A magnetic bead diluent and kit for the detection of 25-hydroxyvitamin D

Abstract

This invention discloses a magnetic bead diluent and kit for the detection of 25-hydroxyvitamin D. The magnetic bead diluent can dissociate 25(OH)D from vitamin D binding protein to a great extent while minimizing damage to the antibodies on the coated magnetic beads. When combined with enzyme-labeled working solution, it is used to prepare a 25(OH)D detection kit. The kit has the advantages of high low-end sensitivity, good specificity, wide linear range, high stability, and high accuracy in the detection of 25(OH)D.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more specifically, to a magnetic bead diluent and kit for the detection of 25-hydroxyvitamin D. Background Technology

[0002] Vitamin D is a fat-soluble steroid derived from cholesterol. Besides regulating calcium and phosphorus metabolism and maintaining bone health, it also participates in cell differentiation, proliferation, and immune regulation. Vitamin D deficiency is a global problem affecting all ages, with particularly pronounced impacts on children, pregnant women, and the elderly. The main causes are insufficient direct sunlight exposure and a lack of vitamin D in the diet. 25-Vitamin D deficiency is associated with an increased risk of various chronic diseases, including bone health, cardiovascular disease, diabetes, hypertension, certain cancers, autoimmune diseases, and infectious diseases. 25-hydroxyvitamin D (25(OH)D) is the main form of vitamin D and reflects vitamin D status; therefore, 25(OH)D testing is of great significance.

[0003] In the human body, approximately 85%–90% of 25(OH)D is bound to vitamin D-binding protein (VDBP) in the bloodstream, with a binding constant as high as Ka = 6 × 10⁻⁶. 5 M -1 10%–15% is bound to albumin (easily dissociated), with less than 1% free. 25(OH)D binds to VDBP in the following ways:

[0004] (1) Intermolecular interactions include hydrophobic interactions, hydrogen bonding interactions and ionic bonding interactions.

[0005] Hydrophobic interactions: The 25(OH)D molecule contains certain hydrophobic regions, and VDBP also has hydrophobic binding sites. Hydrophobic interactions play an important role in their binding, attracting 25(OH)D and VDBP together through mutual attraction between hydrophobic molecules. Hydrogen bonding: During the binding process, hydrogen bonds may form between 25(OH)D and VDBP. Hydrogen bonds are a weak intermolecular force formed between hydrogen atoms and atoms with higher electronegativity (such as oxygen and nitrogen). For example, the hydroxyl group (-OH) in the 25(OH)D molecule can form hydrogen bonds with certain amino acid residues on VDBP (such as asparagine and glutamine), further stabilizing their binding. Ionic bonding: Although this interaction is relatively weak, it may also participate in the binding of 25(OH)D and VDBP under certain conditions. For example, certain charged groups in the 25-hydroxyvitamin D molecule may attract oppositely charged groups on VDBP through ionic bonds, enhancing the binding affinity.

[0006] (2) Structural adaptability includes shape complementarity and the role of amino acid residues. Shape complementarity: The molecular shape of 25(OH)D is adapted to the shape of the binding pocket of VDBP. VDBP has a specific three-dimensional structure, which contains a binding pocket that can accommodate 25(OH)D. The shape and size of this binding pocket match the molecular structure of 25(OH)D, so that 25(OH)D can be inserted into it just like a key into a lock, achieving a tight binding. The role of amino acid residues: Specific amino acid residues on VDBP play a key role in the binding process. These amino acid residues can form a direct interaction with 25-hydroxyvitamin D, or regulate the binding by affecting the conformation of the protein.

[0007] (3) Dynamic nature of the binding process: Binding and dissociation equilibrium: The binding of 25(OH)D with VDBP is a dynamic equilibrium process. In the blood, 25(OH)D continuously binds to and dissociates from VDBP. This dynamic equilibrium is affected by a variety of factors, such as the concentration of 25(OH)D and VDBP, temperature, pH value, etc.

