25-hydroxyvitamin D dissociation liquid, detection kit and application

Abstract

The invention provides a 25-hydroxyvitamin D dissociation liquid, a detection kit and application. The 25-hydroxyvitamin D dissociation liquid comprises 10-100mM of a buffer solution with the pH value of 7.2-7.4, 0.1%-5% of long-chain alkane carboxylic acid and 0.1%-1% of an organic solvent. The 25-hydroxyvitamin D detection kit is prepared by developing a new 25-hydroxyvitamin D dissociation agent capable of replacing perfluorooctanoic acid and combining a double-antibody sandwich method, and has the characteristics of high accuracy, high sensitivity, good stability, simplicity and convenience in operation, high detection efficiency, low cost, safety, environmental friendliness and the like; and the method has relatively high economic value and social benefit.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to a 25-hydroxyvitamin D dissociation solution, a detection kit and applications thereof. Background Art

[0002] In addition to regulating calcium and phosphorus metabolism and maintaining plasma calcium and phosphorus levels, vitamin D is also closely related to autoimmune diseases, diabetes, cardiovascular disease, cell growth and differentiation, etc. VD deficiency can cause rickets and osteomalacia in newborns, osteoporosis in the elderly, and other diseases. 25-hydroxyvitamin D (25-OH VD) is an important indicator of whether the body has sufficient vitamin D, the most important of which are 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

[0003] Currently, the methods for 25-OH VD on the market can be divided into two categories: mass spectrometry and immunoassay. Mass spectrometry is the "gold standard" for detecting 25-OH VD. Its advantages are high measurement accuracy and the ability to distinguish between 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. However, mass spectrometry has high requirements for instruments and operators, high testing costs, and long testing processes, making this method difficult to implement in ordinary hospitals and testing institutions.

[0004] Compared with mass spectrometry, immunoassay has the advantages of high throughput and rapid detection. With the rise of chemiluminescence technology, the detection of 25-OH VD on fully automatic biochemical analyzers has gradually become popular. It has the advantages of simple operation, high sensitivity, strong specificity, and accurate results, which can meet the needs of daily clinical testing. The first generation of chemiluminescence immunoassay reagents mainly used competitive immunoassays, represented by Germany's Roche Diagnostics, the United States' Abbott, and Italy's DiaSorin. Although the first generation of reagents met the needs of clinical testing, they still had disadvantages such as low sensitivity and high price, and the correlation with the mass spectrometry results was low, with a correlation coefficient R 2 <0.8. The second-generation 25-hydroxyvitamin D test mainly introduced the small molecule sandwich method, using the double antibody sandwich antigen method, which not only improved the sensitivity of the reagent, but also had a better relative clinical compliance rate due to the better specificity of the double antibody sandwich.

[0005] The first step of 25-OH VD serum detection is generally the dissociation step, which releases 25-OH VD from vitamin D binding protein (VDBP) through a specific dissociation method. Commonly used methods can be divided into five categories: acid-base method, reduction method, enzyme digestion method, organic solvent extraction and replacement method. Among them, the acid-base method and the reduction method both denature VDBP to make 25-hydroxyvitamin D free. The disadvantage of this method is that strong acids or strong reducing agents usually have a great impact on antibodies, while weak acids or weak reducing agents cannot completely dissociate free 25-OH VD. The enzyme digestion method is to specifically cut VDBP by protease. The reaction of this method is mild, but the introduction of protease increases the cost of the reagent and requires higher stability of the reagent. Organic solvent extraction is a pre-treatment step for mass spectrometry. Its characteristic is that it can completely release free 25-OH VD. However, most organic solvents have the effect of denaturing proteins and affect the activity of antibodies. The replacement method is a relatively new method, which is characterized by the release of free 25-OH VD by competing with 25-OH VD for VDBP through 25-OH VD structural analogs. This method has the advantages of simple and mild reaction and high replacement efficiency. Currently, the commonly used replacement substances are fluoroalkanoic acids, fluoro complete salts, etc., among which perfluorooctanoic acid or perfluorooctanoic acid sulfonate are the most commonly used. However, perfluorooctanoic acid has been considered a Class 2 carcinogen, and its structure is stable and the degradation cycle is long, which is not conducive to environmental protection. Summary of the invention

[0006] In view of the above problems, the present invention has developed a new 25-hydroxyvitamin D dissociator that can replace perfluorooctanoic acid. At the same time, combined with the double antibody sandwich method, a 25-hydroxyvitamin D detection kit is prepared. The kit has the characteristics of high accuracy, high sensitivity, good stability, simple operation, high detection efficiency, low cost, safety and environmental protection, and has high economic value and social benefits.

[0007] To achieve the above-mentioned purpose, the present invention provides a 25-hydroxyvitamin D dissociation solution, which comprises: a buffer solution of 10-100 mM and pH 7.2-7.4, 0.1%-5% long-chain alkane carboxylic acid, and 0.1%-1% organic solvent.

[0008] Furthermore, the buffer comprises at least one of Tris-HCl buffer, PBs buffer and HEPES buffer.

[0009] Furthermore, the long-chain alkane carboxylic acid includes at least one of octanoic acid, nonanoic acid and decanoic acid.

[0010] Furthermore, the organic solvent includes at least one of DMSO, DMF, methanol, ethanol, ethylene glycol, and acetone.

