25-hydroxyvitamin d dissociation solution, detection kit and application

Abstract

The application provides a 25-hydroxyvitamin D dissociation solution, a detection kit and application, and the 25-hydroxyvitamin D dissociation solution comprises: 10-100 mM and pH 7.2-7.4 buffer solution, 0.1%-5% long-chain alkane carboxylic acid, 0.1%-1% organic solvent. The application develops a new 25-hydroxyvitamin D dissociation agent which can replace perfluorooctanoic acid, and combines a double-antibody sandwich method to prepare a 25-hydroxyvitamin D detection kit, which 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.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and more specifically, to a 25-hydroxyvitamin D dissociation solution, a detection kit, and its applications. Background Technology

[0002] Besides regulating calcium and phosphorus metabolism and maintaining plasma calcium and phosphorus levels, vitamin D is also closely related to autoimmune diseases, diabetes, cardiovascular diseases, and cell growth and differentiation. Vitamin D deficiency can cause rickets and osteomalacia in newborns, and osteoporosis in the elderly. 25-hydroxyvitamin D (25-OH VD) is an important indicator of vitamin D sufficiency in the body, with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 being the most important.

[0003] Currently, methods for detecting 25-OH vitamin D can be broadly categorized into mass spectrometry and immunoassay. Mass spectrometry is considered the "gold standard" for detecting 25-OH vitamin D, offering advantages such as high accuracy and the ability to distinguish between 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. However, its high requirements for equipment and operators, high cost, and lengthy procedures make it difficult to implement in ordinary hospitals and testing institutions.

[0004] Immunoassay offers advantages over mass spectrometry, including high throughput and rapid detection. With the rise of chemiluminescence technology, the detection of 25-OH VD on fully automated biochemical analyzers has become increasingly common, offering advantages such as ease of operation, high sensitivity, strong specificity, and accurate results, meeting routine clinical testing needs. First-generation chemiluminescent immunoassay reagents primarily employed competitive immunoassays, with representative companies including Roche Diagnostics (Germany), Abbott (USA), and DiaSorin (Italy). While these first-generation reagents met clinical testing requirements, they still suffered from drawbacks such as low sensitivity at low values, high cost, and relatively low correlation with mass spectrometry results (correlation coefficient R0). 2 <0.8. The detection of second-generation 25-hydroxyvitamin D mainly introduces a small molecule sandwich method, using a double antibody sandwich antigen method. This not only improves the sensitivity of the reagent, but also has a better clinical concordance rate due to the better specificity of the double antibody sandwich.

[0005] The first step in 25-OH vitamin D serum detection is generally a dissociation step, releasing 25-OH vitamin D from vitamin D-binding protein (VDBP) through a specific dissociation method. Common methods can be categorized into five main types: acid-base methods, reduction methods, enzymatic digestion methods, organic solvent extraction, and displacement methods. Acid-base and reduction methods both denature VDBP, releasing 25-hydroxyvitamin D into a free state. A drawback of these methods is that strong acids or strong reducing agents often significantly affect antibodies, while weak acids or weak reducing agents cannot completely dissociate free 25-OH vitamin D. Enzymatic digestion uses proteases to specifically cleave VDBP. This method is mild; however, the introduction of proteases increases reagent costs and requires higher reagent stability. Organic solvent extraction is a pretreatment step in mass spectrometry, characterized by the complete release of free 25-OH vitamin D. However, most organic solvents denature proteins, affecting antibody activity. The substitution method is a relatively new approach. Its key feature is that it uses a 25-OH VD structural analogue to compete with 25-OH VD for VDBP, thereby releasing free 25-OH VD. This method has the advantages of simple and mild reaction, and high substitution efficiency. Commonly used substitution materials include fluoroalkyl acids and fluorocomplete salts, with perfluorooctanoic acid (PFOA) or PFOA sulfonate being the most frequently used. However, PFOA is already considered a Group 2 carcinogen; its stable structure and long degradation cycle are detrimental to environmental protection. Summary of the Invention

[0006] In view of the above problems, this invention has developed a new 25-hydroxyvitamin D dissociation agent that can replace perfluorooctanoic acid. At the same time, combined with the double antibody sandwich method, a 25-hydroxyvitamin D detection kit was prepared, which 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 objectives, the present invention provides a 25-hydroxyvitamin D dissociation solution, comprising: a 10-100 mM buffer solution with a pH of 7.2-7.4, 0.1%-5% long-chain alkane carboxylic acid, and 0.1%-1% organic solvent.

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

[0009] Furthermore, long-chain alkane carboxylic acids include 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 assay kit, characterized in that the 25-hydroxyvitamin D assay 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.

