aniline-1-naphthalenesulfonic acid, mesna, tris(2-carbonylethyl)phosphine hydrochloride, dimethyl sulfoxide and N,N-dimethylformamide, and one or more of dithiothreitol, methanol, ethanol, perfluorobutyric acid, perfluorononanoic acid and perfluorooctanoic acid.

[0026] Optionally, in the sample dissociation solution, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450 and lauryl alcohol polyoxyethylene ether;

[0027] Optionally, in the sample dissociation solution, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives and ProClin series preservatives.

[0028] In some embodiments of the present application, the solvent of the R1 reagent includes a buffer and 0.5wt%-5wt% of sodium chloride, 0.05wt%-5wt% of a sugar protective agent, 0.02wt%-1wt% of a surfactant, 0.05wt%-2wt% of a protein protective agent, and 0.05wt%-0.5wt% of a preservative; wherein the buffer includes one or more of a PBS buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a MES buffer, a HEPES buffer, a PIPES buffer, a TES buffer, a TAPSO buffer, and a MOPS buffer;

[0029] Optionally, in the solvent of the R1 reagent, the sugar protective agent includes one or more of sucrose and trehalose;

[0030] Optionally, in the solvent of the R1 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100 and Triton X-450;

[0031] Optionally, in the solvent of the R1 reagent, the protein protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, glycerol, mannitol, sorbitol, alanine, glycine, sarcosine, L-tyrosine and phenylalanine;

[0032] Optionally, in the solvent of the R1 reagent, the preservative includes one or more of proclin 300, Bronidox-L, gentamicin sulfate, NaN3 and KroVin series;

[0033] Optionally, the concentration of the binding molecule 1-solid phase conjugate in the R1 reagent is 0.3 mg / mL-2 mg / mL.

[0034] In some embodiments of the present application, the solvent of the R2 reagent includes a buffer and 0.01wt%-5wt% of a protective agent, 0.02wt%-1.5wt% of a surfactant, 0.05wt%-0.5wt% of a blocking agent, and 0.01wt%-5wt% of a preservative; wherein the buffer includes one or more of a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer, and a TES buffer;

[0035] Optionally, in the solvent of the R2 reagent, the protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, sodium chloride, sucrose, trehalose and mannitol;

[0036] Optionally, in the solvent of the R2 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan;

[0037] Optionally, in the solvent of the R2 reagent, the blocking agent includes anti-biotin antibody, MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, sheep IgG, goat IgG, HBR-5, HBR-7, TRU Block TM , HBR-1, HBR-X and P-heterophilic antibodies;

[0038] Optionally, in the solvent of the R2 reagent, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives and ProClin series preservatives;

[0039] Optionally, the concentration of the antibody marker 1 in the R2 reagent is 0.5 μg / mL-2.0 μg / mL.

[0040] In some embodiments of the present application, the solvent of the R3 reagent includes a buffer and 0.5wt%-5wt% of sodium chloride, 0.5wt%-5wt% of a sugar protective agent, 0.02wt%-1wt% of a surfactant, 0.05wt%-2wt% of a protein protective agent, and 0.05wt%-0.5wt% of a preservative; wherein the buffer includes one or more of a PBS buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a MES buffer, a HEPES buffer, a PIPES buffer, a TES buffer, a TAPSO buffer, and a MOPS buffer;

[0041] Optionally, in the solvent of the R3 reagent, the sugar protective agent includes one or more of trehalose, sucrose and dextran;

[0042] Optionally, in the solvent of the R3 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100 and Triton X-450;

[0043] Optionally, in the solvent of the R3 reagent, the protein protective agent includes one or more of bovine serum albumin and plant-derived recombinant human serum albumin, glycerol, mannitol, sorbitol, alanine, glycine, sarcosine, L-tyrosine and phenylalanine;

[0044] Optionally, in the solvent of the R3 reagent, the preservative includes one or more of proclin 300, Bronidox-L, gentamicin sulfate, NaN3 and KroVin series;

[0045] Optionally, the concentration of the antibody-solid phase conjugate in the R3 reagent is 0.3 mg / mL-2 mg / mL.

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

[0047] (1) The solid phases in the binding molecule 1-solid phase conjugate and the antibody-solid phase conjugate are each independently magnetic beads with a diameter of 0.1 μm to 3 μm;

[0048] (2) the molecular binding pairs include one or more of the following molecular binding pairs: streptavidin and biotin, fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody;

[0049] (3) The tracer marker includes one or more of terpyridine ruthenium, horseradish peroxidase, alkaline phosphatase, acridinium ester, luminol and its derivatives, isoluminol and its derivatives, and adamantane.

[0050] In some embodiments of the present application, the detection kit further comprises a calibrator and / or a quality control product;

[0051] The calibrator and the quality control product each independently include 25-hydroxyvitamin D antigen and an antigen solvent;

[0052] Optionally, the antigen solvent includes a buffer and 1wt%-5wt% of a sugar protective agent, 0.02wt%-1wt% of a surfactant, and 0.05wt%-0.5wt% of a preservative; wherein the buffer includes one or more of a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer, and a TES buffer;

[0053] Optionally, in the antigen solvent, the sugar protective agent includes one or more of sucrose, trehalose, lactose and mannitol;

[0054] Optionally, in the antigen solvent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, TritonX-100, Triton X-450, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan;

[0055] Optionally, in the antigen solvent, the preservative includes proclin 300.

[0056] One or more embodiments of the present application provide a method for detecting 25-hydroxyvitamin D, which comprises: using the 25-hydroxyvitamin D detection kit to detect 25-hydroxyvitamin D in a sample to be tested.

[0057] In some embodiments of the present application, the sample to be tested is serum or plasma.

[0058] Compared with the traditional technology, this application has the following beneficial effects:

[0059] The detection kit of the present application, based on the design of working reagent 1 and working reagent 2, can detect 25-hydroxyvitamin D by non-competitive double antibody sandwich method on a highly sensitive electrochemiluminescence platform, effectively avoiding the result deviation caused by human operation, with high sensitivity, and also having the characteristics of large linear range, good repeatability, good correlation with mass spectrometry results and short time consumption. Details of one or more embodiments of the present application are presented in the following description, and other features, purposes and advantages of the present application will become apparent from the specification and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0061] Figure 1 The results are from the competition method and mass spectrometry. DETAILED DESCRIPTION

[0062] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present 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 present application, and it should be understood that the present application can be implemented without one or more of these details.