[0011] The present invention also provides a 25-hydroxyvitamin D detection kit, characterized in that the 25-hydroxyvitamin D detection kit comprises: a solid phase reagent, a luminescent reagent, and a 25-hydroxyvitamin D dissociation solution as described in any one of claims 1 to 4.

[0012] Furthermore, both the solid phase reagent and the luminescent reagent are provided with a treatment solution, which includes: 10-100mM and pH7.0-7.4 reagent buffer, 0.5%-2% inorganic salt, 0.2%-5% protein protective agent, 0.01%-0.1% surfactant, 0.01%-0.1% preservative, and blocking agent.

[0013] Furthermore, the reagent buffer includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer; and / or the inorganic salt includes at least one of NaCl, KCl, and Na2SO4; and / or the protein protective agent includes at least one of BSA, fish skin gelatin, and trehalose; and / or the surfactant includes at least one of PEG6000, Tween-20, and Triton X-100; and / or the preservative includes at least one of PC300, kanamycin, and sodium azide.

[0014] Furthermore, the preparation method of the solid phase reagent includes: after fully mixing the carboxyl magnetic beads, taking 1 ml of 10 mg / ml magnetic beads and placing them in a centrifuge tube; using an activation buffer to wash the magnetic beads 3 times, then adding 1 ml of the activation buffer to resuspend them for use; weighing EDC and sulfo-NHS respectively, and adding the activation buffer to adjust their concentrations to 10 mg / ml; adding EDC and sulfo-NHS to the standby magnetic beads, mixing them thoroughly and placing them on a shaker for reaction for 20 minutes; washing the activated magnetic beads with the activation buffer 3 times, then adding the cross-linking buffer to resuspend them to a concentration of 10 mg / ml, measuring the antibody and adding it to the magnetic beads, mixing them thoroughly and placing them on a shaker for reaction at room temperature for 2 hours; after the reaction is completed, washing the magnetic beads with the cross-linking buffer 3 times, adding 1 ml of the blocking buffer to resuspend them, and reacting at room temperature for 1 hour; after the blocking reaction is completed, washing the magnetic beads with the blocking buffer 4 times, and finally resuspending the magnetic beads to a concentration of 10 mg / ml; adding the magnetic beads coated with the antibody to the treatment solution, mixing them thoroughly to obtain the solid phase reagent.

[0015] Furthermore, the preparation method of the luminescent reagent includes: dissolving the acridinium ester activated substance with anhydrous DMSO to a concentration of 5 mg / mL; adding the acridinium ester activated substance and the labeled antibody in a molar ratio of 15:1 to the cross-linking buffer, and placing it at room temperature in the dark for 2 hours; after the reaction is completed, adding 1 / 10 volume of lysine solution to terminate the reaction for 0.5 hours; separating and purifying the reaction solution with a Sephadex G25 column balanced with a 0.1 mol / L and pH 7.4 PB buffer to obtain an acridinium ester-labeled labeled antibody, and then adding an equal volume of glycerol, and storing the labeled antibody at -20°C for standby use; adding the labeled antibody to the treatment solution, and mixing it thoroughly to obtain the luminescent reagent.

[0016] The present invention also provides an application of a 25-hydroxyvitamin D dissociation solution as described in any one of claims 1 to 4 or a 25-hydroxyvitamin D kit as described in any one of claims 5 to 9, characterized in that the application is to detect the 25-hydroxyvitamin D content in a sample.

[0017] After adopting the technical solution of the present invention, the following technical effects can be achieved: (1) Efficient dissociation: The dissociation solution can effectively release free 25-OH VD, ensuring that the sample used in subsequent testing is in a free form, thereby improving the sensitivity and accuracy of the test; (2) Mild operating conditions: The components of the dissociation solution work under mild conditions and will not cause adverse effects on samples or reagents, ensuring the stability of the experiment and the consistency of the results; (3) Avoid environmental pollution: Compared with the use of traditional dissociation agents such as perfluorooctanoic acid, the long-chain alkane carboxylic acid substitute provided by the present invention is more environmentally friendly and complies with environmental regulations, especially in the context of the ban on perfluorooctanoic acid in some countries / regions; (4) Low cost and easy operation: The dissociation solution has simple components, low cost, and simple operation procedures, making it easy to promote and apply on a large scale in clinical laboratories or scientific research environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 The 25-hydroxyvitamin D master calibration curve provided in Example 4 of the present invention; Figure 2 A linear curve graph of the reagent provided in Example 5 of the present invention in the range of 2.0-150 ng / mL; Figure 3 The test results of the 25-hydroxyvitamin D kit provided in Example 5 of the present invention are compared with the test results of the mass spectrometry method. DETAILED DESCRIPTION

[0019] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] An embodiment of the present invention provides a 25-hydroxyvitamin D dissociation solution, which includes: a buffer solution of 10-100 mM and pH 7.2-7.4, 0.1%-5% long-chain alkane carboxylic acid, and 0.1%-1% organic solvent.