[0012] Furthermore, both the solid-phase reagent and the luminescent reagent are provided with a treatment solution, which includes: a 10-100mM reagent buffer solution with a pH of 7.0-7.4, 0.5%-2% inorganic salts, 0.2%-5% protein protectant, 0.01%-0.1% surfactant, 0.01%-0.1% preservative, and blocking agent.

[0013] Further, the reagent buffer includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer; and / or inorganic salts include at least one of NaCl, KCl, and Na2SO4; and / or protein protectants include at least one of BSA, fish skin gelatin, and trehalose; and / or surfactants include at least one of PEG6000, Tween-20, and Triton X-100; and / or preservatives include at least one of PC300, kanamycin, and sodium azide.

[0014] Further, the preparation method of the solid-phase reagent includes: thoroughly mixing carboxyl magnetic beads, taking 1 ml of 10 mg / ml magnetic beads and placing them in a centrifuge tube; washing the magnetic beads three times with activation buffer, then resuspending them in 1 ml of activation buffer for later use; weighing EDC and sulfo-NHS separately, and adding activation buffer to adjust their concentration to 10 mg / ml; adding EDC and sulfo-NHS to the prepared magnetic beads, thoroughly mixing, and reacting on a shaker for 20 min; washing the activated magnetic beads three times with activation buffer, then resuspending them in crosslinking buffer to a concentration of 10 mg / ml; adding antibody to the magnetic beads, thoroughly mixing, and reacting on a shaker at room temperature for 2 h; after the reaction, washing the magnetic beads three times with crosslinking buffer, adding 1 ml of blocking buffer to resuspend them, and reacting at room temperature for 1 h; after the blocking reaction, washing the magnetic beads four times with blocking buffer, and finally resuspending the magnetic beads to a concentration of 10 mg / ml; adding the antibody-coated magnetic beads to the treatment solution, thoroughly mixing, and obtaining the solid-phase reagent.

[0015] Further, the preparation method of the luminescent reagent includes: dissolving the acridine ester activator in anhydrous DMSO to a concentration of 5 mg / mL; adding the acridine ester activator and the labeled antibody to a cross-linking buffer at a molar ratio of 15:1 and reacting at room temperature in the dark for 2 h; after the reaction is completed, adding 1 / 10 volume of lysine solution to terminate the reaction for 0.5 h; separating and purifying the reaction solution using a Sephadex G25 column equilibrated with 0.1 mol / L PB buffer at pH 7.4 to obtain the acridine ester-labeled antibody, then adding an equal volume of glycerol and storing the labeled antibody at -20℃ for later use; adding the labeled antibody to the processing solution and mixing thoroughly to obtain the luminescent reagent.

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

[0017] By adopting the technical solution of the present invention, the following technical effects can be achieved:

[0018] (1) High efficiency dissociation: This dissociation solution can effectively release free 25-OH VD, ensuring that the sample used in subsequent detection is in free form, thereby improving the sensitivity and accuracy of detection;

[0019] (2) Mild operating conditions: The components of the dissociation solution work under mild conditions and will not have an adverse effect on the sample or reagent, ensuring the stability of the experiment and the consistency of the results;

[0020] (3) Avoid environmental pollution: Compared with traditional dissociation agents such as perfluorooctanoic acid, the long-chain alkane carboxylic acid substitutes provided by this invention are more environmentally friendly and comply with environmental regulations, especially in the context of some countries / regions banning perfluorooctanoic acid;

[0021] (4) Low cost and easy operation: The components of the dissociation solution are simple and the cost is low. The operation process is not complicated, which makes it easy to promote and apply on a large scale in clinical laboratories or scientific research environments. Attached Figure Description

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0023] Figure 1 The master calibration curve of 25-hydroxyvitamin D provided in Example 4 of this invention;

[0024] Figure 2 Linearity curve of the reagent provided in Example 5 of the present invention in the range of 2.0-150 ng / mL;

[0025] Figure 3 The detection results of the 25-hydroxyvitamin D kit provided in Example 5 of this invention are compared with the detection results of mass spectrometry. Detailed Implementation

[0026] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] An embodiment of the present invention provides a 25-hydroxyvitamin D dissociation solution, which comprises: a 10-100 mM buffer solution with a pH of 7.2-7.4, 0.1%-5% long-chain alkane carboxylic acid, and 0.1%-1% organic solvent.

[0028] This invention provides a 25-hydroxyvitamin D dissociation solution, wherein a buffer solution is used to provide a suitable environment, maintaining the pH value of the solution within the range of 7.2 to 7.4, ensuring the stability and effectiveness of the dissociation process. The buffer solution controls the pH of the liquid, preventing instability of other components or poor dissociation due to pH changes. Long-chain alkane carboxylic acids have strong hydrophobicity and can interact with vitamin D-binding protein (VDBP), promoting the effective dissociation of 25-hydroxyvitamin D from VDBP. The concentration of the long-chain alkane carboxylic acid is set between 0.1% and 5%, and its concentration can be optimized to control the efficiency and rate of dissociation. Organic solvents can further promote the release of 25-hydroxyvitamin D and help improve dissociation efficiency; the concentration of the organic solvent used is low to avoid negatively impacting the activity of reagents or antibodies.

