Magnetic bead diluent and kit for detecting 25-hydroxyvitamin D

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a magnetic bead diluent and a kit for detecting 25-hydroxyvitamin D. Background Art

[0002] Vitamin D is a fat-soluble steroid derived from cholesterol. In addition to regulating calcium and phosphorus metabolism and maintaining bone health, it is also involved in the differentiation, proliferation and immune regulation of tissue cells. Vitamin D deficiency is a global problem that affects people of all ages, especially children, pregnant women and the elderly. The main reasons are insufficient direct sunlight exposure and lack of vitamin D in the diet. 25-Vitamin D deficiency is associated with an increased risk of a variety of 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 the status of vitamin D. Therefore, 25(OH)D testing is of great significance.

[0003] In the human body, about 85% to 90% of 25(OH)D is bound to vitamin D binding protein (VDBP) in the blood circulation, with a binding constant of up to Ka = 6×10 5 M -1 , 10% to 15% is bound to albumin (easily dissociated), and the free portion is less than 1%. 25(OH)D and VDBP are combined in the following ways:

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

[0005] Hydrophobic interaction: 25(OH)D molecules contain certain hydrophobic regions, and VDBP also has hydrophobic binding sites. Hydrophobic interaction plays an important role in the binding of the two. Through the mutual attraction between hydrophobic molecules, 25(OH)D and VDBP are brought close to each other and bind. Hydrogen bonding: During the binding process, hydrogen bonds may form between 25(OH)D and VDBP. Hydrogen bonds are a weak intermolecular interaction force formed between hydrogen atoms and atoms with high electronegativity (such as oxygen, nitrogen, etc.). 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, glutamine, etc.), further stabilizing the binding of the two. Ionic bonding: Although this effect 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, thereby enhancing the affinity of the binding.

[0006] (2) Structural adaptability includes shape complementarity and the role of amino acid residues. Shape complementarity: The molecular shape of 25(OH)D is compatible with 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, allowing 25(OH)D to fit neatly into it, just like a key inserted into a lock, achieving 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 binding by affecting the conformation of the protein.

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

[0008] The detection of 25(OH)D is generally carried out by a competitive method, and since the detection of 25(OH)D includes the total 25(OH)D of free and bound, the dissociation of 25(OH)D from VDBP is a key step in the detection. Therefore, its related kits usually include a dissociation agent in addition to conventional reagent components. General dissociation methods include strong acid and strong alkali treatment, organic solvent action, heavy metal ion action, enzymatic hydrolysis, salting out, etc. For example, after the sample is pretreated by the strong acid and strong alkali treatment method, in order to prevent the strong acid and strong alkali from destroying the key raw material antibody in the reagent component, a second component is required to neutralize the pH of the reagent to protect the antibody. Therefore, the reagent components are generally set to four or even five components, including sample pretreatment liquid, pH adjustment buffer, coating component, labeling component, and antibody component. The strong acid and strong alkali treatment method has many components, which is inconvenient for reagent production and will increase the cost, and add a burden to the instrument test. In addition, strong acids and strong alkalis are highly corrosive and will also cause damage to the components of the fully automatic luminometer. The organic solvents commonly used in the organic solvent method include acetone, mercaptoethanol, dimethyl sulfoxide, lower alcohols, etc., which are more volatile than inorganic solvents and have side effects on the stability of the reagents. In addition, some organic solvents are highly toxic, which limits the application of the reagents. The principles of the heavy metal ion method, enzymatic hydrolysis method, and salting-out method are all to release 25(OH)D by destroying vitamin-binding proteins, but the key component of the reagent, antibodies, are also proteins, so the formula and dosage need to be carefully coordinated. The dissociation agents in the above methods are generally added during the sample processing stage, and the reagent component formulation method and usage steps are relatively cumbersome, so it is necessary to develop reagent components that are easier to detect.

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

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

[0011] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[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, the steroid includes pregnenolone; the protein denaturing agent includes thiourea and guanidine hydrochloride; and the reducing agent includes L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride.

[0014] In order to dissociate 5(OH)D from VDBP, the present invention adds a steroid substance to the magnetic bead diluent, so that it replaces 25(OH)D and binds to VDBP, thereby releasing the analyte 25(OH)D. The steroid substance can be pregnenolone or other common steroid substances, but pregnenolone is more preferred.