[0021] The present invention provides a 25-hydroxyvitamin D dissociation solution, wherein a buffer is used to provide a suitable environment, maintain the pH value of the solution within the range of 7.2 to 7.4, and ensure stability and effectiveness during the dissociation process; the function of the buffer is to control the pH value of the liquid to avoid instability of other components or poor dissociation effect due to pH changes. Long-chain alkane carboxylic acids have strong hydrophobicity and can interact with vitamin D binding protein (VDBP) to promote the effective dissociation of 25-hydroxyvitamin D from VDBP; the concentration of long-chain alkane carboxylic acids is set between 0.1% and 5%, and the efficiency and speed of dissociation can be controlled by optimizing its concentration. Organic solvents can further promote the release of 25-hydroxyvitamin D and help improve the dissociation efficiency; the organic solvent used is at a low concentration to avoid the negative impact of the solvent on the activity of the reagent or antibody.

[0022] The design of this dissociation solution is based on a deep understanding of the dissociation mechanism of 25-hydroxyvitamin D, especially its binding and dissociation process with vitamin D binding protein. By using a combination of long-chain alkane carboxylic acids and organic solvents, this dissociation solution can efficiently release free 25-OH VD under mild conditions without damaging antibodies or other important molecular structures.

[0023] In some embodiments of the present application, the buffer includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer.

[0024] Tris-HCl buffer has a strong buffering capacity and can maintain the pH stability of the solution in the neutral or slightly alkaline range. PBS buffer is a phosphate-containing buffer and usually contains a certain concentration of salt. Because PBS buffer is close to the salt concentration and pH value in the body, it is suitable for maintaining the stability of biological molecules such as cells and proteins in experiments. HEPES buffer is more adaptable to temperature changes and can maintain good buffering capacity in a wider pH range.

[0025] Using one or more of Tris-HCl, PBS, and HEPES buffers as the buffer system can effectively maintain the pH value of the solution stable and avoid experimental result errors caused by pH fluctuations during the experiment. Different buffers have different buffering capacities and pH ranges. Combining them can further enhance the overall buffering capacity of the system, thereby providing stronger pH stability and higher experimental accuracy during complex experiments. Especially in the case of large changes in temperature, ionic strength, or other environmental factors, the use of these buffers can effectively reduce the impact of the external environment and improve the reliability and stability of the experiment.

[0026] In some embodiments of the present application, the long-chain alkane carboxylic acid includes at least one of octanoic acid, nonanoic acid, and decanoic acid.

[0027] Long-chain alkane carboxylic acids act as dissociating agents, working together with other solution components to promote the dissociation of 25-hydroxyvitamin D from the bound state. Long-chain alkane carboxylic acids refer to fatty acid molecules with longer carbon chains, including octanoic acid, nonanoic acid, decanoic acid, etc. These acids are hydrophobic and can interact with specific molecules in the solution, thereby changing the dissociation state of these molecules.

[0028] Specifically, octanoic acid is a long-chain alkane carboxylic acid with 8 carbon atoms, which has strong hydrophobicity and can form hydrophobic interactions with protein or fat molecules, thereby promoting the dissociation of certain molecules from the binding substances. Nonanoic acid is a long-chain alkane carboxylic acid with 9 carbon atoms, which is used to promote dissociation or improve the solubility of molecules. Its molecular structure is similar to that of octanoic acid and also has strong hydrophobicity. Decanoic acid is a long-chain alkane carboxylic acid with 10 carbon atoms, which has stronger hydrophobicity than octanoic acid and nonanoic acid.

[0029] These long-chain alkane carboxylic acids can effectively promote the dissociation of 25-hydroxyvitamin D from its conjugates by binding to the hydrophobic region of vitamin D binding protein VDBP. Depending on the buffer system used and the concentration control, these acids can improve the dissociation efficiency while maintaining the stability of the experiment.

[0030] In some embodiments of the present application, the organic solvent includes at least one of DMSO, DMF, methanol, ethanol, ethylene glycol, and acetone.

[0031] DMSO, as a polar solvent, has a strong dissolving power and can dissolve a variety of polar and non-polar substances. It can penetrate biological membranes and enter cells. DMF, as a polar solvent, can dissolve a variety of organic and inorganic compounds. Methanol is a low molecular weight organic solvent with a low boiling point and good dissolving power. Ethanol has the characteristics of strong dissolving power and low volatility. Ethylene glycol has a high dissolving power and can dissolve a variety of water-insoluble organic compounds. Acetone, as a polar organic solvent, can effectively dissolve fats, oils, resins and a variety of organic compounds.

[0032] These organic solvents have strong dissolving power and can dissolve a variety of polar and non-polar compounds. By using these solvents, the efficiency of the reaction system and the solubility of the reactants can be improved, thereby accelerating the reaction process. These solvents have good dissolving properties and different polarities, and can be flexibly selected according to experimental requirements. Ethanol, methanol, DMSO, etc. not only have strong dissolving power, but also have certain biocompatibility and low toxicity, and can be safely used in biological research.

[0033] An embodiment of the present invention further provides a 25-hydroxyvitamin D detection kit, characterized in that the 25-hydroxyvitamin D detection kit comprises: a solid phase reagent, a luminescent reagent, and a 25-hydroxyvitamin D dissociation solution as described in any one of claims 1-4.