[0008] Detection of 25(OH)D is generally performed using a competitive assay. Since the detection of 25(OH)D includes both free and bound total 25(OH)D, dissociating 25(OH)D from VDBP is a crucial step. Therefore, related kits typically include dissociation agents in addition to standard reagent components. Common dissociation methods include strong acid / base treatment, organic solvent reaction, heavy metal ion reaction, enzymatic digestion, and salting out. For example, in the strong acid / base treatment method, after sample pretreatment, a second component is needed to neutralize the pH of the reagent to protect the antibody, a critical raw material in the reagent components, to prevent damage from the strong acid / base. Therefore, the reagent components are generally four or even five, including sample pretreatment solution, pH-adjusting buffer, coating component, labeling component, and antibody component. The strong acid / base treatment method involves many components, which is inconvenient for reagent production and increases costs, while also increasing the burden on instrument testing. Furthermore, strong acids and bases are highly corrosive and can cause damage to components of fully automated chemiluminescence analyzers. Organic solvent methods commonly use organic solvents such as acetone, mercaptoethanol, dimethyl sulfoxide, and lower alcohols. These solvents are more volatile than inorganic solvents, which can negatively impact reagent stability. Furthermore, some organic solvents are highly toxic, limiting their application. Heavy metal ion methods, enzymatic methods, and salting-out methods all work by disrupting vitamin-binding proteins to release 25(OH)D. However, antibodies, a key component of these reagents, are also proteins, requiring careful formulation and dosage adjustments. The dissociation agents in these methods are typically added during sample processing, making reagent formulation and usage procedures cumbersome. Therefore, it is necessary to develop reagents that are easier to detect.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The purpose of this invention is to provide a magnetic bead diluent and kit for the detection of 25-hydroxyvitamin D (25(OH)D). The kit prepared with this magnetic bead diluent not only has simple components, but also has high sensitivity and specificity for the detection of 25(OH)D, and at the same time has high reagent stability.

[0011] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0012] In a first aspect, the present invention provides a magnetic bead diluent for 25(OH)D detection, comprising a steroid, a protein denaturant, and a reducing agent;

[0013] Among them, steroids include pregnenolone; protein denaturants include thiourea and guanidine hydrochloride; reducing agents include L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride.

[0014] To dissociate 5(OH)D from VDBP, this invention adds a steroid substance to the magnetic bead diluent, causing it to replace 25(OH)D in binding with VDBP, thereby releasing the analyte 25(OH)D. The steroid substance can be pregnenolone or other common steroid substances, but pregnenolone is preferred.

[0015] In this invention, the protein denaturant guanidine hydrochloride causes VDBP protein to randomly coil by forming and dissociating peptide bonds in the protein structure, resulting in the protein losing its active structure and undergoing reversible denaturation. Thiourea also has the ability to denature proteins, interacting with hydrogen bonds and hydrophobic interactions in protein molecules, thus disrupting the structural stability of proteins. Although the denaturing ability of thiourea is relatively weak, it can still effectively denature proteins in certain situations; moreover, experiments have shown that thiourea and guanidine hydrochloride have a synergistic effect, enabling a more complete dissociation of 25(OH)D from VDBP.

[0016] L-cysteine ​​and tris(2-carboxyethyl)phosphonic acid hydrochloride are reducing agents that can prevent 25(OH)D from being oxidized, thus providing protection.

[0017] In some embodiments, the magnetic bead diluent includes 1-10 mM pregnenolone. Specifically, the concentration of pregnenolone can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM, or any value between 1 and 10 mM.

[0018] In some embodiments, the magnetic bead diluent includes 10-100 mM thiourea and 10-100 mM guanidine hydrochloride. Specifically, the concentration of thiourea can be 10 mM, 20 mM, 30 mM, 40 mM, 5 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM, or any value between 10 and 100 mM; the concentration of guanidine hydrochloride can be 10 mM, 20 mM, 30 mM, 40 mM, 5 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM, or any value between 10 and 100 mM.

[0019] In some embodiments, the magnetic bead diluent includes 1-10 mM L-cysteine ​​and 1-10 mM tris(2-carboxyethyl)phosphine hydrochloride. Specifically, the concentration of L-cysteine ​​can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM, or any value between 1 and 10 mM; the concentration of tris(2-carboxyethyl)phosphine hydrochloride can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM, or any value between 1 and 10 mM.

[0020] In some embodiments, the magnetic bead diluent further includes a buffer solution.

[0021] In some embodiments, the buffer includes, but is not limited to, tris(hydroxymethyl)aminomethane buffer, and may also be other conventional buffers in the art, such as HEPES buffer, PB buffer, PBS buffer, Tris buffer, or phosphate buffer.

[0022] In some embodiments, the magnetic bead diluent may also include one or more of the following: protective proteins, surfactants, chelating agents, salts, and preservatives.

[0023] In some embodiments, the protective proteins include, but are not limited to, fetal bovine serum, calf serum, horse serum, bovine serum albumin, human serum albumin, and casein.

[0024] In some embodiments, the surfactants include, but are not limited to, Triton series surfactants, Tween series surfactants, and polyethylene glycol surfactants.