[0015] In the present invention, the protein denaturant guanidine hydrochloride forms and dissociates peptide bonds in the protein structure to cause random curling of the VDBP protein, causing the protein to lose its active structure and undergo reversible denaturation. Thiourea also has the ability to denature the protein and can interact with hydrogen bonds and hydrophobic interactions in the protein molecules to destroy the structural stability of the protein. Although the denaturation ability of thiourea is relatively weak, it can still effectively denature the protein in some cases; and it is found in experiments that thiourea and guanidine hydrochloride have a synergistic effect, which can make the dissociation of 25(OH)D and VDBP more thorough.

[0016] L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride are reducing agents that can prevent 25(OH)D from being oxidized and play a protective role.

[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 1-10 mM.

[0018] In some embodiments, the magnetic bead diluent includes 10-100mM thiourea and 10-100mM guanidine hydrochloride. Specifically, the concentration of thiourea can be 10mM, 20mM, 30mM, 40mM, 5m0M, 60mM, 70mM, 80mM, 90mM and 100mM, or any value between 10-100mM; the concentration of guanidine hydrochloride can be 10mM, 20mM, 30mM, 40mM, 5m0M, 60mM, 70mM, 80mM, 90mM and 100mM, or any value between 10-100mM.

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

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

[0021] In some embodiments, the buffer includes but is not limited to Tris 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 further comprises one or more of a protective protein, a surfactant, a chelating agent, a salt and a preservative.

[0023] In some embodiments, 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 surfactant includes, but is 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 ethylenediaminetetraacetate, 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 also includes 50-200mM Tris buffer at pH 7.4, 10-200g / L serum albumin, 1-20v / v% animal serum, 0.1-1v / v% Triton series surfactant or Tween series surfactant, 5-10g / L polyethylene glycol surfactant, 1-10mM chelating agent, 50-150mM salt and preservative.

[0029] The concentration of the Tris buffer may 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 50-200 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 0.5-20%.

[0031] The concentration of the surfactant may 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%, and may be any value between 0.1-1%.

[0032] The concentration of the chelating agent may 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, and may be any value between 1 and 10 mM.

[0033] The concentration of the salt may be, but is not limited to, 50 mM, 60 mM, 80 mM, 100 mM, 120 mM and 150 mM, or any value between 50-150 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 capture proteins.

[0035] In a third aspect, the present invention provides a kit for detecting 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 the magnetic beads in the magnetic bead working solution is 0.2-0.5 mg / mL. Specifically, the concentration of the magnetic beads in the prepared magnetic bead 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 above-mentioned kit further includes one or more of coated antibodies, magnetic beads, coating fluid, blocking fluid, sample diluent, washing fluid, enzyme-labeled antibodies, enzyme-labeled diluent, enzyme-labeled working fluid and standard / quality control products.

[0038] The detection principle of the detection kit prepared by the present invention is different from the common competition method, but a small molecule sandwich method. The competition method is a reagent-limited type, and generally the low-end sensitivity and linear range are difficult to meet the standards, while the sandwich method is a reagent-sufficient type, which has great advantages in low-end sensitivity and linear range. The sandwich method kit includes two major components: a capture antibody coated on magnetic beads and a detection antibody labeled with a marker. The analyte in the sample to be tested (the present invention is 25-hydroxyvitamin D) is combined with the capture antibody on the magnetic beads to form a "magnetic bead-capture antibody-25-hydroxyvitamin D" complex, non-specific binding is removed by washing, and a detection antibody labeled with a marker is further added to form a "magnetic bead-capture antibody-25-hydroxyvitamin D-detection antibody-marker" complex. The concentration of the analyte in the sample can be obtained by detecting the amount of the marker (the amount of the marker is positively correlated with the concentration of the analyte in the sample within a certain range).