[0034] The 25-hydroxyvitamin D detection kit provided by the present invention is mainly used to detect 25-hydroxyvitamin D in a sample, and is suitable for clinical, scientific research and other occasions where vitamin D levels need to be monitored. Among them, the solid phase reagent can bind or adsorb the target molecule. On this basis, the solid phase reagent can be a solid surface coated with an anti-25-hydroxyvitamin D antibody, such as a microplate, a magnetic bead or other suitable solid matrix; the reagent is used to capture 25-hydroxyvitamin D or its derivatives in the sample so that it can be further analyzed. The luminescent reagent refers to a reagent that can emit a measurable light signal under specific conditions such as adding a substrate, being activated by laser light or a chemical reaction. In the present invention, the luminescent reagent reacts with the binding site of 25-hydroxyvitamin D, and the intensity of the luminescent reaction is proportional to the concentration of 25-hydroxyvitamin D in the sample, thereby achieving detection. The role of the 25-hydroxyvitamin D dissociation solution is to promote the dissociation of 25-hydroxyvitamin D from its binding form. By using the dissociation solution, 25-hydroxyvitamin D can be effectively released from the binding protein in blood, urine or other samples. The dissociated 25-hydroxyvitamin D can enter the reaction area bound to the solid phase reagent, so as to carry out subsequent detection and analysis.

[0035] The use of dissociation solution to release 25-hydroxyvitamin D can effectively reduce its interference with binding proteins, allowing 25-hydroxyvitamin D to fully participate in subsequent detection reactions, which helps to improve the sensitivity and accuracy of the test, especially when the vitamin D concentration in the sample is low. The kit simplifies the operation steps by integrating solid phase reagents, luminescent reagents and dissociation solution into a complete detection system. Users only need to follow the instructions and add the corresponding samples and reagents to obtain clear test results, which greatly improves the convenience and consistency of the test. The use of solid phase reagents enables large-scale, high-throughput detection. For example, solid phase matrices such as microplates or magnetic beads can process multiple samples at the same time, thereby improving the efficiency of the experiment. The kit can be widely used in biological samples such as blood and urine, and through luminescent signal detection, there is no need for complex equipment or long analysis processes, which makes the detection of vitamin D levels faster and more efficient.

[0036] In some embodiments of the present application, both the solid phase reagent and the luminescent reagent are provided with a treatment solution, which includes: 10-100 mM reagent buffer with a pH of 7.0-7.4, 0.5%-2% inorganic salt, 0.2%-5% protein protective agent, 0.01%-0.1% surfactant, 0.01%-0.1% preservative, and blocking agent.

[0037] The purpose of designing the treatment solution is to optimize the stability, performance and reaction conditions of the solid phase reagent and the luminescent reagent, thereby improving the efficiency and accuracy of the detection process. The role of the reagent buffer is to maintain the pH stability of the reaction system. Within the pH range of 7.0-7.4, the buffer can maintain the stability of the reaction environment and ensure that the solid phase reagent and the luminescent reagent work under suitable conditions, thereby improving the accuracy and consistency of the detection. The addition of inorganic salts is mainly to adjust the ionic strength of the reaction system, which helps to enhance the interaction between reagents, optimize the solubility of reagents, and improve the efficiency of the solid phase reagent binding to 25-hydroxyvitamin D. Protein protectants are used to prevent the protein in the solid phase reagent and the luminescent reagent from denaturing or degrading during storage and reaction. Proteins are usually very sensitive to conditions such as temperature and pH. The addition of protein protectants can extend the shelf life of the reagents, maintain their stability and activity, thereby improving the service life of the reagents and the reliability of the detection results. The addition of surfactants can reduce the surface tension of the solution, help the solid phase reagent and the luminescent reagent to be better dispersed in the solution, and avoid the failure of the reagents due to aggregation or precipitation. In addition, surfactants can also help remove nonspecific adsorption between reagents and improve the binding specificity of target molecules. The role of preservatives is to prevent the growth and contamination of microorganisms, ensure the long-term storage stability of reagents, prevent bacteria or fungi and other microorganisms from affecting the quality of reagents, and thus ensure the accuracy of test results. Blockers are mainly used to prevent non-specific binding reactions that may exist in solid phase reagents and luminescent reagents. By binding to non-target substances in the reagents, blockers can reduce background noise, improve the signal-to-noise ratio of the test, and ensure the accuracy of the measurement results.

[0038] The use of reagent buffers, protein protectants and preservatives can significantly improve the stability of solid phase reagents and luminescent reagents, extend their shelf life, and reduce the risk of denaturation, degradation or contamination during storage and operation, thereby improving the reliability of the test results. By adding reasonable salt concentrations, surfactants and blockers, the interaction between solid phase reagents and luminescent reagents can be optimized, nonspecific binding or interference reactions can be reduced, and the binding efficiency of target molecules and reagents can be improved. This helps to improve the sensitivity and specificity of the test. The addition of blockers and surfactants can effectively reduce the interference of background signals, reduce the possibility of nonspecific binding, and improve the clarity and accuracy of the final luminescent signal; especially in complex biological samples, the reduction of background noise is crucial for the accurate determination of the results. The formulation of the treatment solution can reduce the impact of interfering substances and nonspecific reactions, thereby improving the detection sensitivity of 25-hydroxyvitamin D. By optimizing the reaction conditions of the reagents, accurate test results can still be obtained when low concentrations of target molecules are present.

[0039] In some embodiments of the present application, the reagent buffer includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer; and / or the inorganic salt includes at least one of NaCl, KCl, and Na2SO4; and / or the protein protective agent includes at least one of BSA, fish skin gelatin, and trehalose; and / or the surfactant includes at least one of PEG6000, Tween-20, and Triton X-100; and / or the preservative includes at least one of PC300, kanamycin, and sodium azide.