[0029] 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 processes with vitamin D-binding proteins. 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 the antibody or other important molecular structures.

[0030] In some embodiments of this application, the buffer solution includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer.

[0031] Tris-HCl buffer has strong buffering capacity, maintaining pH stability within a neutral or slightly alkaline range. PBS buffer is a phosphate-containing buffer, typically containing a certain concentration of salt. Because PBS buffer closely approximates the salt concentration and pH value found in living organisms, it is suitable for maintaining the stability of cells and proteins in experiments. HEPES buffer is even more adaptable to temperature changes and maintains good buffering capacity over a wide pH range.

[0032] Using one or more of Tris-HCl, PBS, and HEPES buffers as a buffering system can effectively maintain the stability of the solution's pH value, avoiding experimental errors caused by pH fluctuations during the experiment. Different buffers have different buffering capacities and pH ranges; using them in combination can further enhance the overall buffering capacity of the system, thus providing stronger pH stability and higher experimental accuracy in complex experiments. Especially under conditions of significant temperature, ionic strength, or other environmental variations, using these buffers can effectively reduce the influence of the external environment and improve the reliability and stability of the experiment.

[0033] In some embodiments of this application, the long-chain alkane carboxylic acid includes at least one of octanoic acid, nonanoic acid, and decanoic acid.

[0034] Long-chain alkane carboxylic acids act as dissociation agents, interacting with other solution components to promote the dissociation of 25-hydroxyvitamin D from its bound state. Long-chain alkane carboxylic acids refer to fatty acid molecules with relatively long carbon chains, including octanoic acid, nonanoic acid, and decanoic acid. These acids are hydrophobic and can interact with specific molecules in solution, thereby altering the dissociation state of these molecules.

[0035] Specifically, octanoic acid is an 8-carbon long-chain alkane carboxylic acid with strong hydrophobicity, capable of forming hydrophobic interactions with protein or fat molecules, thereby promoting the dissociation of certain molecules from their bound substances. Nonanoic acid is a 9-carbon long-chain alkane carboxylic acid used to promote dissociation or improve molecule solubility; its molecular structure is similar to octanoic acid, and it also has strong hydrophobicity. Decanoic acid is a 10-carbon long-chain alkane carboxylic acid with even stronger hydrophobicity than octanoic acid and nonanoic acid.

[0036] These long-chain alkane carboxylic acids can effectively promote the dissociation of 25-hydroxyvitamin D from its conjugates by binding to hydrophobic regions of the vitamin D-binding protein VDBP. Depending on the buffer system used and the concentration control, these acids can enhance dissociation efficiency while maintaining experimental stability.

[0037] In some embodiments of this application, the organic solvent includes at least one of DMSO, DMF, methanol, ethanol, ethylene glycol, and acetone.

[0038] DMSO, as a polar solvent, possesses strong dissolving power, capable of dissolving a variety of polar and non-polar substances, and can penetrate biological membranes to 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 is characterized by strong dissolving power and low volatility. Ethylene glycol has 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.

[0039] These organic solvents possess extremely strong dissolving power, capable of dissolving a wide variety of polar and nonpolar compounds. Using these solvents can improve the efficiency of the reaction system and the solubility of reactants, thereby accelerating the reaction process. These solvents have excellent solubility properties and varying polarities, allowing for flexible selection based on experimental needs. Ethanol, methanol, DMSO, and others not only have strong dissolving power but also exhibit certain biocompatibility and low toxicity, making them safe for use in biological research.

[0040] An embodiment of 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 1-4.

[0041] The 25-hydroxyvitamin D assay kit provided by this invention is mainly used to detect 25-hydroxyvitamin D in samples and is suitable for clinical, research, and other occasions requiring monitoring of vitamin D levels. The solid-phase reagent can bind or adsorb target molecules. This solid-phase reagent can be a solid surface coated with anti-25-hydroxyvitamin D antibodies, such as a microplate, magnetic beads, or other suitable solid matrix. This reagent is used to capture 25-hydroxyvitamin D or its derivatives in the sample, enabling further analysis. The luminescent reagent is a reagent that emits a measurable light signal under specific conditions, such as the addition of a substrate, activation by laser, or chemical reaction. In this invention, the luminescent reagent reacts with the binding site of 25-hydroxyvitamin D. The intensity of the luminescence reaction is proportional to the concentration of 25-hydroxyvitamin D in the sample, thus achieving detection. The 25-hydroxyvitamin D dissociation solution promotes the dissociation of 25-hydroxyvitamin D from its bound form. By using the dissociation solution, 25-hydroxyvitamin D can be effectively released from binding proteins in blood, urine, or other samples. The dissociated 25-hydroxyvitamin D can then enter the reaction zone bound to the solid-phase reagent for subsequent detection and analysis.