[0025] In some embodiments, the chelating agent includes, but is not limited to, disodium EDTA, and may also be other conventional chelating agents in the art.

[0026] In some embodiments, the salt includes, but is not limited to, sodium chloride, and may also be other conventional salts in the art, such as KCl, NH4Cl, MgCl2, MgSO4 and CaCl2.

[0027] In some embodiments, the preservative includes, but is not limited to, Proclin 300, and may also be other conventional preservatives in the art, such as sodium azide.

[0028] In some embodiments, the magnetic bead diluent further includes 50-200 mM of pH 7.4 tris(hydroxymethyl)aminomethane buffer, 10-200 g / L serum albumin, 1-20 v / v% animal serum, 0.1-1 v / v% Triton series surfactants or Tween series surfactants, 5-10 g / L polyethylene glycol surfactant, 1-10 mM chelating agent, 50-150 mM salt, and preservative.

[0029] The concentration of the tris(hydroxymethyl)aminomethane buffer can be, but is not limited to, 50 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM and 200 mM, or any value between 50 and 200 mM.

[0030] The concentration of the protective protein can be, but is not limited to, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 5%, 8%, 10%, 15%, and 20%, or any value between 0.5% and 20%.

[0031] The concentration of the surfactant can be, but is not limited to, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%, or any value between 0.1% and 1%.

[0032] The concentration of the chelating agent can be, but is not limited to, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM and 10 mM, or any value between 1 and 10 mM.

[0033] The salt concentration can be, but is not limited to, 50 mM, 60 mM, 80 mM, 100 mM, 120 mM and 150 mM, or any value between 50 and 150 mM.

[0034] In a second aspect, the present invention provides a magnetic bead working solution, comprising the above-mentioned magnetic bead diluent and a magnetic bead-protein complex, wherein the magnetic bead-protein complex is prepared by coating magnetic beads with a protein-capturing agent.

[0035] Thirdly, the present invention provides a kit for the detection of 25-hydroxyvitamin D, comprising: the above-mentioned magnetic bead diluent or a magnetic bead working solution prepared from the above-mentioned magnetic bead diluent and magnetic beads.

[0036] In some embodiments, the concentration of magnetic beads in the working solution is 0.2-0.5 mg / mL. Specifically, the concentration of magnetic beads in the prepared working solution can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, or 0.5 mg / mL, or any value between 0.2-0.5 mg / mL.

[0037] In some embodiments, the kit may further include one or more of the following: coated antibody, magnetic beads, coating solution, blocking solution, sample diluent, washing solution, enzyme-labeled antibody, enzyme-labeled diluent, enzyme-labeled working solution, and standards / quality control products.

[0038] The detection principle of the test kit prepared in this invention differs from common competitive methods; it employs a small-molecule sandwich method. Competitive methods are reagent-limited, generally struggling to meet low-end sensitivity and linearity requirements. The sandwich method, however, utilizes sufficient reagents, offering significant advantages in both low-end sensitivity and linearity. The sandwich method kit comprises two main components: a capture antibody coated on magnetic beads and a labeled detection antibody. The analyte in the sample (25-hydroxyvitamin D in this invention) binds to the capture antibody on the magnetic beads, forming a "magnetic bead-capture antibody-25-hydroxyvitamin D" complex. After washing to remove non-specific bindings, the labeled detection antibody is further added, forming a "magnetic bead-capture antibody-25-hydroxyvitamin D-detection antibody-label" complex. The concentration of the analyte in the sample can be determined by detecting the amount of the labeled antibody (the amount of the labeled antibody is positively correlated with the concentration of the analyte in the sample within a certain range).

[0039] Fourthly, the present invention provides the use of the above-mentioned magnetic bead diluent, magnetic bead working solution, or kit in the following (I) or (II):

[0040] (I) Detection of 25-hydroxyvitamin D for non-diagnostic or therapeutic purposes, using the sandwich method;

[0041] (II) Preparation of a product for detecting 25-hydroxyvitamin D, wherein the product is tested for 25-hydroxyvitamin D by sandwich method.