[0040] The present embodiment relates to a reagent composition, specifically including a combination of multiple buffers, inorganic salts, protein protective agents, surfactants and preservatives, which is intended to improve the performance and stability of the reagent in a specific biological experiment. Different buffers can provide more suitable pH stability according to the detection needs, avoiding the adverse effects of changes in the acid-base environment on the reaction results. Inorganic salts play a role in regulating ionic strength, helping dissolution and improving the conductivity of the solution in the reagent. Proteins are often prone to denaturation or degradation during the treatment process. The addition of protein protective agents can effectively prevent these phenomena. Protective agents such as BSA, fish skin gelatin, and trehalose are often used to maintain the natural conformation of proteins to prevent enzyme activity loss or aggregation precipitation. Surfactants such as PEG6000, Tween-20, and Triton X-100 can reduce the surface tension of the solution and promote the dissolution or dispersion of the solute. Preservatives such as PC300, kanamycin and sodium azide are used to prevent the growth of microorganisms in the solution and ensure the long-term stability of the reagent.

[0041] In some embodiments of the present application, the preparation method of the solid phase reagent includes: after the carboxyl magnetic beads are fully mixed, 1 ml of 10 mg / ml magnetic beads are placed in a centrifuge tube; the magnetic beads are washed 3 times with an activation buffer, and then 1 ml of activation buffer is added to resuspend them for use; EDC and sulfo-NHS are weighed respectively, and the activation buffer is added to adjust their concentrations to 10 mg / ml; EDC and sulfo-NHS are added to the standby magnetic beads, and after fully mixing, they are placed on a shaker for reaction for 20 minutes; the activated magnetic beads are washed 3 times with an activation buffer, and then the cross-linking buffer is added to resuspend their concentration to 10 mg / ml, the antibody is added to the magnetic beads, and after fully mixing, they are placed on a shaker for reaction at room temperature for 2 hours; after the reaction is completed, the magnetic beads are washed 3 times with a cross-linking buffer, 1 ml of a blocking buffer is added to resuspend them, and they are reacted at room temperature for 1 hour; after the blocking reaction is completed, the magnetic beads are washed 4 times with a blocking buffer, and finally the magnetic beads are resuspended to a concentration of 10 mg / ml; the magnetic beads coated with the antibody are added to the treatment solution, and the solid phase reagent is obtained after fully mixing.

[0042] This embodiment relates to a method for preparing a solid phase reagent, which is mainly used to fix antibodies on the surface of magnetic particles with carboxyl functional groups by covalent bonding. The specific method includes the following steps: Activation of magnetic particles: The surface of magnetic particles is activated by a two-step method of EDC and sulfo-NHS, so that the surface carboxyl groups are converted into active ester intermediates. This activation process can ensure the smooth progress of the subsequent antibody cross-linking reaction. Antibody cross-linking: According to a specific ratio, that is, 1 mg of magnetic particles and 10 μg of antibodies, the antibodies are combined with the activated magnetic particles, and the primary amine groups in the antibodies react with the activated ester groups on the surface of the magnetic particles to form a stable covalent bonding structure. This reaction can ensure that the antibodies are stably attached to the surface of the magnetic particles and ensure their effectiveness in subsequent applications. Blocking reaction: In order to prevent the unreacted activated ester groups from continuing to react, a blocking agent is used for blocking treatment. This step can prevent the redundant active groups on the surface of the magnetic particles from affecting the subsequent experimental results or performance. Washing and final concentration adjustment: by-products and unreacted reagents produced during the reaction are removed by multiple washings, and the concentration of the solid phase reagent is finally adjusted to 0.1-0.5 mg / mL to complete the preparation of the solid phase reagent.

[0043] In some embodiments of the present application, the preparation method of the luminescent reagent includes: dissolving the acridinium ester activator with anhydrous DMSO to a concentration of 5 mg / mL; adding the acridinium ester activator and the labeled antibody in a molar ratio of 15:1 to the cross-linking buffer, and placing it at room temperature in the dark for 2 hours; after the reaction is completed, adding 1 / 10 volume of lysine solution to terminate the reaction for 0.5 hours; separating and purifying the reaction solution with a Sephadex G25 column balanced with 0.1 mol / L and pH 7.4 PB buffer to obtain an acridinium ester-labeled labeled antibody, and then adding an equal volume of glycerol, and storing the labeled antibody at -20°C for standby use; adding the labeled antibody to the treatment solution, and mixing it thoroughly to obtain the luminescent reagent.

[0044] This embodiment relates to a method for preparing a luminescent reagent, the core of which is to prepare a labeled antibody with luminescent properties by reacting an acridinium ester with a 25-hydroxyvitamin D labeled antibody, and finally form a luminescent reagent. The specific steps include: dissolution and activation of the acridinium ester, labeling reaction of the antibody, quenching of lysine, purification, and finally preparing a luminescent working solution.