[0063] 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.

[0064] the term

[0065] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0066] 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").

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

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

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

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

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

[0078] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the 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.

[0079] In recent years, small molecule non-competitive methods have developed rapidly, including hapten open non-competitive immunoassay, detection based on enzyme-labeled analyte-analyte antibody site replacement non-competitive method, sandwich method research based on other recognition materials, and double antibody sandwich method based on anti-immune complex antibodies. With the development of phage display technology and autonomous diversified library technology, the problem of accurate identification of new antigenic epitopes of antigen-antibody complexes has been effectively solved, and anti-immune complex antibodies have been developed more efficiently and accurately, making them have good application prospects in small molecule sandwich immunoassays. The present invention selects highly sensitive and specific monoclonal antibodies developed based on anti-immune complexes, and develops a sensitive and reliable 25-OHVitamin D immunoassay method on an electrochemiluminescence platform.

[0080] In a first aspect of an embodiment of the present application, a 25-hydroxyvitamin D detection kit is provided, the detection kit comprising a working reagent 1 and a working reagent 2;

[0081] in,

[0082] The working reagent 1 includes R1 reagent and R2 reagent, or includes R3 reagent; the R1 reagent includes binding molecule 1-solid phase conjugate, the R2 reagent includes antibody marker 1, the antibody marker 1 includes binding molecule 2 and antibody 1 that specifically binds to 25-hydroxyvitamin D, and the binding molecule 1 and the binding molecule 2 constitute a molecular binding pair;

[0083] The R3 reagent includes an antibody-solid phase conjugate, wherein the antibody in the antibody-solid phase conjugate is an antibody 1 that specifically binds to 25-hydroxyvitamin D;

[0084] The working reagent 2 includes an antibody marker 2, and the antibody marker 2 includes a tracer marker and an antibody 2 that specifically binds to 25-hydroxyvitamin D.

[0085] The detection kit of the present application, based on the design of working reagent 1 and working reagent 2, can detect 25-hydroxyvitamin D by a non-competitive double antibody sandwich method on a highly sensitive electrochemiluminescence platform, effectively avoiding the result deviation caused by human operation, with high sensitivity, and also has the characteristics of a large linear range, good repeatability, good correlation with mass spectrometry results, and short time consumption.

[0086] The detection kit of the present application detects 25-hydroxyvitamin D by sandwich method based on antibody 1 and antibody 2. In the detection kit of the present application, the solid phase (microspheres, magnetic beads) is directly coated with antibodies (including R3 reagents) or indirectly coated with antibodies (including R1 and R2 reagents).

[0087] The detection kit of the present application is used with a fully automatic electrochemiluminescence instrument to realize automatic sample addition, incubation, cleaning and detection steps, which can avoid the result deviation caused by human operation. The sample to be tested and the sample dissociation solution react fully, and 25-hydroxyvitamin D is released from the binding protein; then it forms a sandwich immune complex with working reagent 1 and working reagent 2, and is fixed to the solid phase. The electrochemiluminescence system of the fully automatic electrochemiluminescence instrument emits a continuous and stable light signal. The light signal calculates the concentration of the sample through the standard curve, and the signal value is proportional to the concentration.

[0088] The high-sensitivity electrochemiluminescence platform combined with the non-competitive double antibody sandwich method for testing 25-hydroxyvitamin D has effectively solved the problem of low sensitivity of the 25-hydroxyvitamin D competitive method, making the detection limit of 25-hydroxyvitamin D reach 0.15ng / mL, the linear range expanded to 0.15-150ng / mL, the repeatability CV <5%, and the reaction time shortened (for example, 9min, 18min). The newly developed VD sandwich reagent has a better correlation with the mass spectrometry results, with a correlation coefficient r>0.975.

[0089] In some examples, the solvent of the working reagent 2 includes a buffer and 0.01wt%-5wt% (e.g., 0.01wt%, 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of a protective agent, 0.02wt%-1wt% (e.g., 0.02wt%, 0.04wt%, 0.06wt%) of a protective agent. %, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%) of surfactant, 0.05wt%-0.5wt% (for example, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.4 5wt%, 0.5wt%) of blocking agent and 0.01wt%-5wt% (for example, 0.01wt%, 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of preservative; wherein the buffer solution includes a pH value of 6.0-9.0 (for example, 6.0, 6.5, 7.0, 7.5, 8.0, 8. 5, 9.0), one or more of phosphate buffer, Bis-Tris buffer, HEPES buffer, MES buffer, citrate buffer, MOPSO buffer, PIPES buffer and TES buffer with a concentration of 0.01 mol / L-0.2 mol / L (for example, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.2 mol / L).

[0090] In some examples, the blocking agent in the solvent of the working reagent 2 includes anti-biotin antibody, MAK33 Poly, HBR-5, HBR-7, TRU Block TM , HBR-1, HBR-X, rabbit IgG, mouse IgG, bovine IgG, sheep IgG, goat IgG and P-heterophilic antibody. Further optionally, the blocking agent includes anti-biotin antibody, HBR-5, HBR-7, TRUBlock TM At least one of HBR-1, HBR-X and MAK33 Poly, and one or more of P-heterophilic antibody, rabbit IgG, mouse IgG, bovine IgG, sheep IgG and goat IgG. The active blocker and the passive blocker are used together to improve the detection sensitivity of the kit.

[0091] In some examples, in the solvent of the working reagent 2, the protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, sodium chloride, sucrose, trehalose, lactose and mannitol.

[0092] In some examples, in the solvent of the working reagent 2, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, Pluronic F-68, Pluronic F-127, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan.

[0093] In some examples, the preservative in the solvent of the working reagent 2 includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives (eg, KroVin100, KroVin500) and ProClin series preservatives (eg, Proclin150, Proclin 300, Proclin 950).

[0094] Optionally, the concentration of the antibody marker 2 in the working reagent 2 is 0.5 μg / mL-2.0 μg / mL (for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 μg / mL).

[0095] In some examples, the detection kit further includes a sample dissociation solution;

[0096] The sample dissociation solution includes a buffer and 0.02wt%-1wt% (for example, 0.02wt%, 0.04wt%, 0.06wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%) of a surfactant, 1wt%-20wt% (for example, 1wt%, 2wt%, 4wt%, 6wt%, 8wt%, 10wt%, 12wt%, 14wt%, 16wt%, 18wt%, 20wt%) of a dissociation agent and 0.01wt%-5wt% (for example, 0.01wt%, 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1. The preservative is preferably a preservative having a pH value of 3.0-7.5 (for example, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7, 7.5) and a concentration of 0.01 mol / L-0.2 mol / L (for example, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.2 mol / L), HEPES buffer, citric acid buffer, acetate buffer, phosphate buffer, Bis-Tris buffer, MOPSO buffer, PIPES buffer and TES buffer. The sample dissociation solution of the present application plays a dissociation role. VD binds to the VD binding protein in the serum and needs to be dissociated before detection.