[0039] In a fourth aspect, 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, the detection method being a sandwich method;

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

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

[0043] The present invention uses pregnenolone, thiourea, guanidine hydrochloride, L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride as main components, and designs a new magnetic bead diluent, which can dissociate 25(OH)D from vitamin D binding protein to a great extent and has little damage to the antibodies coated on the magnetic beads, so that no additional treatment of the sample is required, and the detection process is simpler and faster. In addition, the detection performance of the magnetic bead diluent of the present invention is also good, which meets the 25(OH)D detection standard, has good reactivity, low detection limit (detection limit is 1 ng / mL), good detection linearity (|r|=0.997 in the range of 1 to 10 ng / mL), good repeatability (CV<5%), and high determination accuracy (relative deviation does not exceed ±15%). The magnetic bead working solution prepared by the magnetic bead diluent of the present invention also has the advantage of good stability, the thermal stability test decrease is within 10%, and it can be effectively airborne for 30 days, which is of great significance for more effective and convenient detection of serum 25(OH) vitamin D. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0045] Figure 1 The linear analysis results of the reagents prepared with different magnetic bead diluents were clinically compared. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0047] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0048] Example 1

[0049] Since the molecular weight of 25(OH)D is very small, it is difficult for two epitopes to be immunologically bound to two antibodies at the same time. Therefore, the sandwich method used in the kit of this embodiment is a special sandwich method. The specific principle is: the magnetic beads are coated with anti-25(OH)D antibodies (coated antibodies), and the antibodies (enzyme markers) against the "anti-25(OH)D antibody-25(OH)D antigen" complex are labeled with alkaline phosphatase (AP). The antibody of the complex is characterized by binding only to the "VD antibody and VD antigen complex" and not to the single anti-25(OH)D antibody. Therefore, if 25(OH)D (the object to be detected) exists in the sample, it will form an immune complex of the structure of "anti-25(OH)D antibody-25(OH)D antigen" with the coated antibodies on the magnetic beads. The unbound object to be detected is removed by washing, and then the enzyme marker is added, and an immune complex of the structure of "coated antibody-object to be detected-enzyme marker" is formed. The unbound enzyme marker is removed by washing, and then the substrate solution is added. The substrate emits light under the catalysis of the enzyme. The luminescence intensity is detected, and the luminescence intensity is positively correlated with the concentration of the analyte in the sample within a certain range.

[0050] This embodiment is a kit for detecting 25-hydroxyvitamin D and a preparation method thereof. The kit includes a magnetic bead working solution and an enzyme label working solution. The specific preparation methods of the magnetic bead working solution and the enzyme label working solution are as follows:

[0051] 1. Magnetic bead working solution

[0052] S1. Preparation of basic solution:

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

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

[0055] 3) Weigh 10 g bovine serum albumin, 5 g polyethylene glycol 6000, 8.77 g sodium chloride, and 3.72 g disodium ethylenediaminetetraacetate respectively, add them into a container, and 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, make up to 1 L, and store at 2-8°C.

[0057] S2. Preparation of magnetic bead diluent:

[0058] Prepare 100 mL of the base 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)phosphine hydrochloride, respectively. Mix well and homogenize by ultrasonication to ensure that all compounds are dissolved. Finally, filter with a 0.22 μm filter and store at 2-8°C.

[0059] S3. Magnetic beads coating:

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

[0061] 2) Magnetic bead coating: resuspend the magnetic beads with reaction buffer and add an appropriate amount of coating antibody (Bioventix-anti-25-hydroxyvitamin D monoclonal antibody-catalog number vitD3.5H10), then place on a rotating 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 blocking: Wash the magnetic beads three times with washing solution before blocking;

[0063] 4) Magnetic bead blocking: add appropriate amount of magnetic bead blocking solution and place on a rotating mixer, incubate in a constant temperature incubator, incubation conditions: temperature: 37±1.5℃, rotation speed 30 times / min, time 16-24h;

[0064] 5) Post-blocking wash: Wash the magnetic beads three times with post-blocking wash buffer.

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

[0066] Use the obtained magnetic bead diluent to dilute the magnetic beads to 0.2-0.5 mg / mL to prepare a magnetic bead working solution, which is stored at 2-8°C.

[0067] 2. Enzyme labeling working solution

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

[0069] The kit of this embodiment detects 25-hydroxyvitamin D in a sample by a sandwich method.

[0070] Comparative Example 1

[0071] The difference from Example 1 is that 0.316 g of pregnenolone in Example 1 is replaced by 0.096 g of ergosterol, and the rest of the formula is the same.

[0072] Comparative Example 2

[0073] The difference from Example 1 is that 0.316 g of pregnenolone in Example 1 is replaced by 0.384 g of 7-dehydrocholesterol, and the rest of the formula is the same.

[0074] Comparative Example 3

[0075] The difference from Example 1 is that pregnenolone is not contained, and the rest of the formula is the same.