[0042] Using a dissociation buffer to release 25-hydroxyvitamin D effectively reduces its interference with binding proteins, allowing 25-hydroxyvitamin D to fully participate in subsequent detection reactions. This helps improve the sensitivity and accuracy of the detection, especially when the vitamin D concentration in the sample is low. The kit integrates solid-phase reagents, luminescent reagents, and dissociation buffers into a complete detection system, simplifying the operation. Users only need to follow the instructions, add the appropriate sample and reagents, and obtain clear detection results, greatly improving 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 simultaneously, thereby improving experimental efficiency. This kit can be widely used for biological samples such as blood and urine, and detection via luminescent signals eliminates the need for complex equipment or lengthy analysis processes, making vitamin D level detection faster and more efficient.

[0043] In some embodiments of this application, both the solid-phase reagent and the luminescent reagent are provided with a treatment solution, which includes: a 10-100mM reagent buffer solution with a pH of 7.0-7.4, 0.5%-2% inorganic salts, 0.2%-5% protein protectant, 0.01%-0.1% surfactant, 0.01%-0.1% preservative, and an inhibitor.

[0044] The purpose of the processing solution is to optimize the stability, performance, and reaction conditions of the solid-phase and luminescent reagents, thereby improving the efficiency and accuracy of the detection process. The reagent buffer maintains pH stability within the reaction system. Within the pH range of 7.0-7.4, the buffer helps maintain the stability of the reaction environment, ensuring that the solid-phase and luminescent reagents operate under suitable conditions, thus 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 enhance the interaction between reagents, optimize reagent solubility, and improve the efficiency of the solid-phase reagent binding to 25-hydroxyvitamin D. Protein protectants are used to prevent the denaturation or degradation of proteins in the solid-phase and luminescent reagents during storage and reaction. Proteins are generally very sensitive to conditions such as temperature and pH; adding protein protectants can extend the shelf life of the reagents, maintain their stability and activity, thereby improving the reagent's lifespan and the reliability of the detection results. The addition of surfactants can reduce the surface tension of the solution, helping the solid-phase and luminescent reagents to be better dispersed in the solution, avoiding reagent failure due to aggregation or precipitation. In addition, surfactants can also help remove non-specific adsorption between reagents, improving the binding specificity of target molecules. Preservatives prevent the growth and contamination of microorganisms, ensuring the long-term storage stability of reagents and preventing bacteria or fungi from affecting reagent quality, thereby ensuring the accuracy of test results. Blocking agents are mainly used to prevent non-specific binding reactions that may exist in solid-phase and luminescent reagents. By binding to non-target substances in the reagent, blocking agents can reduce background noise, improve the signal-to-noise ratio of the detection, and ensure the accuracy of measurement results.

[0045] The use of reagent buffers, protein protectants, and preservatives can significantly improve the stability of solid-phase and luminescent reagents, extend their shelf life, and reduce the risk of denaturation, degradation, or contamination during storage and handling, thereby improving the reliability of detection results. By adding appropriate salt concentrations, surfactants, and blockers, the interaction between solid-phase and luminescent reagents can be optimized, reducing non-specific binding or interfering reactions and improving the binding efficiency of target molecules to reagents. This helps improve the sensitivity and specificity of detection. The addition of blockers and surfactants can effectively reduce background signal interference, lower the possibility of non-specific binding, and improve the clarity and accuracy of the final luminescent signal; especially in complex biological samples, reducing background noise is crucial for accurate result interpretation. The formulation of the processing solution can reduce the influence of interfering substances and non-specific reactions, thereby improving the detection sensitivity of 25-hydroxyvitamin D. By optimizing the reaction conditions of the reagents, accurate detection results can still be obtained in the presence of low concentrations of target molecules.

[0046] In some embodiments of this application, the reagent buffer includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer; and / or inorganic salts include at least one of NaCl, KCl, and Na2SO4; and / or protein protectants include at least one of BSA, fish skin gelatin, and trehalose; and / or surfactants include at least one of PEG6000, Tween-20, and Triton X-100; and / or preservatives include at least one of PC300, kanamycin, and sodium azide.