[0097] In some examples, in the sample dissociation solution, the dissociation agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, mesna, tris(2-carbonylethyl)phosphine hydrochloride, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, perfluorobutyric acid, perfluorononanoic acid and perfluorooctanoic acid. The present application selects a special dissociation agent, improves the dissociation effect of the sample, and improves the accuracy of sample detection (the dissociation agent selected in the present application makes the sample dissociated sufficiently, reduces missed detection; makes the effects of different samples consistent, the measured values ​​are more stable, and the repeatability is good). Further optionally, the dissociation agent includes at least one of 8-aniline-1-naphthalenesulfonic acid, mesna, tris(2-carbonylethyl)phosphine hydrochloride, dimethyl sulfoxide and N,N-dimethylformamide, and one or more of dithiothreitol, methanol, ethanol, perfluorobutyric acid, perfluorononanoic acid and perfluorooctanoic acid.

[0098] In some examples, in the sample dissociation solution, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450 and lauryl alcohol polyoxyethylene ether.

[0099] In some examples, in the sample dissociation solution, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives (eg, KroVin100, KroVin500) and ProClin series preservatives (eg, Proclin 150, Proclin300, Proclin950).

[0100] In some examples, the solvent of the R1 reagent includes a buffer and 0.5wt%-5wt% (e.g., 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of sodium chloride, 0.05wt%-5wt% (e.g., 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of sugar. Protective agent, 0.02wt%-1wt% (for example, 0.02wt%, 0.04wt%, 0.06wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%) of surfactant, 0.05wt%-2wt% (for example, 0.05wt%, 0.1wt%, 0.5wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%) of surfactant t%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2wt%) of protein protective agent, 0.05wt%-0.5wt% (for example, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%) of preservative; wherein the buffer solution comprises a pH value of 6.0- 9.0 (for example, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0), one or more of PBS buffer, Bis-Tris buffer, MES buffer, HEPES buffer, PIPES buffer, TES buffer, TAPSO buffer and MOPS buffer with a concentration of 0.01mol / L-0.2mol / L (for example, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.2mol / L).

[0101] In some examples, in the solvent of the R1 reagent, the sugar protective agent includes one or more of sucrose and trehalose.

[0102] In some examples, in the solvent of the R1 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100 and Triton X-450.

[0103] In some examples, in the solvent of the R1 reagent, the protein protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, glycerol, mannitol, sorbitol, alanine, glycine, sarcosine, L-tyrosine and phenylalanine.

[0104] In some examples, the preservative in the solvent of the R1 reagent includes one or more of proclin 300, Bronidox-L, gentamicin sulfate, NaN3 and KroVin series.

[0105] In some examples, the concentration of the binding molecule 1-solid phase conjugate in the R1 reagent is 0.3 mg / mL-2 mg / mL (e.g., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 μg / mL).

[0106] In some examples, the solvent of the R2 reagent includes a buffer and 0.01wt%-5wt% (e.g., 0.01wt%, 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of a protective agent, 0.02wt%-1.5wt% (e.g., 0.02wt%, 0.04wt%, 0.06wt%, 0.08wt%) of a protective agent. %, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%) of surfactant, 0.05wt%-0.5wt% (for example, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%) of blocking agent and 0.01wt%-5wt% (for example, 0.01wt%, 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of preservative; wherein the buffer solution comprises a pH value of 6.0-9.0 (for example, 6.0, 6.5, 7.0, 7.5). .5, 8.0, 8.5, 9.0), phosphate buffer with a concentration of 0.01mol / L-0.2mol / L (for example, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.2mol / L), one or more of Bis-Tris buffer, HEPES buffer, MES buffer, citrate buffer, MOPSO buffer, PIPES buffer and TES buffer.

[0107] In some examples, in the solvent of the R2 reagent, the protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, sodium chloride, sucrose, trehalose, lactose and mannitol;

[0108] In some examples, in the solvent of the R2 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan.

[0109] In some examples, the blocking agent in the solvent of the R2 reagent includes anti-biotin antibody, MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, sheep IgG, goat IgG, HBR-5, HBR-7, TRU Block TM , HBR-1, HBR-X and P-heterophilic antibodies.

[0110] In some examples, the preservative in the solvent of the R2 reagent includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives (KroVin100, KroVin500) and ProClin series preservatives (Proclin 150, Proclin 300, Proclin 950).

[0111] In some examples, the concentration of the antibody marker 1 in the R2 reagent is 0.5 μg / mL-2.0 μg / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 μg / mL).

[0112] In some examples, the solvent of the R3 reagent includes a buffer and 0.5wt%-5wt% (e.g., 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of sodium chloride, 0.5wt%-5wt% (e.g., 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of sodium chloride, %) of a sugar protective agent, 0.02wt%-1wt% (e.g., 0.02wt%, 0.04wt%, 0.06wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%) of a surfactant, 0.05wt%-2wt% (e.g., 0.05wt%, 0.1wt%, 0.5wt%, 1.0wt%, 1 .1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2wt%) of protein protective agent, 0.05wt%-0.5wt% (for example, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%) of preservative; wherein, the slow The flushing solution includes one or more of PBS buffer, Bis-Tris buffer, MES buffer, HEPES buffer, PIPES buffer, TES buffer, TAPSO buffer and MOPS buffer with a pH value of 6.0-9.0 and a concentration of 0.01 mol / L-0.2 mol / L (for example, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.2 mol / L).

[0113] In some examples, in the solvent of the R3 reagent, the sugar protective agent includes one or more of trehalose, sucrose and dextran.

[0114] In some examples, in the solvent of the R3 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100 and Triton X-450.

[0115] In some examples, in the solvent of the R3 reagent, the protein protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, glycerol, mannitol, sorbitol, alanine, glycine, sarcosine, L-tyrosine and phenylalanine.

[0116] In some examples, the preservative in the solvent of the R3 reagent includes one or more of proclin 300, Bronidox-L, gentamicin sulfate, NaN3 and KroVin series.