[0076] Comparative Example 4

[0077] The difference from Example 1 is that thiourea is not contained, and the rest of the formula is the same.

[0078] Comparative Example 5

[0079] The difference from Example 1 is that guanidine hydrochloride is not contained, and the rest of the formula is the same.

[0080] Comparative Example 6

[0081] The difference from Example 1 is that 0.076 g of thiourea and 0.095 g of guanidine hydrochloride are not contained, and the rest of the formula is the same.

[0082] Comparative Example 7

[0083] The difference from Example 1 is that L-cysteine ​​is not contained, and the rest of the formula is the same.

[0084] Comparative Example 8

[0085] The difference from Example 1 is that tris(2-carboxyethyl)phosphine hydrochloride is not contained, and the rest of the formula is the same.

[0086] Comparative Example 9

[0087] The difference from Example 1 is that tris(2-carboxyethyl)phosphine hydrochloride and L-cysteine ​​are not contained, and the rest of the formula is the same.

[0088] Comparative Example 10

[0089] The difference from Example 1 is that tris(2-carboxyethyl)phosphine hydrochloride and L-cysteine ​​are not contained, 0.307 g of glutathione is added, and the rest of the formula is the same.

[0090] Comparative Example 11

[0091] The difference from Example 1 is that tris(2-carboxyethyl)phosphine hydrochloride and L-cysteine ​​are not contained, 0.154 g of dithiothreitol is added, and the rest of the formula is the same.

[0092] Example 2

[0093] This example verifies the effect of the main components of the magnetic bead diluent on the reactivity, that is, the luminescence values ​​of samples of different concentrations (the samples are pure products prepared by serum dilution) of Example 1 and Comparative Examples 1-11 are respectively detected, and the results are shown in Table 1:

[0094] Table 1 Luminescence value results of gradient concentration samples prepared with different magnetic bead diluent formulas

[0095]

[0096] According to 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 are consistent with the fact that the higher the sample concentration, the higher the luminescence value.

[0097] Comparative Example 3 was analyzed. The formula did not contain steroids. The main function of steroids was to replace the binding of dissociated 25(OH)D with VDBP. It was speculated that the main reason for the unresponsiveness of Comparative Example 3 was the lack of 25(OH)D substitute. Even if 25(OH)D and VDBP were separated in a short period of time, they were in dynamic equilibrium and could continue to bind due to their strong affinity. Although Comparative Examples 1 and 2 are reactive, they are significantly lower than Comparative Example 1, so pregnenolone is screened out as a suitable 25(OH)D substitute to participate in the reaction; Comparative Example 6 is analyzed, the formula does not contain protein-destroying substances, and 25(OH)D cannot be separated from VDBP, so the data show no reactivity; Comparative Example 9 is analyzed, there is no reducing agent to protect 25(OH)D in the formula, because 25(OH)D is easily oxidized, so the data show no reactivity, and when the reducing agents L-cysteine ​​and tris(2-carboxyethyl)phosphine hydrochloride are replaced with glutathione or dithiothreitol (Comparative Examples 10 and 11), there is reactivity, but it is significantly weaker than Example 1, Comparative Examples 7 and 8.

[0098] In summary, from the reactivity comparison of 12 groups of data, it can be concluded that Example 1 and Comparative Examples 4, 5, 7, and 8 can be used as preferred formulations, and the clinical comparison correlation and stability of the reagents will be further studied.

[0099] Example 3

[0100] This example verifies the effect of the main components of the magnetic bead diluent on the correlation of clinical comparison. 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 detected by the kits of Example 1, Comparative Examples 4, 5, 7, and 8, and the correlation was calculated. The test results are shown in Tables 2 and 3. Figure 1 As shown;

[0101] Table 2 Results of clinical sample testing using reagents prepared with different magnetic bead diluent formulas

[0102]

[0103] The results show that the formula groups with correlation r greater than 0.90 for the five magnetic bead diluent formulas are Example 1, Comparative Example 7, and Comparative Example 8, especially Example 1, where the correlation r reaches 0.972. In Comparative Examples 4 and 5, the correlation r is less than 0.90, and Comparative Examples 4 and 5, respectively, add less thiourea or guanidine hydrochloride than Example 1. It is possible that thiourea and guanidine hydrochloride can work synergistically to make the dissociation of 25(OH)D and VDBP more thorough. In summary, preferred Examples 1, Comparative Examples 7, and Comparative Examples 8 were subjected to stability experiments to further screen the formulas.