[0047] This embodiment relates to a reagent composition, specifically a combination of various buffer solutions, inorganic salts, protein protectants, surfactants, and preservatives, designed to improve the performance and stability of the reagent in specific biological experiments. Different buffer solutions can provide more suitable pH stability according to the detection requirements, avoiding adverse effects of changes in the acid-base environment on the reaction results. Inorganic salts in the reagent play a role in regulating ionic strength, aiding dissolution, and improving solution conductivity. Proteins are often prone to denaturation or degradation during processing; the addition of protein protectants can effectively prevent these phenomena. Protectants such as BSA, fish skin gelatin, and trehalose are commonly used to maintain the native conformation of proteins, preventing loss of enzyme activity or aggregation and precipitation. Surfactants such as PEG6000, Tween-20, and Triton X-100 can reduce the surface tension of the solution, promoting the dissolution or dispersion of solutes. Preservatives such as PC300, kanamycin, and sodium azide are used to prevent the growth of microorganisms in the solution, ensuring the long-term stability of the reagent.

[0048] In some embodiments of this application, the preparation method of the solid-phase reagent includes: thoroughly mixing carboxyl magnetic beads, placing 1 ml of 10 mg / ml magnetic beads in a centrifuge tube; washing the magnetic beads three times with activation buffer, then resuspending them in 1 ml of activation buffer for later use; weighing EDC and sulfo-NHS separately, and adding activation buffer to adjust their concentration to 10 mg / ml; adding EDC and sulfo-NHS to the prepared magnetic beads, thoroughly mixing, and reacting on a shaker for 20 min; washing the activated magnetic beads three times with activation buffer, then resuspending them in crosslinking buffer to a concentration of 10 mg / ml; adding antibody to the magnetic beads, thoroughly mixing, and reacting on a shaker at room temperature for 2 h; after the reaction, washing the magnetic beads three times with crosslinking buffer, adding 1 ml of blocking buffer to resuspend them, and reacting at room temperature for 1 h; after the blocking reaction, washing the magnetic beads four times with blocking buffer, and finally resuspending the magnetic beads to a concentration of 10 mg / ml; adding the antibody-coated magnetic beads to the treatment solution, thoroughly mixing, and obtaining the solid-phase reagent.

[0049] This embodiment relates to a method for preparing a solid-phase reagent, mainly used to immobilize antibodies on the surface of magnetic microparticles with carboxyl functional groups via covalent bonding. The specific method includes the following steps: Activation of magnetic microparticles: The surface of the magnetic microparticles is activated using a two-step method of EDC and sulfo-NHS, converting the surface carboxyl groups into active ester intermediates. This activation process ensures the smooth progress of subsequent antibody cross-linking reactions. Antibody cross-linking: The antibody is bound to the activated magnetic microparticles at a specific ratio of 1 mg of magnetic microparticles to 10 μg of antibody. The primary amine groups in the antibody react with the activated ester groups on the surface of the magnetic microparticles, forming a stable covalently bonded structure. This reaction ensures stable adhesion of the antibody to the surface of the magnetic microparticles, guaranteeing its effectiveness in subsequent applications. Blocking reaction: To prevent unreacted activated ester groups from continuing to react, a blocking agent is used for blocking. This step prevents excess active groups on the surface of the magnetic microparticles from affecting subsequent experimental results or performance. Cleaning and final concentration adjustment: By repeatedly cleaning, the byproducts and unreacted reagents generated during the reaction are removed. Finally, the concentration of the solid reagent is adjusted to 0.1-0.5 mg / mL to complete the preparation of the solid reagent.

[0050] In some embodiments of this application, the preparation method of the luminescent reagent includes: dissolving the acridine ester activator in anhydrous DMSO to a concentration of 5 mg / mL; adding the acridine ester activator and the labeled antibody to a cross-linking buffer at a molar ratio of 15:1 and reacting at room temperature in the dark for 2 h; after the reaction is completed, adding 1 / 10 volume of lysine solution to terminate the reaction for 0.5 h; separating and purifying the reaction solution using a Sephadex G25 column equilibrated with 0.1 mol / L PB buffer at pH 7.4 to obtain the acridine ester-labeled antibody, then adding an equal volume of glycerol and storing the labeled antibody at -20℃ for later use; adding the labeled antibody to the processing solution and mixing thoroughly to obtain the luminescent reagent.

[0051] This embodiment relates to a method for preparing a luminescent reagent. The core of this method involves reacting acridine ester with a 25-hydroxyvitamin D-labeled antibody to prepare a labeled antibody with luminescent properties, ultimately forming the luminescent reagent. Specific steps include: dissolving and activating acridine ester, the antibody labeling reaction, lysine quenching, purification, and the final preparation of the luminescent working solution.