[0117] In some examples, the concentration of the antibody-solid phase conjugate in the R3 reagent is 0.3 mg / mL-2 mg / mL (e.g., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 μg / mL).

[0118] In some examples, the detection kit meets one or more of the following conditions:

[0119] (1) The solid phases in the binding molecule 1-solid phase conjugate and the antibody-solid phase conjugate are each independently a magnetic bead (or microsphere) with a diameter of 0.1 μm-3 μm (for example, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3 μm), and the surface of the magnetic bead carries a variety of functional groups, including but not limited to -OH, -COOH, -NH2, and tosyl;

[0120] (2) the molecular binding pairs include one or more of the following molecular binding pairs: streptavidin and biotin, fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody;

[0121] (3) The tracer marker includes one or more of terpyridine ruthenium, horseradish peroxidase, alkaline phosphatase, acridinium ester, luminol and its derivatives, isoluminol and its derivatives, and adamantane.

[0122] In some examples, the detection kit further includes a calibrator and / or a quality control product; the calibrator and the quality control product each independently include a 25-hydroxyvitamin D antigen and an antigen solvent.

[0123] In some examples, the antigen solvent includes a buffer and 1wt%-5wt% (e.g., 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%) of a sugar protective agent, 0.02wt%-1wt% (e.g., 0.02wt%, 0.04wt%, 0.06wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%) of a surfactant and 0.05wt%-0.5wt% (e.g., 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%) of a surfactant. The invention relates to a preservative comprising: a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01 mol / L-0.2 mol / L (for example, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.2 mol / L), a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer and a TES buffer.

[0124] In some examples, in the antigen solvent, the sugar protective agent includes one or more of sucrose, trehalose, lactose and mannitol.

[0125] In some examples, in the antigen solvent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan.

[0126] In some examples, the preservative in the antigen solvent includes proclin 300.

[0127] A second aspect of an embodiment of the present application provides a method for detecting 25-hydroxyvitamin D, the method comprising: using the 25-hydroxyvitamin D detection kit to detect 25-hydroxyvitamin D in a sample to be tested.

[0128] The detection method of the present application mainly adopts the above-mentioned 25-hydroxyvitamin D detection kit to detect 25-hydroxyvitamin D in the sample to be tested. The detection method can detect 25-hydroxyvitamin D by a non-competitive double antibody sandwich method on a highly sensitive electrochemiluminescence platform, effectively avoiding the result deviation caused by human operation, with high sensitivity, and also has the characteristics of large linear range, good repeatability, good correlation with mass spectrometry results and short time consumption.

[0129] Furthermore, the detection method includes: the sample to be tested and the sample dissociation solution are fully reacted, and 25-hydroxyvitamin D is released from the binding protein; then, a sandwich immune complex is formed with working reagent 1 and working reagent 2, and fixed to a solid phase, and a continuous and stable light signal is emitted through the electrochemiluminescence system of a fully automatic electrochemiluminescence instrument. The light signal is used to calculate the concentration of the sample through a standard curve, and the signal value is proportional to the concentration.

[0130] In some examples, the sample to be tested is serum or plasma.

[0131] 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.

[0132] 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.

[0133] Example 1

[0134] 25-OH VD detection kit using a streptavidin (SA) and biotin system for indirect labeling

[0135] 1.1 Preparation of 25-OH VD sample dissociation solution

[0136] The 25-OH VD sample dissociation solution includes Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 0.05 wt% 80, 0.05wt% Brij-35, 10wt% methanol, 2wt% dimethyl sulfoxide (DMSO), 1wt% dithiothreitol (DTT), 2wt% perfluorooctanoic acid and 0.05wt% proclin300.

[0137] 1.2 Preparation of Streptavidin-coupled Magnetic Microparticle Solution

[0138] The streptavidin-coupled magnetic beads solution was removed from the supernatant using a magnetic stand, washed twice with HEPES buffer at pH 7.2 and 0.2 mol / L, and resuspended in solvent 1, with the concentration of streptavidin-coupled magnetic particles being 0.75 mg / mL. Solvent 1 contained: HEPES at pH 7.2 and 0.2 mol / L, 0.15 M sodium chloride, 0.1 wt% sucrose, 0.05 wt% 20, 2wt% bovine serum albumin, and 0.05wt% proclin300.

[0139] 1.3 Preparation of Biotinylated 25-OH VD Antibody 1 Solvent 2

[0140] The solvent 2 of the biotin-labeled 25-OH VD antibody 1 includes a Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 1 wt% of trehalose, 0.5 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of mouse IgG, 0.2 wt% of P-heterophilic antibody, and 0.2 wt% of TRUBlock TM (Manufacturer: meridian, the raw materials mentioned below are all from here, no further details are given), 0.1wt% MAK33Poly, 0.05wt% Proclin 300 and 0.05wt% gentamicin.

[0141] 1.4 Preparation of 25-OH VD Antibody 2 Solvent 3 Labeled with Ruthenium Trispyridine

[0142] The solvent 3 of the 25-OH VD antibody 2 labeled with ruthenium trispyridine includes: Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 2 wt% of trehalose, 1 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of anti-biotin antibody, 0.1 wt% of mouse IgG, 0.2 wt% of P-heterophil antibody, and 0.2 wt% of TRU Block. TM , 0.1wt% MAK33 Poly, 0.05wt% Proclin 300 and 0.05wt% gentamicin.

[0143] 1.5 Preparation of biotinylated 25-OH VD antibody 1 working solution

[0144] Take 25-OH VD antibody 1 in a pre-treated ultrafiltration tube, use pH 8.0, 25mM Tris-HCl buffer for ultrafiltration centrifugation for purification three times, transfer the purified antibody 1 to a collection tube, test the concentration and adjust it to an appropriate concentration; add biotin at a molar ratio of 1:30, and react at room temperature for 30 minutes; after the reaction, transfer the reaction solution to a pre-treated ultrafiltration tube; ultrafiltration purification three times to obtain biotin-labeled 25-OH VD antibody 1.

[0145] The biotin-labeled 25-OH VD antibody 1 was diluted to 1.0 μg / mL using the solvent 2 prepared in step 1.3 of this example to prepare a biotin-labeled 25-OH VD antibody 1 working solution.