[0042] The present invention has the following beneficial effects:

[0043] This invention designs a novel magnetic bead diluent using pregnenolone, thiourea, guanidine hydrochloride, L-cysteine, and tris(2-carboxyethyl)phosphonic acid hydrochloride as the main components. This diluent effectively dissociates 25(OH)D from vitamin D-binding proteins while minimizing damage to the antibodies on the coated magnetic beads, thus eliminating the need for additional sample processing and simplifying and accelerating the detection process. Furthermore, the magnetic bead diluent exhibits good detection performance, meeting 25(OH)D detection standards, and possesses advantages such as good reactivity, low detection limit (1 ng / mL), excellent linearity (|r| = 0.997 in the range of 1–10 ng / mL), good repeatability (CV < 5%), and high accuracy (relative deviation not exceeding ±15%). The working solution prepared with the magnetic bead diluent also demonstrates good stability, with a thermal stability degradation of less than 10%, and can be effectively carried on air for 30 days. This is of great significance for more effective and convenient detection of serum 25(OH) vitamin D. Attached Figure Description

[0044] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 Clinical comparative linear analysis results of reagents prepared with different magnetic bead diluents. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0047] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0048] Example 1

[0049] Because the molecular weight of 25(OH)D is very small, it is difficult for two epitopes to simultaneously bind to two antibodies. Therefore, the sandwich method used in this kit is a special sandwich method. The specific principle is as follows: magnetic beads are coated with anti-25(OH)D antibody (coating antibody), and an antibody (enzyme label) against the "anti-25(OH)D antibody-25(OH)D antigen" complex is labeled with alkaline phosphatase (AP). This antibody complex is characterized by binding only to the "VD antibody and VD antigen complex," not to the anti-25(OH)D antibody alone. Therefore, if 25(OH)D (the analyte) is present in the sample, it will form an immune complex with the coating antibody on the magnetic beads, exhibiting an "anti-25(OH)D antibody-25(OH)D antigen" structure. Washing removes unbound analyte, and then adding the enzyme label forms an immune complex with a "coating antibody-analyte-enzyme label" structure. Unbound enzyme-labeled material is removed by washing, followed by the addition of substrate solution. The substrate then emits light under the catalysis of the enzyme. The light emission intensity is detected, and it is positively correlated with the concentration of the analyte in the sample within a certain range.

[0050] This embodiment describes a kit for the detection of 25-hydroxyvitamin D and its preparation method. The kit includes a magnetic bead working solution and an enzyme-labeled working solution; the specific preparation methods for the magnetic bead working solution and the enzyme-labeled working solution are as follows:

[0051] 1. Magnetic bead working fluid

[0052] S1. Preparation of the basic solution:

[0053] 1) Add 500 mL of pure water to the reaction vessel;

[0054] 2) Weigh 6.06g of tris(hydroxymethyl)aminomethane into a container and mix well; adjust the pH to 7.4±0.1 at room temperature;

[0055] 3) Weigh out 10g of bovine serum albumin, 5g of polyethylene glycol 6000, 8.77g of sodium chloride, and 3.72g of disodium ethylenediaminetetraacetate and add them to a container, then mix well;

[0056] 4) Finally, measure 200 mL of fetal bovine serum, 5 mL of Triton X100, and 0.5 mL of preservative Proclin 300 into a container, mix well, and bring the volume up to 1 L. Store at 2-8℃.

[0057] S2. Preparation of magnetic bead diluent:

[0058] Prepare 100 mL of basic solution and add it to a suitable container. Add 0.316 g of pregnenolone, 0.095 g of guanidine hydrochloride, 0.076 g of thiourea, 0.135 g of L-cysteine, and 0.25 g of tris(2-carboxyethyl)phosphonic acid hydrochloride, mix well, and sonicate to homogenize to ensure that all compounds are dissolved. Finally, filter through a 0.22 μm filter and store at 2-8 °C.

[0059] S3. Magnetic bead coating:

[0060] 1) Washing before coating: Take an appropriate amount of magnetic beads (JSR, Tosyl MS160) and wash 3 times with PBS buffer;

[0061] 2) Magnetic bead coating: After resuspending the magnetic beads in reaction buffer, add an appropriate amount of coating antibody (Bioventix-anti-25-hydroxyvitamin D monoclonal antibody-catalog number vitD3.5H10), then place it on a rotary mixer and incubate in a constant temperature incubator. Incubation conditions: temperature: 37±1.5℃, rotation speed 30 times / min, time 16-24h.

[0062] 3) Washing before sealing: Wash the magnetic beads 3 times with pre-sealing washing solution;

[0063] 4) Magnetic bead blocking: After adding an appropriate amount of magnetic bead blocking solution, place it on a rotary mixer and incubate it in a constant temperature incubator. Incubation conditions: temperature: 37±1.5℃, rotation speed 30 times / min, time 16-24h.