[0052] Specifically, acridine ester dissolution and activation: Acridine ester was dissolved in anhydrous dimethyl sulfoxide (DMSO) to a concentration of 5 mg / mL and stored under light-protected and dry conditions to maintain its stability. Antibody labeling reaction: 25-hydroxyvitamin D-labeled antibody and acridine ester were added to crosslinking buffer (0.05 mol / L phosphate buffer, pH 9.5) at a molar ratio of 1:15 and reacted at room temperature in the dark for 2 hours. Reaction termination and quenching: A 10-100 g / L lysine solution was added to the reaction system, with the volume of lysine added being 1 / 10 of the reaction solution. The reaction was continued for 0.5 hours to terminate the labeling reaction. Purification step: The labeled antibody was purified by Sephadex G25 column chromatography, equilibrated with phosphate buffer at pH 7.4, and separated. Preparation of luminescent reagent: The purified labeled antibody was mixed with an appropriate amount of processing solution to obtain the luminescent reagent working solution, which can finally be used for detection or analytical applications.

[0053] The embodiments of the present invention also provide an application of the 25-hydroxyvitamin D dissociation solution as described in any one of claims 1-4 or the 25-hydroxyvitamin D kit as described in any one of claims 5-9, characterized in that the application is for detecting the 25-hydroxyvitamin D content in a sample.

[0054] The 25-hydroxyvitamin D dissociation solution is used to effectively release 25-hydroxyvitamin D from the sample, making subsequent detection 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 a sample can be quantitatively or qualitatively detected.

[0055] This invention develops a novel 25-hydroxyvitamin D dissociation solution, which can replace traditional perfluorooctanoic acid (PFOA), and combines it with a double-antibody sandwich method to prepare a 25-hydroxyvitamin D detection kit. The kit uses a double-antibody sandwich method for detection, and the specific procedure is as follows: First, 10 μL of serum sample is aspirated, and 50 μL of dissociation solution is added at minute 0, and incubated at 37°C for 5 minutes; then, 50 μL of solid-phase reagent and 50 μL of luminescent reagent are added, and incubation continues at 37°C for 10 minutes; finally, the photoluminescence value is measured.

[0056] The advantages of the 25-hydroxyvitamin D assay kit provided by this invention are that the dissociation solution used is mainly composed of long-chain alkane carboxylic acids, which has a good displacement effect and can provide high accuracy, high sensitivity, and good stability. Furthermore, the kit is easy to operate on automated chemiluminescence immunoassay analyzers and has high detection efficiency. Compared with traditional perfluorooctanoic acid and its analogues, the reagent of this invention not only reduces production costs but also has better safety and environmental friendliness.

[0057] In this embodiment, the coating antibody was purchased from Nanjing Oukai Biotechnology, the detection antibody was purchased from Jiangsu Boya Biotechnology, and the 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 standards were purchased from Sigma. Other components not specifically mentioned in this embodiment are commercially available.

[0058] Example 1

[0059] Solid-phase reagent buffer (treatment solution): 50 mmol / L phosphate buffer at pH 7.2, 9 g / L sodium chloride, 20 g / L bovine serum albumin, 20 g / L trehalose, 0.8 g / L Tween 20, 1 g / L sodium azide, 1 g / L mouse IgG.

[0060] The present invention provides a method for preparing a solid-phase reagent, namely, magnetic microparticles coated with 25-hydroxyvitamin D antibody, specifically including the following steps:

[0061] After thoroughly mixing the aM-270 carboxylated magnetic beads, take out 1 ml (10 mg / ml) and place it in a centrifuge tube;

[0062] b. Wash the magnetic beads three times with activation buffer (10 mmol / L MES, pH 5.5), resuspend in 1 ml of activation buffer, and set aside for later use;

[0063] c. Weigh out a certain amount of activator EDC and sulfo-NHS respectively, and then add the corresponding volume of activation buffer to a concentration of 10 mg / ml;

[0064] d. Add 150 μl of Sulfo-NHS and 100 μl of EDC to the prepared magnetic beads, mix thoroughly, and then place on a shaker for 20 minutes to activate the reaction.

[0065] 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) to resuspend to a concentration of 10 mg / ml, accurately measure 0.2 mg of coated antibody and add it to the prepared magnetic beads and mix thoroughly. Place in a shaker at room temperature for 3 hours.

[0066] f. After the reaction is complete, wash the magnetic beads three times with cross-linking buffer, resuspend them in 1 ml of blocking buffer (50 mmol / L Tris, 1% BSA, 0.01% Triton X-100, 0.09% NaN3, pH 8.0), and react at room temperature for 1 hour.

[0067] g. After blocking, wash the magnetic beads four times with blocking buffer and finally resuspend them in 1 ml, which is a concentration of 10 mg / ml.

[0068] h. Take 0.5 ml of the antibody-coated magnetic beads and add them to 25 ml of solid-phase reagent buffer. Mix thoroughly and set aside. This completes the preparation of the magnetic microparticle working solution.

[0069] Example 2

[0070] The luminescent reagent buffer (treatment solution) consists of 50 mmol / L phosphate buffer at pH 7.0, 9 g / L sodium chloride, 20 g / L bovine serum albumin, 1 g / L Tween 20, 1 g / L sodium azide, and 1 g / L mouse IgG.