[0146] 1.6 Preparation of terpyridine-ruthenium labeled 25-OH VD antibody 2 working solution

[0147] The 25-OH VD antibody 2 was placed in a pre-treated ultrafiltration tube, and ultrafiltration centrifuged using a pH 7.4, 25 mM PBS buffer for purification three times. The purified antibody 2 was transferred to a collection tube, and the concentration was tested and adjusted to an appropriate concentration. Activated terpyridine ruthenium was added at a molar ratio of 1:30. The reaction was allowed to proceed at room temperature for 30 minutes. After the reaction was completed, the reaction solution was transferred to a pre-treated ultrafiltration tube. The terpyridine ruthenium labeled 25-OH VD antibody 2 was obtained by ultrafiltration purification three times.

[0148] The ruthenium-labeled 25-OH VD antibody 2 was diluted to 1.2 μg / mL using the solvent 3 prepared in step 1.4 of this example to prepare the terpyridine ruthenium-labeled 25-OH VD antibody 2 working solution.

[0149] 1.7 Preparation of Calibrator and Control Solution

[0150] Prepare the diluent for calibration quality control: HEPES buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 1 wt% 20, 5% trehalose, 0.25wt% proclin 300.

[0151] Use this diluent to prepare 25-OH VD antigen calibration working solution and quality control working solution.

[0152] 1.8 Packaging and Assembly of Kits

[0153] 25-Hydroxyvitamin D Assay Kit (Electrochemiluminescence Method): 10 mL / bottle of the sample dissociation solution prepared in step 1.1, 5.0 mL / bottle of the streptavidin-coupled magnetic microparticle solution prepared in step 1.2, 10 mL / bottle of the biotin-labeled 25-OH VD antibody 1 working solution prepared in step 1.5, 10 mL / bottle of the terpyridine ruthenium-labeled 25-OH VD antibody 2 working solution prepared in step 1.6, and 1.0 mL / bottle of the calibrator working solution and the quality control working solution prepared in step 1.7, respectively, are packaged together and stored at 2-8°C.

[0154] 1.9 Kit-based detection methods

[0155] 1. Sample pretreatment: Pre-treat the sample to be tested (such as serum, plasma or artificially prepared samples) accordingly according to the actual situation; if the serum or plasma is turbid, centrifuge it at 4℃5000rpm for 5min and take the supernatant for testing.

[0156] 2. Before using the reagent, put it into the Pumen Company's fully automatic electrochemiluminescence analyzer 30 minutes in advance to automatically mix the magnetic beads and keep them in a suspended state.

[0157] 3. Calibration and quality control of the test kit: Use the matching calibrators to perform calibration tests and determine the accuracy of the current system through the quality control results.

[0158] 4. Sample testing:

[0159] (1) After the user applies for the test, the first step is that the system automatically draws 10 μL of sample and 50 μL of sample dissociation solution and mixes them for 9 minutes; the sample 25-OH VD is dissociated and released. The second step is that the system automatically draws 35 μL of streptavidin-coupled magnetic particle solution, 60 μL of biotin-labeled 25-OH VD antibody 1 working solution, and 50 μL of terpyridine ruthenium-labeled 25-OH VD antibody 2 working solution and adds them to the reaction cup, and automatically incubates at 37°C for 9 minutes to form an antigen-antibody sandwich complex, and then the entire complex is bound to the magnetic particles under the interaction of biotin and streptavidin.

[0160] (2) After the incubation is completed, the system automatically draws the reaction mixture into the measuring cell. The magnetic particles are adsorbed to the electrode by the magnet, and the unbound substances are washed away. When voltage is applied to the electrode, chemiluminescence is generated. The generated light signal is detected by a photomultiplier tube, and the detection result is automatically found by the instrument from the calibration curve (this calibration curve is obtained by the instrument through a two-point calibration of the main calibration curve obtained by scanning).

[0161] Example 2

[0162] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0163] The 25-OH VD sample dissociation solution includes Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 0.05 wt% 80, 0.05wt% Brij-35, 10wt% methanol, 0.1wt% 8-aniline-1-naphthalenesulfonic acid (ANS), 2wt% dimethyl sulfoxide (DMSO), 1.5wt% perfluorobutyric acid and 0.05wt% proclin 300. The remaining reagents and operations are the same as those in Example 1.

[0164] Example 3

[0165] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0166] The 25-OH VD sample dissociation solution includes Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 0.05 wt% Tween 80, 0.05 wt % of Brij-35, 10 wt % of ethanol, 1 wt % of tris(2-carbonylethyl)phosphine hydrochloride, 2 wt % of N,N-dimethylformamide and 0.05 wt % of proclin 300. The remaining reagents and operations were the same as those in Example 1.

[0167] Example 4

[0168] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0169] The 25-OH VD sample dissociation solution includes a pH of 7.2, a concentration of 0.2 mol / L Bis-Tris buffer, and 0.05 wt% Tween 80, 0.05 wt % of Brij-35, 10 wt % of methanol, 1 wt % of dithiothreitol (DTT), 2 wt % of perfluorooctanoic acid and 0.05 wt % of proclin 300. The remaining reagents and operations were the same as those in Example 1.

[0170] Example 5

[0171] This embodiment is substantially the same as Embodiment 2, with the main changes being:

[0172] The 25-OH VD sample dissociation solution includes a pH of 7.2, a concentration of 0.2 mol / L Bis-Tris buffer, 0.05 wt% 80, 0.05wt% Brij-35, 0.1wt% 8-aniline-1-naphthalenesulfonic acid (ANS), 2wt% dimethyl sulfoxide (DMSO), 1.5wt% perfluorobutyric acid and 0.05wt% proclin 300. The remaining reagents and operations are the same as those in Example 2.

[0173] Example 6

[0174] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0175] The 25-OH VD sample dissociation solution includes Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 0.05 wt% 80, 0.05 wt % of Brij-35, 10 wt % of methanol and 0.05 wt % of proclin 300. The remaining reagents and operations were the same as those in Example 2.

[0176] Example 7

[0177] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0178] The 25-OH VD sample dissociation solution includes Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 0.1 wt % Brij-35, 10 wt % methanol, 2 wt % dimethyl sulfoxide (DMSO), 1 wt % dithiothreitol (DTT), 2 wt % perfluorooctanoic acid and 0.05 wt % proclin 300. The remaining reagents and operations are the same as those in Example 1.

[0179] Example 8

[0180] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0181] The 25-OH VD sample dissociation solution includes Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 0.1 wt% 80, 10 wt % methanol, 2 wt % dimethyl sulfoxide (DMSO), 1 wt % dithiothreitol (DTT), 2 wt % perfluorooctanoic acid and 0.05 wt % proclin 300. The remaining reagents and operations were the same as those in Example 1.