[0045] Specifically, acridinium ester dissolution and activation: acridinium ester is dissolved in anhydrous dimethyl sulfoxide (DMSO) to a concentration of 5 mg / mL and stored in a dark and dry condition to maintain its stability. Antibody labeling reaction: 25-hydroxyvitamin D labeled antibody and acridinium ester are added to the cross-linking buffer, i.e., 0.05 mol / L and pH 9.5 phosphate buffer, at a molar ratio of 1:15, and reacted at room temperature in the dark for 2 hours. Reaction termination and quenching: Add a lysine solution with a concentration of 10-100 g / L, add lysine to the reaction system in a volume of 1 / 10 of the reaction solution, and continue to react for 0.5 hours to terminate the labeling reaction. Purification step: Separate and purify the labeled antibody by Sephadex G25 column chromatography, balance with pH 7.4 phosphate buffer. Preparation of luminescent reagent: Mix the purified labeled antibody with an appropriate amount of treatment solution to obtain a luminescent reagent working solution, which can eventually be used for detection or analysis applications.

[0046] An embodiment of the present invention also provides an application of a 25-hydroxyvitamin D dissociation solution as described in any one of claims 1 to 4 or a 25-hydroxyvitamin D kit as described in any one of claims 5 to 9, characterized in that it is used to detect the 25-hydroxyvitamin D content in a sample.

[0047] The 25-hydroxyvitamin D dissociation solution is used to effectively release 25-hydroxyvitamin D from the sample, making subsequent testing more efficient and accurate. The 25-hydroxyvitamin D kit contains relevant chemical reagents that can be used in conjunction with the dissociation solution to form a complete detection system. In this way, the content of 25-hydroxyvitamin D in the sample can be quantitatively or qualitatively detected.

[0048] The present invention has developed a new type of 25-hydroxyvitamin D dissociation solution, which can replace the traditional perfluorooctanoic acid, and combined with the double antibody sandwich method to prepare a 25-hydroxyvitamin D detection kit. The kit uses the double antibody sandwich method for detection, and the specific process is: first, take 10μL of serum sample, add 50μL of dissociation solution at the 0th minute, and incubate at 37℃ for 5 minutes; then add 50μL of solid phase reagent and 50μL of luminescent reagent, continue to incubate at 37℃ for 10 minutes, and finally detect the light value.

[0049] The 25-hydroxyvitamin D detection kit provided by the present invention has the advantages that the dissociation solution used is mainly composed of long-chain alkane carboxylic acids, has a good replacement effect, and can provide high accuracy, high sensitivity and good stability. In addition, the kit is easy to operate on an automated chemiluminescent immunoassay instrument and has high detection efficiency. Compared with traditional perfluorooctanoic acid and its analogs, the reagent of the present invention not only reduces production costs, but also has better safety and environmental protection.

[0050] The coating antibody of this example was purchased from Nanjing Okey Biotech, the detection antibody was purchased from Jiangsu Boya Biotech, the 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 standards were purchased from Sigma, and other components not specifically described in this example are commercially available.

[0051] Example 1 Solid phase reagent buffer (treatment solution): 50mmol / L phosphate buffer with pH 7.2, 9g / L sodium chloride, 20g / L bovine serum albumin, 20g / L trehalose, 0.8g / L Tween 20, 1g / L sodium azide, 1g / L mouse IgG.

[0052] The embodiment of the present invention provides a method for preparing a solid phase reagent, that is, magnetic particles coated with 25-hydroxyvitamin D antibody, which specifically comprises the following steps: After fully mixing, take out 1 ml (10 mg / ml) of aM-270 carboxyl magnetic beads and place it in a centrifuge tube; b. Wash the beads three times with activation buffer (10mmol / L MES, pH5.5), resuspend in 1ml activation buffer and set aside; c. Weigh a certain amount of activating agents EDC and sulfo-NHS respectively, and then add the corresponding volume of activation buffer to a concentration of 10 mg / ml; d. Add 150μl Sulfo-NHS and 100μl EDC to the reserved magnetic beads, mix thoroughly and place on a shaker for activation reaction for 20 minutes; e. Wash the activated magnetic beads three times with activation buffer, add 1 ml of cross-linking buffer (25 mmol / L MES, pH 6.0, 0.05% Tween-20) and resuspend to a concentration of 10 mg / ml, accurately measure 0.2 mg of coated antibody and add it to the spare magnetic beads and mix thoroughly, and place it on a shaker at room temperature for 3 hours; f. After the reaction, wash the beads three times with cross-linking buffer, add 1 ml of blocking buffer (50 mmol / L Tris, 1% BSA, 0.01% Triton X-100, 0.09% NaN3, pH 8.0), resuspend, and react at room temperature for 1 hour.

[0053] g. After blocking, wash the beads 4 times with blocking buffer and resuspend to 1 ml, i.e. the concentration is 10 mg / ml.

[0054] h. Take 0.5 ml of antibody-coated magnetic beads and add them into 25 ml of solid phase reagent buffer. Mix thoroughly and set aside. The preparation of magnetic particle working solution is completed.

[0055] Example 2 Luminescent reagent buffer (treatment solution): 50mmol / L phosphate buffer with pH 7.0, 9g / L sodium chloride, 20g / L bovine serum albumin, 1g / L Tween 20, 1g / L sodium azide, 1g / L mouse IgG.