[0104] Example 4

[0105] This example verifies the effect of the main components of the magnetic bead diluent on stability.

[0106] The three formulation reagents selected in Example 3 were evaluated for stability. The first was thermal stability evaluation: the reagents were packed into boxes and divided into two portions, one portion was placed in a 37°C constant temperature oven for 7 days, and after 7 days, it was taken out and placed in a 2-8°C refrigerator for at least 4 hours of equilibrium, and the other portion was kept in a 2-8°C refrigerator. After the expiration, the decrease in the luminescence value of the gradient concentration sample before and after thermal acceleration was measured. The thermal acceleration results are shown in Table 3 below:

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

[0108]

[0109] Analysis of the thermal acceleration results shows that Example 1 meets the requirement of a decrease of less than 10%, which is better than Comparative Examples 7 and 8. Compared with Example 1, Comparative Examples 7 and 8 respectively add less L-cysteine ​​or tris(2-carboxyethyl)phosphine hydrochloride, which may be due to the synergistic effect of the two, making the reagent more stable.

[0110] Furthermore, an airborne stability test was performed on Example 1, and the method was as follows: the reagent was loaded into the corresponding reagent compartment in the instrument (the reagent compartment was 2-8° C.), and 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 detection value from the 0th day was calculated. The test results are shown in Table 4 below:

[0111] Table 4 Example 1 Airborne luminous value results on different days

[0112]

[0113] From the airborne stability results of Example 1, it can be seen that the reagent kit of Example 1 decreased by no more than 10% after being airborne for 7 days, 14 days, 21 days, and 30 days, respectively. It can be considered that the reagent can be airborne for 30 days with good stability.

[0114] Example 5

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

[0116] 1. Detection limit

[0117] Five detection limit reference substances (concentration of about 1 ng / mL) were tested respectively, and each sample was measured 5 times.

[0118] Table 5 Detection limit test results

[0119]

[0120] From the results in Table 5, the number of test results below the blank limit of 0.5 ng / mL is 0, so 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 respectively, each sample was tested 3 times, and the relative deviation of each test result was calculated. The results are shown in Table 6.

[0123] Table 6 Detection accuracy results

[0124]

[0125] It can be seen from Table 6 that the relative deviation of the kit of Example 1 does not exceed ±15%.

[0126] 3. Linear:

[0127] The linear samples L1 to L7 were tested, each sample was tested 3 times, and the linear correlation was calculated. The results are shown in Table 7.

[0128] Table 7 Linear correlation coefficient test results

[0129]

[0130]

[0131] It can be seen from Table 7 that the linear correlation coefficient |r| of the kit of Example 1 is not less than 0.990 within the concentration range of [1,150] ng / mL.

[0132] 4. Repeatability:

[0133] The low-value and high-value samples of the repeatability reference were tested, and each sample was tested 10 times to examine the repeatability of the reagent. The results are shown in Table 8.

[0134] Table 8 Repeatability test results

[0135] Number of detections Repeatability reference low value (ng / mL) Repeatability reference high value (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] It can be seen from Table 8 that the coefficient of variation (CV) of the results obtained by testing the repeatability reference material is less than 5%, which meets the requirement of not more than 8%.

[0137] Example 6

[0138] This embodiment is a basic solution study, which is as follows:

[0139] The basic solution of the magnetic bead diluent includes the basic components of buffer, protective protein, surfactant, chelating agent, salt and preservative. The common components are arbitrarily replaced and the component concentration is adjusted to form basic buffers of different formulas (see Table 9 for the specific formula) to study the effect of each formula of basic buffer on the reactivity of the reagent. After the basic buffer is prepared according to the formula, 100mL of the basic solution is prepared and added to a suitable container, and 0.316g of pregnenolone, 0.095g of guanidine hydrochloride, 0.076g of thiourea, 0.135g of L-cysteine, and 0.25g of tris(2-carboxyethyl)phosphine hydrochloride are added respectively. After mixing, ultrasonic homogenization is performed to ensure that all compounds are dissolved, and finally filtered with a 0.22μm filter and stored at 2-8°C.