[0182] Example 9

[0183] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0184] The solvent 2 of the biotin-labeled 25-OH VD antibody 1 includes a Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 1 wt% of trehalose, 0.5 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of mouse IgG, 0.2 wt% of P-heterophilic antibody, 0.2 wt% of rabbit IgG, 0.1 wt% of MAK33 Poly, 0.05 wt% of Proclin 300 and 0.05 wt% of gentamicin. The remaining reagents and operations are consistent with those in Example 1.

[0185] Example 10

[0186] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0187] The solvent 2 of the biotin-labeled 25-OH VD antibody 1 includes a Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 1 wt% of trehalose, 0.5 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of mouse IgG, 0.2 wt% of rabbit IgG, and 0.2 wt% of TRUBlock TM , 0.1wt% MAK33 Poly, 0.05wt% Proclin 300 and 0.05wt% gentamicin. The remaining reagents and operations were the same as those in Example 1.

[0188] Embodiment 11

[0189] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0190] The solvent 2 of the biotin-labeled 25-OH VD antibody 1 includes a Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 1 wt% of trehalose, 0.5 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of HBR-5, 0.2 wt% of P-heterophilic antibody, and 0.2 wt% of TRUBlock TM , 0.1wt% MAK33 Poly, 0.05wt% Proclin 300 and 0.05wt% gentamicin. The remaining reagents and operations were the same as those in Example 1.

[0191] Example 12

[0192] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0193] The solvent 2 of the biotin-labeled 25-OH VD antibody 1 includes a Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 1 wt% of trehalose, 0.5 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.3 wt% of mouse IgG, 0.3 wt% of rabbit IgG, 0.05 wt% of Proclin300 and 0.05 wt% of gentamicin. The remaining reagents and operations are consistent with those in Example 1.

[0194] Embodiment 13

[0195] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0196] The solvent 3 of the 25-OH VD antibody 2 labeled with terpyridine ruthenium includes: Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 2 wt% of trehalose, 1 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of anti-biotin antibody, 0.1 wt% of mouse IgG, 0.2 wt% of P-heterophil antibody, 0.2 wt% of rabbit IgG, 0.1 wt% of MAK33 Poly, 0.05 wt% of Proclin300 and 0.05 wt% of gentamicin. The remaining reagents and operations are consistent with those in Example 1.

[0197] Embodiment 14

[0198] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0199] The solvent 3 of the 25-OH VD antibody 2 labeled with ruthenium trispyridine includes: Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 2 wt% of trehalose, 1 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of anti-biotin antibody, 0.1 wt% of mouse IgG, 0.2 wt% of rabbit IgG, and 0.2 wt% of TRU Block TM , 0.1 wt% of MAK33 Poly, 0.05 wt% of Proclin 300 and 0.05 wt% of gentamicin. The remaining reagents and operations were the same as those in Example 1.

[0200] Embodiment 15

[0201] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0202] The solvent 3 of the 25-OH VD antibody 2 labeled with ruthenium trispyridine includes: Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 2 wt% of trehalose, 1 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of anti-biotin antibody, 0.1 wt% of HBR-7, 0.2 wt% of P-heterophil antibody, and 0.2 wt% of TRU Block. TM , 0.1wt% MAK33 Poly, 0.05wt% Proclin 300 and 0.05wt% gentamicin. The remaining reagents and operations were the same as those in Example 1.

[0203] Example 16

[0204] This embodiment is substantially the same as Embodiment 1, with the following main changes:

[0205] The solvent 3 of the 25-OH VD antibody 2 labeled with terpyridine ruthenium includes: Bis-Tris buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 2 wt% of plant-derived recombinant human serum albumin, 2 wt% of trehalose, 1 wt% of mannitol, 0.05 wt% of Brij-35, 0.05 wt% of PEG4000, 0.1 wt% of anti-biotin antibody, 0.3 wt% of mouse IgG, 0.3 wt% of rabbit IgG, 0.05 wt% of Proclin 300 and 0.05 wt% of gentamicin. The remaining reagents and operations are consistent with those in Example 1.

[0206] Performance test of the detection kit of each embodiment

[0207] 1. Limit of Detection (LoD)

[0208] 1. Samples to be tested: Use matrix to dilute antigen and prepare 5 samples close to the LoD concentration value, where the LoD concentration value is set to 0.15 ng / mL.

[0209] Each of the above-mentioned prepared samples, in addition to the corresponding test substance (i.e., 25-OH VD), also contains the following components: the solvent is the calibration quality control diluent (HEPES buffer with a pH of 7.2 and a concentration of 0.2 mol / L, 1 wt% 20, 5wt% trehalose, 0.25wt% proclin 300).

[0210] 2. Use the kits of the above embodiments to test the samples to be tested in step 1. Specifically, 5 samples close to the LoD concentration value are tested using 3 batches of reagents for 3 days, with each sample repeated 3 times per day. After multiple batches of verification of the above multiple samples, the test results can meet the LoD.

[0211] For specific reference, please refer to "Performance Evaluation Methods for In Vitro Diagnostic Test Systems Part 3: Detection Limit and Quantification Limit in the Pharmaceutical Industry Standard of the People's Republic of China YY1789.3-2022".

[0212] As shown in Table 1, the detection limit (LoD) of the kit can reach 0.15 ng / mL.

[0213] Table 1. Detection limits of the detection kits of various embodiments

[0214] Example Detection limit of the kit (ng / mL) 1 0.150 2 0.168 3 0.175 4 0.168 5 0.195 6 0.185 7 0.163 8 0.166 9 0.164 10 0.172 11 0.179 12 0.190 13 0.166 14 0.175 15 0.178 16 0.187

[0215] 2. Intra-batch precision

[0216] Select three concentration samples of low, medium and high concentrations (i.e., low value samples, median value samples and high value samples) for detection, and use the same batch number of the test kit (i.e., the test kit of Example 1) to repeat the measurement 10 times, calculate the mean M of the measured value, calculate the standard deviation (SD) and the coefficient of variation (CV), and the coefficient of variation (CV) should be less than 5%. Among them, the low value sample, the median value sample and the high value sample are clinical blood samples whose 25-OH VD content meets the range requirements of 5.800±0.100ng / mL, 28.00±0.700ng / mL and 100.00±5.000ng / mL for clinical detection.

[0217] The test results are shown in Table 2.