[0064] 5) Washing after sealing: Wash the magnetic beads 3 times with the sealing washing solution.

[0065] S4. Preparation of magnetic bead working solution:

[0066] Dilute the magnetic beads to 0.2-0.5 mg / mL using the obtained magnetic bead diluent to prepare a magnetic bead working solution, and store it at 2-8℃.

[0067] 2. Enzyme-labeled working solution

[0068] Commercially available alkaline phosphatase-labeled anti-25(OH)D antibody (Zhuhai Lihe Medical Diagnostic Products Co., Ltd., catalog number: VD-AP, which specifically binds to the complex composed of the coated antibody and 25(OH)D) was dissolved in enzyme-labeled diluent (enzyme-labeled diluent pH 6.5, containing 0.1 mol / L morpholine ethanesulfonic acid buffer, 3% glycerol, 20 g / L bovine serum albumin, and 15 g / L casein sodium salt) to prepare enzyme-labeled working solution with a final concentration of 50-200 ng / ml. The solution was filtered through a 0.22 μm filter and stored at 2-8℃.

[0069] The kit in this embodiment detects 25-hydroxyvitamin D in samples using a sandwich method.

[0070] Comparative Example 1

[0071] The difference from Example 1 is that 0.316g of pregnenolone was replaced with 0.096g of ergosterol, while the rest of the formulation remained the same.

[0072] Comparative Example 2

[0073] The difference from Example 1 is that 0.316g of pregnenolone was replaced with 0.384g of 7-dehydrocholesterol, while the rest of the formula remained the same.

[0074] Comparative Example 3

[0075] The difference from Example 1 is that it does not contain pregnenolone, but the rest of the formulation is the same.

[0076] Comparative Example 4

[0077] The difference from Example 1 is that it does not contain thiourea, but the rest of the formulation is the same.

[0078] Comparative Example 5

[0079] The difference from Example 1 is that it does not contain guanidine hydrochloride, but the rest of the formulation is the same.

[0080] Comparative Example 6

[0081] The difference from Example 1 is that it does not contain 0.076g thiourea and 0.095g guanidine hydrochloride, but the rest of the formulation is the same.

[0082] Comparative Example 7

[0083] The difference from Example 1 is that it does not contain L-cysteine, but the rest of the formulation is the same.

[0084] Comparative Example 8

[0085] The difference from Example 1 is that it does not contain tris(2-carboxyethyl)phosphine hydrochloride, but the rest of the formulation is the same.

[0086] Comparative Example 9

[0087] The difference from Example 1 is that it does not contain tris(2-carboxyethyl)phosphine hydrochloride and L-cysteine, but the rest of the formulation is the same.

[0088] Comparative Example 10

[0089] The difference from Example 1 is that it does not contain tris(2-carboxyethyl)phosphine hydrochloride and L-cysteine, and 0.307g of glutathione is added, while the rest of the formulation is the same.

[0090] Comparative Example 11

[0091] The difference from Example 1 is that it does not contain tris(2-carboxyethyl)phosphine hydrochloride and L-cysteine, and 0.154g of dithiothreitol is added, while the rest of the formulation is the same.

[0092] Example 2

[0093] This embodiment verifies the effect of the main components of the magnetic bead diluent on reactivity. Specifically, the luminescence values ​​of samples of different concentrations (prepared by diluting pure products with serum) from Example 1 and Comparative Examples 1-11 were measured. The results are shown in Table 1.

[0094] Table 1. Results of luminescence values ​​of reagent samples prepared with different magnetic bead dilution formulations at gradient concentrations.

[0095]

[0096] Based on the principle of immune response, the higher the sample concentration, the higher the luminescence value. Analysis of the above data shows that: Example 1 and Comparative Examples 1, 2, 4, 5, 7, 8, 10, and 11 all show that the higher the sample concentration, the higher the luminescence value.

[0097] Analysis of Comparative Example 3 shows that the formulation does not contain steroids. The main function of steroids is to replace the binding of the dissociated 25(OH)D with VDBP. It is speculated that the reason why Comparative Example 3 is unreactive is mainly due to the lack of a 25(OH)D substitute. Even if 25(OH)D separates from VDBP in a short time, it is in dynamic equilibrium. Due to the strong affinity between the two, they can still continue to bind. Although Comparative Examples 1 and 2 showed reactivity, it was significantly lower than that of Comparative Example 1. Therefore, pregnenolone was selected as a suitable substitute for 25(OH)D to participate in the reaction. Analysis of Comparative Example 6 showed that the formulation did not contain substances that damage proteins and could not separate 25(OH)D from VDBP, so the data showed no reactivity. Analysis of Comparative Example 9 showed that the formulation did not contain a reducing agent to protect 25(OH)D, and since 25(OH)D is easily oxidized, the data showed no reactivity. However, when the reducing agents L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride were replaced with glutathione or dithiothreitol (Comparative Examples 10 and 11), there was reactivity, but it was significantly weaker than that of Example 1, Comparative Examples 7 and 8.