[0056] An embodiment of the present invention provides a method for preparing a luminescent reagent, namely, an acridinium ester-labeled detection antibody, which specifically comprises the following steps: a. Dissolve the activated acridinium ester in anhydrous DMSO to 5 mg / mL; b. Add the acridinium ester activated product and 25-hydroxyvitamin D labeled antibody in a molar ratio of 15:1 to the labeling buffer (0.05 mol / L phosphate buffer, pH 9.5) and place at room temperature in the dark for 2 hours; c. Then 1 / 10 volume of lysine solution was added to terminate the reaction for 0.5 hours; d. The reaction solution was separated and purified by Sephadex G25 column equilibrated with 0.1 mol / L PB buffer at pH 7.0 to obtain acridinium ester-labeled 25-hydroxyvitamin D antibody, and an equal volume of glycerol was added and stored at -20°C for later use; f. Take 200 ml of luminescent reagent buffer, add acridinium ester-labeled 25-hydroxyvitamin D antibody to a concentration of 0.5 μg / ml, mix thoroughly and set aside.

[0057] Example 3 25-Hydroxyvitamin D dissociation solution: 50 mM phosphate buffer at pH 7.2, 10 g / L capric acid, 2 mL / L ethanol, 5 mL / L ethylene glycol, and 3 mL / L DMF.

[0058] An embodiment of the present invention provides a method for preparing a 25-hydroxyvitamin D dissociation solution, which specifically comprises the following steps: a. Measure and mix a certain amount of ethanol, ethylene glycol and DMF; b. Weigh a certain amount of decanoic acid and add it to the organic solvent mixture and mix thoroughly; c. Then add a quantitative volume of 50 mM phosphate buffer at pH 7.2, mix thoroughly and set aside.

[0059] Example 4 25-hydroxyvitamin D detection process: A fully automatic chemiluminescence analyzer is used, and the method is a double antibody sandwich method. The detection process is to draw 10uL of serum sample at 0min, add 50uL of dissociation solution and incubate at 37℃ for 5min, add 50uL of solid phase reagent and 50uL of luminescent reagent, incubate at 37℃ for 10min, and clear and detect the light value RLU.

[0060] The commercial standard 25-hydroxyvitamin D3 was used to prepare the reagent main calibrator and working calibration quality control. The reagents were diluted according to the concentration labeled on the bottle. The 25-hydroxyvitamin D3 was prepared with calibration diluent (0.01-0.05 mol / L PBS, 0.1%-2% BSA, 0.9%-25% NaCl, 0.1% PC-300, pH 7.2) to concentrations of 0 ng / mL, 2.85 ng / mL, 11.91 ng / mL, 30.90 ng / mL, 74.33 ng / mL, and 150.56 ng / mL. The samples were packaged in 1 mL portions in each bottle and stored at -20°C for later use.

[0061] Calibration curve preparation: the concentration of the calibrator is used as the horizontal axis, the luminescence value RLU corresponding to each concentration of the calibrator is used as the vertical axis, and the fitting method is a quadratic polynomial function curve.

[0062] Sample concentration calculation method: Enter the master curve information in the software, fit the master calibration curve, and the instrument automatically calculates the corresponding concentration value. The serum sample results are compared with the traceability reference mass spectrometry detection system for value assignment and traceability.

[0063] Figure 1 This is the master calibration curve for 25-hydroxyvitamin D.

[0064] Example 5 Basic performance test of the kit: (1) Blank LOB Repeat the measurement of 5 25-hydroxyvitamin D zero-value calibrators 5 times each, calculate the average value X and standard deviation SD of the RLU of the 25 measurement results, substitute X+2SD into the standard curve, and the minimum detection limit of this method is 0.47 ng / mL.

[0065] Table 1 Reagent sensitivity test (2) Linear range test Take a high-value calibrator sample H with a concentration of 150 ng / mL and a low-value calibrator sample L with a concentration of 2 ng / mL, mix sample H and sample L in the ratio of 10H, 9H+1L, 8H+2L, 7H+3L, 6H+4L, 5H+5L, 4H+6L, 3H+7L, 2H+8L, 1H+9L, and 10L to make 11 samples, and measure each sample three times in parallel. Compare the measured values ​​with the theoretical values. The results show that this reagent has good detection linearity in the range of 2.0-150 ng / mL.

[0066] Figure 2 This is the linear curve of this reagent in the range of 2.0-150ng / mL.

[0067] (3) Thermal acceleration stability test The 25-hydroxyvitamin D test reagent was placed in a 37°C heat accelerated test for 7 days, and then the calibrator was tested. The results showed that the RLU was only reduced by 10% compared with the reagent that was not subjected to the heat accelerated test, indicating that the reagent has good stability.

[0068] Table 2 Thermal acceleration test (4) Accuracy and uniformity The relative deviation was calculated by comparing the serum disc samples assigned by mass spectrometry with the "gold standard". The mass spectrometry assigned serum disc was provided by Meikang Shengde Medical Laboratory. The serum disc concentrations were: sample A was 8.99 ng / mL, sample B was 13.07 ng / mL, sample C was 21.01 ng / mL, sample D was 25.87 ng / mL, sample E was 33.58 ng / mL, and sample F was 51.97 ng / mL. The relative mass spectrometry value deviation was less than 6%, and the CV was less than 6%.

[0069] Table 3 Accuracy results (5) Anti-interference analysis Fresh serum from healthy people was taken to prepare mixed serum; pure interfering substances were diluted to a certain concentration (hemoglobin was 5 mg / mL, vitamin C was 30 mg / L, unconjugated bilirubin was 0.2 mg / mL, conjugated bilirubin was 0.2 mg / mL, and chyle was 1450 FTU) with calibrator diluent. Interference serum was prepared at a volume ratio of 19:1 for mixed serum: interfering substance, and control serum was prepared at a volume ratio of 19:1 for mixed serum: calibrator diluent. The concentration of 25-hydroxyvitamin D was measured three times in parallel. The results are shown in Table 4, indicating that this detection system has good anti-interference ability against the above-mentioned interfering substances.