[0218] As can be seen from Table 2, the results of the intra-batch precision of the low, medium and high clinical sample tests were all less than 5%, and the intra-batch precision verification was passed.

[0219] Table 2. Intra-batch precision evaluation

[0220]

[0221] 3. Linear range

[0222] The linear range detection was performed using the kit of Example 1.

[0223] Sample X1: a clinical blood sample with a clinical 25-OH VD content of 1.20 ng / mL; Sample X7: a clinical blood sample with a clinical 25-OH VD content of 174.45 ng / mL. Samples X1 and X7 were mixed according to the ratio (volume ratio) in Table 2 to obtain samples X2 to X6, and each concentration was tested twice. The results are shown in Table 3.

[0224] Table 3. Linear evaluation results

[0225]

[0226] As can be seen from Table 3, the linear range of the kit of the present application is 1.2 ng / mL-150 ng / mL, which is significantly wider than the linear range of Roche's 25-hydroxyvitamin D detection kit ((electrochemiluminescence method) Elecsys Vitamin D total III, whose linear range is: 3 ng / mL-100 ng / mL), and the correlation satisfies R>0.99, and the linear range verification is passed.

[0227] 4. Clinical Comparison

[0228] 47 clinical samples were selected and the kit, competitive method and mass spectrometry of Example 1 were used to perform simultaneous testing on the same clinical blood sample. Among them, the competitive method: the instrument used is Roche instrument: Cobase e411; the reagent used is 25-hydroxyvitamin D detection kit (electrochemiluminescence method) Elecsys Vitamin D total III). Mass spectrometry (LC-MS / MC): See the Chinese Geriatrics Society. "Expert consensus on standardization of 25-hydroxyvitamin D detection by liquid chromatography-tandem mass spectrometry." Chinese Journal of Laboratory Medicine (2021). For detailed test results, see Figure 1 .

[0229] from Figure 1 It can be seen that the slope of the sandwich method and mass spectrometry comparison is 0.983; the correlation coefficient R 2 =0.987; r = 0.9935; the slope of the comparison between the competition method and mass spectrometry is 0.715; the correlation coefficient R 2 =0.945; r=0.9721.

[0230] In summary, the detection kit of the above embodiment of the present application can be used with a fully automatic electrochemiluminescence instrument. During the detection process, the sample to be tested and the sample dissociation solution fully react, 25-hydroxyvitamin D is released from the binding protein, and then forms an immune complex with the detection reagent based on the sandwich method, fixed to the solid phase, and a continuous and stable light signal is emitted by the electrochemiluminescence system of the fully automatic electrochemiluminescence instrument. The light signal calculates the concentration of the sample through the standard curve, and the signal value is proportional to the concentration. The embodiment of the present application solves the problem of low sensitivity of the 25-hydroxyvitamin D competition method well, and has a wide linear range, good repeatability, shortened reaction time, and better correlation than mass spectrometry results.

[0231] 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.

[0232] 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 and drawings can be used to explain the content of the claims.

Claims

1. A 25-hydroxyvitamin D detection kit, characterized in that: The detection kit comprises working reagent 1 and working reagent 2; wherein, The working reagent 1 includes R1 reagent and R2 reagent, wherein the R1 reagent includes a binding molecule 1-solid phase conjugate, the R2 reagent includes an antibody marker 1, the antibody marker 1 includes a binding molecule 2 and an antibody 1 that specifically binds to 25-hydroxyvitamin D, and the binding molecule 1 and the binding molecule 2 form a molecular binding pair; or, the working reagent 1 includes R3 reagent, wherein the R3 reagent includes an antibody-solid phase conjugate, and the antibody in the antibody-solid phase conjugate is an antibody 1 that specifically binds to 25-hydroxyvitamin D; The working reagent 2 includes an antibody marker 2, and the antibody marker 2 includes a tracer marker and an antibody 2 that specifically binds to 25-hydroxyvitamin D.

2. The 25-hydroxyvitamin D detection kit according to claim 1, characterized in that The solvent of the working reagent 2 includes a buffer and 0.01wt%-5wt% of a protective agent, 0.02wt%-1wt% of a surfactant, 0.05wt%-0.5wt% of a blocking agent, and 0.01wt%-5wt% of a preservative; wherein the buffer includes one or more of a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer, and a TES buffer; Optionally, in the solvent of the working reagent 2, the blocking agent includes anti-biotin antibody, MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, sheep IgG, goat IgG, HBR-5, HBR-7, TRU Block TM , HBR-1, HBR-X and P-heterophilic antibodies, and further optionally, the blocking agent includes anti-biotin antibody, HBR-5, HBR-7, TRU Block TM , at least one of HBR-1, HBR-X and MAK33 Poly, and one or more of P-heterophilic antibody, rabbit IgG, mouse IgG, bovine IgG, sheep IgG and goat IgG; Optionally, in the solvent of the working reagent 2, the protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, sodium chloride, sucrose, trehalose, lactose and mannitol; Optionally, in the solvent of the working reagent 2, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, Pluronic F-68, Pluronic F-127, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan; Optionally, in the solvent of the working reagent 2, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives and ProClin series preservatives; Optionally, the concentration of the antibody marker 2 in the working reagent 2 is 0.5 μg / mL-2.0 μg / mL.

3. The 25-hydroxyvitamin D detection kit according to claim 1, characterized in that The detection kit also includes a sample dissociation solution; The sample dissociation solution includes a buffer solution and 0.02wt%-1wt% of a surfactant, 1wt%-20wt% of a dissociation agent, and 0.01wt%-5wt% of a preservative; wherein the buffer solution includes one or more of a HEPES buffer solution having a pH value of 3.0-7.5 and a concentration of 0.01mol / L-0.2mol / L, a citric acid buffer solution, an acetate buffer solution, a phosphate buffer solution, a Bis-Tris buffer solution, a MOPSO buffer solution, a PIPES buffer solution, and a TES buffer solution; Optionally, in the sample dissociation solution, the dissociation agent includes one or more of 8-aniline-1-naphthalenesulfonic acid, dithiothreitol, mesna, tris(2-carbonylethyl)phosphine hydrochloride, methanol, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, perfluorobutyric acid, perfluorononanoic acid and perfluorooctanoic acid. Further optionally, the dissociation agent includes at least one of 8-aniline-1-naphthalenesulfonic acid, mesna, tris(2-carbonylethyl)phosphine hydrochloride, dimethyl sulfoxide and N,N-dimethylformamide, and one or more of dithiothreitol, methanol, ethanol, perfluorobutyric acid, perfluorononanoic acid and perfluorooctanoic acid. Optionally, in the sample dissociation solution, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450 and lauryl alcohol polyoxyethylene ether; Optionally, in the sample dissociation solution, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives and ProClin series preservatives.