[0071] The present invention provides a method for preparing a luminescent reagent, namely an acridinium ester-labeled detection antibody, specifically including the following steps:

[0072] a. Dissolve the acridine ester activator in anhydrous DMSO to a concentration of 5 mg / mL;

[0073] b. Add the acridinium ester activator and the 25-hydroxyvitamin D-labeled antibody to the labeling buffer (0.05 mol / L phosphate buffer, pH 9.5) at a molar ratio of 15:1, and incubate at room temperature in the dark for 2 hours.

[0074] c. Then add 1 / 10 volume of lysine solution to terminate the reaction for 0.5 hours;

[0075] d. The reaction solution was purified by separation using a Sephadex G25 column equilibrated with 0.1 mol / L PB buffer at pH 7.0 to obtain acridinium ester-labeled 25-hydroxyvitamin D antibody. An equal volume of glycerol was added and the solution was stored at -20°C for later use.

[0076] f. Take 200 ml of luminescent reagent buffer, add acrid ester-labeled 25-hydroxyvitamin D antibody to a concentration of 0.5 μg / ml, mix thoroughly and set aside.

[0077] Example 3

[0078] 25-hydroxyvitamin D dissociation solution: 50 mM phosphate buffer at pH 7.2, 10 g / L decanoic acid, 2 mL / L ethanol, 5 mL / L ethylene glycol, 3 mL / L DMF.

[0079] An embodiment of the present invention provides a method for preparing a 25-hydroxyvitamin D dissociation solution, specifically including the following steps:

[0080] a. Measure and mix a fixed amount of ethanol, ethylene glycol, and DMF;

[0081] b. Weigh a measured amount of decanoic acid and add it to the organic solvent mixture, then mix thoroughly;

[0082] c. Then add a fixed volume of 50 mM phosphate buffer at pH 7.2, mix thoroughly, and set aside.

[0083] Example 4

[0084] 25-hydroxyvitamin D detection procedure: A fully automated chemiluminescence analyzer was used, employing a double-antibody sandwich method. The procedure involved aspirating 10 μL of serum sample at 0 min, adding 50 μL of dissociation buffer, and incubating at 37°C for 5 min. Then, 50 μL of solid-phase reagent and 50 μL of luminescent reagent were added, and the mixture was incubated at 37°C for 10 min. The RLU (Radiative Luminescence Unit) value was then clearly defined and detected.

[0085] The commercially available standard 25-hydroxyvitamin D3 was used to prepare the master calibrator and working calibrator quality control sample. The samples were diluted according to the bottle label concentrations. Using calibrator diluent (0.01-0.05 mol / L PBS, 0.1%-2% BSA, 0.9%-25% NaCl, 0.1% PC-300, pH 7.2), 25-hydroxyvitamin D3 was prepared 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 solutions were dispensed in 1 mL vials and stored at -20°C for later use.

[0086] Calibration curve construction: The concentration of calibrator is used as the x-axis, and the luminescence value RLU corresponding to each concentration of calibrator is used as the y-axis. The fitting method is a quadratic polynomial function curve.

[0087] Sample concentration calculation method: Input the master curve information into the software, fit the master calibration curve, and the instrument automatically calculates the corresponding concentration value. The concentration is then assigned for traceability by comparing the serum sample results with those from the traceability reference mass spectrometry detection system.

[0088] Figure 1 The master calibration curve for 25-hydroxyvitamin D.

[0089] Example 5

[0090] Basic performance tests of the reagent kit:

[0091] (1) Blank limit LOB

[0092] Five 25-hydroxyvitamin D zero-value calibrators were measured five times each. The mean value (X) and standard deviation (SD) of the RLU of the 25 measurements were calculated. Substituting X + 2SD into the standard curve, the limit of detection of this method was found to be 0.47 ng / mL.

[0093] Table 1 Reagent sensitivity test

[0094]

[0095] (2) Linear range test

[0096] High-value calibrator sample H (150 ng / mL) and low-value calibrator sample L (2 ng / mL) were taken. Samples H and L were mixed in proportions of 10H, 9H+1L, 8H+2L, 7H+3L, 6H+4L, 5H+5L, 4H+6L, 3H+7L, 2H+8L, 1H+9L, and 10L to prepare 11 samples. Each sample was measured in triplicate, and the measured values ​​were compared with the theoretical values. The results showed that this reagent has good detection linearity in the range of 2.0-150 ng / mL.

[0097] Figure 2 This is a linear curve of this reagent in the range of 2.0-150 ng / mL.

[0098] (3) Thermal accelerated stability test

[0099] The 25-hydroxyvitamin D assay reagent was subjected to accelerated thermal treatment at 37°C for 7 days, followed by testing of the calibrator. The results showed that, compared to the reagent without accelerated thermal treatment, its RLU decreased by only 10%, indicating that this reagent possesses good stability.