[0140] The corresponding magnetic bead working solution was finally prepared according to the method of Example 1, and the same enzyme label working solution was used for testing. The reactivity of the gradient samples was measured on the machine. The results are shown in Table 10.

[0141] Table 9 Basic solution formula

[0142]

[0143] Table 10 Reagent reactivity results of different basic solution formulations

[0144] Sample concentration (ng / mL) Formulation 1 - RLU Formulation 2 - RLU Formulation 3-RLU Formulation 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, so routine adjustments to the base solution compounds (appropriate concentration adjustments or replacement with other common substitutes) have little effect on the reactivity of the reagents.

[0146] Example 7

[0147] This example is a study on the concentration range of the main components of the magnetic bead dilution solution, as follows:

[0148] The amounts of pregnenolone, thiourea, guanidine hydrochloride, L-cysteine, and tris(2-carboxyethyl)phosphine hydrochloride in the formula were changed (the other components remained unchanged, and the basic solution formula used the basic solution of Example 1, see Table 11), and the effects of the concentrations of the relevant components on the reactivity of the reagents were studied. The results of the gradient sample reactivity test on the machine are shown in Table 12.

[0149] Table 11 Magnetic bead dilution formula

[0150] Recipe 1 Recipe 6 Recipe 7 Recipe 8 Basic solution formula 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)phosphine hydrochloride / g 0.25 0.25 0.125 0.025

[0151] Table 12 Reagent reactivity results of different magnetic bead diluent formulas

[0152] Sample concentration (ng / mL) Formulation 1 - RLU Formulation 6-RLU Formula 7-RLU Formulation 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 show that compared with formulation 1, the reactivity of formulations 6-8 changes less, so 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)phosphine hydrochloride) has little effect on the reactivity of the reagent.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

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

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

3. The magnetic bead diluent according to claim 1, characterized in that The magnetic bead diluent includes 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 includes 1-10 mM L-cysteine ​​and 1-10 mM tris(2-carboxyethyl)phosphine hydrochloride.

5. The magnetic bead diluent according to claim 1, characterized in that The magnetic bead diluent also includes a buffer solution; Optionally, the buffer comprises Tris buffer.

6. The magnetic bead diluent according to claim 1, characterized in that The magnetic bead diluent further comprises one or more of protective protein, surfactant, chelating agent, salt and preservative; Optionally, the protective protein includes at least one of fetal bovine serum, calf serum, horse serum, bovine serum albumin, human serum albumin and casein; Optionally, the surfactant includes at least one of a Triton series surfactant, a Tween series surfactant and a polyethylene glycol surfactant; Optionally, the chelating agent comprises disodium ethylenediaminetetraacetate; Optionally, the salt comprises sodium chloride; Optionally, the preservative comprises Proclin300.

7. The magnetic bead diluent according to claim 6, characterized in that The magnetic bead diluent also includes 50-200mM Tris(OH)2M buffer with a pH value of 7.4, 10-200g / L serum albumin, 1-20v / v% animal serum, 0.1-1v / v% Triton series surfactant or Tween series surfactant, 5-10g / L polyethylene glycol surfactant, 1-10mM chelating agent, 50-150mM salt and preservative.

8. A magnetic bead working solution, characterized in that: The invention comprises the magnetic bead diluent and the magnetic bead-protein complex according to any one of claims 1 to 7, wherein the magnetic bead-protein complex is prepared by coating magnetic beads with capture proteins; Optionally, the capture protein is an anti-25(OH)D antibody.

9. A kit for detecting 25-hydroxyvitamin D, characterized in that: include: The magnetic bead diluent according to any one of claims 1 to 7 or the magnetic bead working solution according to claim 8; Optionally, the kit further comprises one or more of coating antibodies, magnetic beads, coating solution, blocking solution, sample diluent, washing solution, enzyme-labeled antibodies, enzyme-labeled diluent, enzyme-labeled working solution and standard / quality control products.

10. Use of the magnetic bead diluent according to any one of claims 1 to 7, the magnetic bead working solution according to claim 8, or the kit according to claim 9 in the following (I) or (II): (I) detecting 25-hydroxyvitamin D for non-diagnostic or therapeutic purposes, wherein the detection method is a sandwich method; (II) preparing a product for detecting 25-hydroxyvitamin D, wherein the product is used for detecting 25-hydroxyvitamin D by a sandwich method.

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