4. The 25-hydroxyvitamin D detection kit according to any one of claims 1 to 3, characterized in that The solvent of the R1 reagent includes a buffer and 0.5wt%-5wt% of sodium chloride, 0.05wt%-5wt% of a sugar protective agent, 0.02wt%-1wt% of a surfactant, 0.05wt%-2wt% of a protein protective agent, and 0.05wt%-0.5wt% of a preservative; wherein the buffer includes one or more of a PBS buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a MES buffer, a HEPES buffer, a PIPES buffer, a TES buffer, a TAPSO buffer, and a MOPS buffer; Optionally, in the solvent of the R1 reagent, the sugar protective agent includes one or more of sucrose and trehalose; Optionally, in the solvent of the R1 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, TritonX-100 and Triton X-450; Optionally, in the solvent of the R1 reagent, the protein protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, glycerol, mannitol, sorbitol, alanine, glycine, sarcosine, L-tyrosine and phenylalanine; Optionally, in the solvent of the R1 reagent, the preservative includes one or more of proclin 300, Bronidox-L, gentamicin sulfate, NaN3 and KroVin series; Optionally, the concentration of the binding molecule 1-solid phase conjugate in the R1 reagent is 0.3 mg / mL-2 mg / mL.

5. The 25-hydroxyvitamin D detection kit according to any one of claims 1 to 3, characterized in that: The solvent of the R2 reagent includes a buffer and 0.01wt%-5wt% of a protective agent, 0.02wt%-1.5wt% of a surfactant, 0.05wt%-0.5wt% of a blocking agent, and 0.01wt%-5wt% of a preservative; wherein the buffer includes one or more of a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer, and a TES buffer; Optionally, in the solvent of the R2 reagent, the protective agent includes one or more of bovine serum albumin, plant-derived recombinant human serum albumin, sodium chloride, sucrose, trehalose and mannitol; Optionally, in the solvent of the R2 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, TritonX-100, Triton X-450, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan; Optionally, in the solvent of the R2 reagent, the blocking agent includes anti-biotin antibody, MAK33 Poly, rabbit IgG, mouse IgG, bovine IgG, sheep IgG, goat IgG, HBR-5, HBR-7, TRU Block TM , HBR-1, HBR-X and P-heterophilic antibodies; Optionally, in the solvent of the R2 reagent, the preservative includes one or more of Bronidox-L, gentamicin sulfate, NaN3, KroVin series preservatives and ProClin series preservatives; Optionally, the concentration of the antibody marker 1 in the R2 reagent is 0.5 μg / mL-2.0 μg / mL.

6. The 25-hydroxyvitamin D detection kit according to any one of claims 1 to 3, characterized in that: The solvent of the R3 reagent includes a buffer and 0.5wt%-5wt% of sodium chloride, 0.5wt%-5wt% of a sugar protective agent, 0.02wt%-1wt% of a surfactant, 0.05wt%-2wt% of a protein protective agent, and 0.05wt%-0.5wt% of a preservative; wherein the buffer includes one or more of a PBS buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a MES buffer, a HEPES buffer, a PIPES buffer, a TES buffer, a TAPSO buffer, and a MOPS buffer; Optionally, in the solvent of the R3 reagent, the sugar protective agent includes one or more of trehalose, sucrose and dextran; Optionally, in the solvent of the R3 reagent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, TritonX-100 and Triton X-450; Optionally, in the solvent of the R3 reagent, the protein protective agent includes one or more of bovine serum albumin and plant-derived recombinant human serum albumin, glycerol, mannitol, sorbitol, alanine, glycine, sarcosine, L-tyrosine and phenylalanine; Optionally, in the solvent of the R3 reagent, the preservative includes one or more of proclin 300, Bronidox-L, gentamicin sulfate, NaN3 and KroVin series; Optionally, the concentration of the antibody-solid phase conjugate in the R3 reagent is 0.3 mg / mL-2 mg / mL.

7. The 25-hydroxyvitamin D detection kit according to any one of claims 1 to 3, characterized in that: The detection kit meets one or more of the following conditions: (1) The solid phases in the binding molecule 1-solid phase conjugate and the antibody-solid phase conjugate are each independently magnetic beads with a diameter of 0.1 μm to 3 μm; (2) the molecular binding pairs include one or more of the following molecular binding pairs: streptavidin and biotin, fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody; (3) The tracer marker includes one or more of terpyridine ruthenium, horseradish peroxidase, alkaline phosphatase, acridinium ester, luminol and its derivatives, isoluminol and its derivatives, and adamantane.

8. The 25-hydroxyvitamin D detection kit according to any one of claims 1 to 3, characterized in that: The detection kit also includes calibrators and / or quality control products; The calibrator and the quality control product each independently include 25-hydroxyvitamin D antigen and an antigen solvent; Optionally, the antigen solvent includes a buffer and 1wt%-5wt% of a sugar protective agent, 0.02wt%-1wt% of a surfactant, and 0.05wt%-0.5wt% of a preservative; wherein the buffer includes one or more of a phosphate buffer having a pH value of 6.0-9.0 and a concentration of 0.01mol / L-0.2mol / L, a Bis-Tris buffer, a HEPES buffer, a MES buffer, a citric acid buffer, a MOPSO buffer, a PIPES buffer, and a TES buffer; Optionally, in the antigen solvent, the sugar protective agent includes one or more of sucrose, trehalose, lactose and mannitol; Optionally, in the antigen solvent, the surfactant includes one or more of Tween 20, Tween 80, Brij-35, Triton X-100, Triton X-450, lauryl alcohol polyoxyethylene ether, polyoxyethylene ether, dodecyl polyethylene glycol, polyethylene glycol, p-isooctylphenyl ether and polyoxyethylene sorbitan; Optionally, in the antigen solvent, the preservative includes proclin 300.

9. A method for detecting 25-hydroxyvitamin D, characterized in that: The detection method comprises: using the 25-hydroxyvitamin D detection kit according to any one of claims 1 to 8 to detect 25-hydroxyvitamin D in a sample to be tested.

10. The method for detecting 25-hydroxyvitamin D according to claim 9, characterized in that: The sample to be tested is serum or plasma.

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