[0098] In summary, based on the reactivity comparison data of 12 sets, Example 1 and Comparative Examples 4, 5, 7, and 8 can be considered as preferred formulations, and further research should be conducted on the clinical correlation and stability of the reagents.

[0099] Example 3

[0100] This example verifies the effect of the main components of the magnetic bead diluent on the correlation of clinical comparisons. A total of 30 clinical serum samples were collected, and their 25-hydroxyvitamin D concentrations were measured by mass spectrometry as reference concentrations. The 25-hydroxyvitamin D concentrations of each sample were further measured using the kits from Example 1 and Comparative Examples 4, 5, 7, and 8, and the correlation was calculated. The results are shown in Table 2. Figure 1 As shown;

[0101] Table 2. Results of clinical sample tests using reagents prepared with different magnetic bead diluent formulations.

[0102]

[0103] The results analysis showed that the formulations with a correlation coefficient (r) greater than 0.90 for the five magnetic bead diluent formulations were Example 1, Comparative Example 7, and Comparative Example 8, with Example 1 showing a correlation coefficient (r) of 0.972. Comparative Examples 4 and 5, however, had correlation coefficients (r) less than 0.90. Compared to Example 1, Comparative Examples 4 and 5 contained less thiourea or guanidine hydrochloride, respectively. This suggests that thiourea and guanidine hydrochloride may work synergistically to achieve a more complete dissociation of 25(OH)D from VDBP. Therefore, Example 1, Comparative Example 7, and Comparative Example 8 were preferred for stability testing to further screen the formulations.

[0104] Example 4

[0105] This embodiment verifies the influence of the main components of the magnetic bead diluent on stability.

[0106] The stability of the three formulations selected in Example 3 was evaluated, starting with thermal stability: the reagents were divided into two portions. One portion was placed in a 37°C oven for 7 days, and then placed in a 2-8°C refrigerator for at least 4 hours to equilibrate. The other portion remained in the 2-8°C refrigerator. After the expiration period, the decrease in luminescence values ​​of the gradient concentration samples before and after thermal acceleration was measured. The results of thermal acceleration are shown in Table 3 below:

[0107] Table 3. Results of luminescence values ​​of reagents prepared with different dilution formulations after 7 days of thermal acceleration.

[0108]

[0109] Analysis of the thermal acceleration results showed that Example 1 met the requirement of a reduction of less than 10%, which was better than Comparative Examples 7 and 8. Compared with Example 1, Comparative Examples 7 and 8 added less L-cysteine ​​or tris(2-carboxyethyl)phosphine hydrochloride, respectively. It is possible that the two played a synergistic role, making the reagent more stable.

[0110] Furthermore, airborne stability testing was conducted on Example 1 using the following method: Reagents were loaded into the corresponding reagent compartments of the instrument (the reagent compartments were at 2-8°C). The same sample was measured at specified time intervals (0 days, 7 days, 14 days, 21 days, and 30 days), and the deviation of the measured value from that on day 0 was calculated. The test results are shown in Table 4 below.

[0111] Table 4 Results of Airborne Light Values ​​under Different Weather Conditions in Example 1

[0112]

[0113] As can be seen from the airborne stability results of Example 1, the reagent kit of Example 1 showed a decrease of no more than 10% after 7, 14, 21 and 30 days of airborne operation, respectively. It can be considered that the reagent can be airborne for 30 days and has good stability.

[0114] Example 5

[0115] The performance indicators of the reagent kit in Example 1 were further tested, as follows:

[0116] 1. Limit of detection

[0117] Five detection limit reference samples (concentration of approximately 1 ng / mL) were tested, with each sample tested five times.

[0118] Table 5 shows the detection results for the detection limit.

[0119]

[0120] Based on the results in Table 5, there were 0 detection results below the blank limit of 0.5 ng / mL, therefore the detection limit can be considered to be 1 ng / mL.

[0121] 2. Accuracy:

[0122] Reference samples C1 and C2 (concentrations shown in Table 6) were tested separately, with each sample tested three times. The relative deviation of each test result was calculated, and the results are shown in Table 6.