[0070] Table 4 Other anti-interference results (6) Clinical relevance analysis The detection kit configured in Example 1 was used for comparison with mass spectrometry to detect 50 clinical serum samples. The results are as follows: Figure 3 The correlation coefficient between the reagent of the present invention and the control reagent is R 2 =905, and the regression equation is y=0.9968x+2.3152. This result shows that the reagent of the present invention and the control reagent have good correlation, specificity and accuracy.

[0071] Figure 3 The test results of 25-hydroxyvitamin D kit were compared with the mass spectrometry test results.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 25-hydroxyvitamin D dissociation solution, characterized in that: The 25-hydroxyvitamin D dissociation solution comprises: a buffer solution of 10-100 mM and pH 7.2-7.4, 0.1%-5% long-chain alkane carboxylic acid, and 0.1%-1% organic solvent.

2. The 25-hydroxyvitamin D dissociation solution according to claim 1, characterized in that The buffer comprises at least one of Tris-HCl buffer, PBs buffer and HEPES buffer.

3. The 25-hydroxyvitamin D dissociation solution according to claim 1, characterized in that The long-chain alkane carboxylic acid includes at least one of octanoic acid, nonanoic acid and decanoic acid.

4. The 25-hydroxyvitamin D dissociation solution according to claim 1, characterized in that The organic solvent includes at least one of DMSO, DMF, methanol, ethanol, ethylene glycol and acetone.

5. A 25-hydroxyvitamin D detection kit, characterized in that: The 25-hydroxyvitamin D detection kit comprises: a solid phase reagent, a luminescent reagent, and a 25-hydroxyvitamin D dissociation solution as described in any one of claims 1-4.

6. The 25-hydroxyvitamin D detection kit according to claim 5, characterized in that: The solid phase reagent and the luminescent reagent are both provided with a treatment solution, which includes: 10-100mM reagent buffer with pH 7.0-7.4, 0.5%-2% inorganic salt, 0.2%-5% protein protective agent, 0.01%-0.1% surfactant, 0.01%-0.1% preservative, and blocking agent.

7. The 25-hydroxyvitamin D detection kit according to claim 6, characterized in that: The reagent buffer comprises at least one of Tris-HCl buffer, PBs buffer and HEPES buffer; and / or The inorganic salt comprises at least one of NaCl, KCl, and Na2SO4; and / or The protein protective agent comprises at least one of BSA, fish skin gelatin and trehalose; and / or The surfactant includes at least one of PEG6000, Tween-20, and Triton X-100; and / or The preservative includes at least one of PC300, kanamycin and sodium azide.

8. The 25-hydroxyvitamin D detection kit according to claim 5, characterized in that: The preparation method of the solid phase reagent comprises: after fully mixing the carboxyl magnetic beads, taking 1 ml of 10 mg / ml magnetic beads and placing them in a centrifuge tube; washing the magnetic beads 3 times with an activation buffer, then adding 1 ml of the activation buffer to resuspend them for standby use; weighing EDC and sulfo-NHS respectively, and adding the activation buffer to adjust their concentrations to 10 mg / ml; adding EDC and sulfo-NHS to the standby magnetic beads, fully mixing them, and placing them on a shaker for reaction for 20 minutes; washing the activated magnetic beads 3 times with the activation buffer, then adding the cross-linking buffer to resuspend them to a concentration of 10 mg / ml, measuring the antibody and adding it to the magnetic beads, fully mixing them, and placing them on a shaker for reaction at room temperature for 2 hours; washing the magnetic beads 3 times with the cross-linking buffer after the reaction is completed, adding 1 ml of the blocking buffer to resuspend them, and reacting them at room temperature for 1 hour; after the blocking reaction is completed, washing the magnetic beads 4 times with the blocking buffer, and finally resuspending the magnetic beads to a concentration of 10 mg / ml; adding the magnetic beads coated with the antibody to the treatment solution, and fully mixing them to obtain the solid phase reagent.

9. The 25-hydroxyvitamin D detection kit according to claim 5, characterized in that: The preparation method of the luminescent reagent comprises: dissolving an acridinium ester activated substance with anhydrous DMSO to a concentration of 5 mg / mL; adding the acridinium ester activated substance and the labeled antibody in a molar ratio of 15:1 into a cross-linking buffer, and placing the mixture at room temperature in the dark for a reaction for 2 hours; adding a 1 / 10 volume of a lysine solution after the reaction is completed to terminate the reaction for 0.5 hours; separating and purifying the reaction solution with a Sephadex G25 column balanced with a 0.1 mol / L PB buffer solution with a pH of 7.4 to obtain an acridinium ester labeled labeled antibody, and then adding an equal volume of glycerol, and storing the labeled antibody at -20°C for standby use; adding the labeled antibody into a treatment solution, and mixing the mixture thoroughly to obtain a luminescent reagent.

10. A use of the 25-hydroxyvitamin D dissociation solution as described in any one of claims 1 to 4 or the 25-hydroxyvitamin D kit as described in any one of claims 5 to 9, characterized in that: The application is to detect the content of 25-hydroxyvitamin D in a sample.

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