[0123] Table 6. Detection Accuracy Results

[0124]

[0125] As shown in Table 6, the relative deviation of the kit in Example 1 does not exceed ±15%.

[0126] 3. Linear:

[0127] The linear samples L1 to L7 were tested, with each sample tested three times, and the linear correlation was calculated. The results are shown in Table 7.

[0128] Table 7 Results of linear correlation coefficient detection

[0129]

[0130]

[0131] As shown in Table 7, the linear correlation coefficient |r| of the kit in Example 1 is not less than 0.990 in the concentration range of [1,150] ng / mL.

[0132] 4. Repeatability:

[0133] The repeatability of the reagents was examined by testing low-value and high-value samples of the repeatability reference material, with each sample tested 10 times. The results are shown in Table 8.

[0134] Table 8 Repeatability Test Results

[0135] Number of tests Low value of repeatability reference (ng / mL) High value of repeatability reference (ng / mL) 1 19.97 38.47 2 20.62 37.90 3 19.98 38.84 4 19.64 38.26 5 19.28 35.14 6 19.74 37.29 7 20.15 37.22 8 18.69 37.11 9 17.60 36.34 10 19.01 35.24 mean 19.47 37.18 Standard deviation 0.87 1.28 Coefficient of variation (CV) 4.5％ 3.4％

[0136] As shown in Table 8, the coefficient of variation (CV) of the results obtained from the test of repeatability reference is less than 5%, which meets the requirement of not exceeding 8%.

[0137] Example 6

[0138] This embodiment focuses on the basic solution study, as detailed below:

[0139] The base solution for magnetic bead diluents includes buffer, protective protein, surfactant, chelating agent, salt, and preservative. Common components were arbitrarily replaced and component concentrations adjusted to create different base buffer formulations (see Table 9 for specific formulations). The effect of each base buffer formulation on reagent reactivity was investigated. After preparing the base buffer according to the formulation, 100 mL of the base solution was added to a suitable container, and 0.316 g of pregnenolone, 0.095 g of guanidine hydrochloride, 0.076 g of thiourea, 0.135 g of L-cysteine, and 0.25 g of tris(2-carboxyethyl)phosphonic acid hydrochloride were added. After mixing, the mixture was sonicated to homogenize, ensuring all compounds were dissolved. Finally, it was filtered through a 0.22 μm filter and stored at 2–8 °C.

[0140] Following the method in Example 1, the corresponding magnetic bead working solution was finally prepared and tested with the same enzyme-labeled working solution. The reactivity of gradient samples was measured on the instrument, and the results are shown in Table 10.

[0141] Table 9 Basic Solution Formulation

[0142]

[0143] Table 10. Reactivity results of reagents prepared with different base solution formulations.

[0144] Sample concentration (ng / mL) Formula 1-RLU Formula 2-RLU Formula 3-RLU Formula 4-RLU Formula 5-RLU 0 781 804 778 757 785 10 9596 9788 9313 9341 9784 20 28455 28591 28102 28770 28947 50 172322 171873 168117 167548 171942 100 677625 687518 685078 662988 683926 150 1275919 1262394 1281405 1242872 1295950

[0145] The analysis results show that, compared with formulation 1, the reactivity of formulations 2-5 changes less. Therefore, routine adjustments to the base solution compounds (adjusting the concentration appropriately or replacing them with other common substitutes) have little impact on reagent reactivity.

[0146] Example 7

[0147] This example studies the concentration range of the main components in the magnetic bead diluent, as detailed below:

[0148] The amounts of pregnenolone, thiourea, guanidine L-cysteine ​​hydrochloride, and tris(2-carboxyethyl)phosphine hydrochloride in the formulation were changed (the remaining components remained unchanged, and the basic solution formulation used the basic solution of Example 1, see Table 11) to study the effect of the concentration of relevant components on the reactivity of the reagent. The results of the gradient sample reactivity measured by the instrument are shown in Table 12.

[0149] Table 11 Magnetic bead diluent formulation

[0150] Formula 1 Formula 6 Formula 7 Formula 8 Basic solution formulation 1 / mL 100 100 100 100 Pregnenolone / g 0.316 0.032 0.158 0.316 Guanidine hydrochloride / g 0.095 0.95 0.425 0.095 Thiourea / g 0.076 0.076 0.38 0.76 L-cysteine / g 0.135 0.014 0.067 0.135 Tris(2-carboxyethyl)phosphonic hydrochloride / g 0.25 0.25 0.125 0.025

[0151] Table 12. Reagent reactivity results prepared with different magnetic bead diluent formulations.

[0152] Sample concentration (ng / mL) Formula 1-RLU Formula 6-RLU Formula 7-RLU Formula 8-RLU 0 781 768 794 804 10 9596 9778 9455 9833 20 28455 29075 27612 28685 50 172322 175441 169806 171994 100 677625 666376 674033 687518 150 1275919 1238662 1309858 1291230

[0153] The analysis results showed that, compared with formulation 1, the reactivity of formulations 6-8 changed less. Therefore, limiting the concentration of the main components of the magnetic bead diluent within the specified range (1-10mM pregnenolone, 10-100mM thiourea, 10-100mM guanidine hydrochloride, 1-10mM L-cysteine, 1-10mM tris(2-carboxyethyl)phosphonic acid hydrochloride) had little impact on the reagent reactivity.

[0154] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A magnetic bead diluent for the detection of 25-hydroxyvitamin D, characterized in that, Including steroids, protein denaturants, and reducing agents; The steroids include pregnenolone; The protein denaturing agents include thiourea and guanidine hydrochloride; The reducing agent includes L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride.

2. The magnetic bead diluent according to claim 1, characterized in that, The magnetic bead diluent contains 1-10 mM pregnenolone.

3. The magnetic bead diluent according to claim 1, characterized in that, The magnetic bead diluent contains 10-100 mM thiourea and 10-100 mM guanidine hydrochloride.

4. The magnetic bead diluent according to claim 1, characterized in that, The magnetic bead diluent contains 1-10 mM L-cysteine ​​and 1-10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride.

5. The magnetic bead diluent according to claim 1, characterized in that, The magnetic bead diluent also includes a buffer solution.

6. The magnetic bead diluent according to claim 5, characterized in that, The buffer solution includes tris(hydroxymethyl)aminomethane buffer.

7. The magnetic bead diluent according to claim 1, characterized in that, The magnetic bead diluent also includes one or more of the following: protective proteins, surfactants, chelating agents, salts, and preservatives.

8. The magnetic bead diluent according to claim 7, characterized in that, The protective protein includes at least one of fetal bovine serum, calf serum, horse serum, bovine serum albumin, human serum albumin, and casein.

9. The magnetic bead diluent according to claim 7, characterized in that, The surfactant includes at least one of the Triton series surfactants, the Tween series surfactants, and polyethylene glycol surfactants.

10. The magnetic bead diluent according to claim 7, characterized in that, The chelating agent includes disodium ethylenediaminetetraacetate.

11. The magnetic bead diluent according to claim 7, characterized in that, The salt includes sodium chloride.

12. The magnetic bead diluent according to claim 7, characterized in that, The preservative includes Proclin 300.

13. The magnetic bead diluent according to claim 7, characterized in that, The magnetic bead diluent also includes 50-200 mM of pH 7.4 tris(hydroxymethyl)aminomethane buffer, 10-200 g / L serum albumin, 1-20 v / v% animal serum, 0.1-1 v / v% Triton series surfactants or Tween series surfactants, 5-10 g / L polyethylene glycol surfactant, 1-10 mM chelating agent, 50-150 mM salt, and preservatives.

14. A magnetic bead working fluid, characterized in that, The invention includes the magnetic bead diluent and the magnetic bead-protein complex as described in any one of claims 1-13, wherein the magnetic bead-protein complex is prepared by coating magnetic beads with a protein-capturing agent.

15. The magnetic bead working fluid according to claim 14, characterized in that, The capture protein is an anti-25(OH)D antibody.

16. A kit for the detection of 25-hydroxyvitamin D, characterized in that, include: The magnetic bead diluent according to any one of claims 1-13 or the magnetic bead working solution according to any one of claims 14-15.

17. The kit according to claim 16, characterized in that, The kit also includes one or more of the following: coated antibody, magnetic beads, coating solution, blocking solution, sample diluent, washing solution, enzyme-labeled antibody, enzyme-labeled diluent, enzyme-labeled working solution, and standards / quality control products.

18. The use of the magnetic bead diluent according to any one of claims 1-13, the magnetic bead working solution according to any one of claims 14-15, or the kit according to any one of claims 16-17 in the following (I) or (II): (I) Detection of 25-hydroxyvitamin D for non-diagnostic or therapeutic purposes, wherein the detection method is the sandwich method; (II) Preparation of a product for detecting 25-hydroxyvitamin D, wherein the product is tested for 25-hydroxyvitamin D by a sandwich method.

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