[0100] Table 2 Thermal Acceleration Experiment

[0101]

[0102] (4) Accuracy and uniformity

[0103] The relative deviations of serum samples were calculated using mass spectrometry-assigned samples, which were compared to the gold standard. The mass spectrometry-assigned serum samples were provided by Meikang Shengde Medical Laboratory. The serum concentrations on the samples were: Sample A 8.99 ng / mL, Sample B 13.07 ng / mL, Sample C 21.01 ng / mL, Sample D 25.87 ng / mL, Sample E 33.58 ng / mL, and Sample F 51.97 ng / mL. The relative deviations from the mass spectrometry measurements were less than 6%, and the CV was less than 6%.

[0104] Table 3 Accuracy Results

[0105]

[0106] (5) Anti-interference analysis

[0107] Fresh serum from healthy individuals was collected and mixed serum was prepared. The interfering substances were diluted to specific concentrations using calibrator diluent (hemoglobin 5 mg / mL, vitamin C 30 mg / L, unconjugated bilirubin 0.2 mg / mL, conjugated bilirubin 0.2 mg / mL, chyle 1450 FTU). Interfering serum was prepared at a mixed serum:interfering substance volume ratio of 19:1, and control serum was prepared at a mixed serum:calibrator diluent volume ratio of 19:1. 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.

[0108] Table 4 Other anti-interference results

[0109]

[0110] (6) Clinical relevance analysis

[0111] The detection kit configured in Example 1 and mass spectrometry were used to test 50 clinical serum samples. The results are as follows: Figure 3 The correlation coefficient between the reagent of this invention and the control reagent is R. 2 =905, and the regression equation is y = 0.9968x + 2.3152. This result indicates that the reagent of this invention has good correlation, specificity, and accuracy with the control reagent.

[0112] Figure 3 Comparison of 25-hydroxyvitamin D assay results with mass spectrometry results.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 by, The 25-hydroxy vitamin D dissociation solution is composed of 10-100 mM and pH 7.2-7.4 buffer, 0.1%-5% long-chain alkane carboxylic acid, and 0.1%-1% organic solvent; The buffer includes at least one of Tris-HCl buffer, PBs buffer, and HEPES buffer; The long-chain alkane carboxylic acid is decanoic acid; The organic solvent includes at least one of DMSO, DMF, methanol, ethanol, ethylene glycol, and acetone.

2. A 25-hydroxy vitamin D test kit, characterized by, The 25-hydroxy vitamin D detection kit includes a solid-phase reagent, a luminescent reagent, and the 25-hydroxy vitamin D dissociation solution of claim 1.

3. The 25-hydroxyvitamin D test kit of claim 2, wherein, The solid-phase reagent and the luminescent reagent are both provided with a treatment solution including 10-100 mM and pH 7.0-7.4 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.

4. The 25-hydroxy vitamin D detection kit of claim 3, wherein 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 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.

5. The 25-hydroxyvitamin D test kit of claim 2, wherein, The preparation method of the solid-phase reagent includes: uniformly mixing carboxyl magnetic beads, taking 1 ml of 10 mg / ml magnetic beads in a centrifuge tube, washing the magnetic beads with an activation buffer for 3 times, then adding 1 ml of the activation buffer for resuspension and standby, weighing EDC and sulfo-NHS respectively, and adding the activation buffer to adjust the concentration to 10 mg / ml, adding EDC and sulfo-NHS to the standby magnetic beads, uniformly mixing, and placing on a shaker for reaction for 20 min, washing the activated magnetic beads with the activation buffer for 3 times, then adding a cross-linking buffer for resuspension to a concentration of 10 mg / ml, weighing the antibody, adding to the magnetic beads, uniformly mixing, and placing on a shaker for reaction for 2 h at room temperature, washing the magnetic beads with the cross-linking buffer for 3 times after the reaction, adding 1 ml of blocking buffer for resuspension, and reacting for 1 h at room temperature, washing the magnetic beads with the blocking buffer for 4 times after the blocking reaction, finally resuspending the magnetic beads to a concentration of 10 mg / ml, and adding the antibody-coated magnetic beads to the treatment solution, uniformly mixing, and obtaining the solid-phase reagent.

6. The 25-hydroxyvitamin D test kit of claim 2, wherein, The preparation method of the luminescent reagent comprises the following steps: dissolving acridine ester active substance in anhydrous DMSO at a concentration of 5 mg / mL; adding the acridine ester active substance and the labeled antibody into a cross-linking buffer at a molar ratio of 15:1, and reacting at room temperature for 2 h in the dark; adding 1 / 10 volume of lysine solution to terminate the reaction for 0.5 h after the reaction is completed; separating and purifying the reaction solution by using a Sephadex G25 column balanced with 0.1 mol / L and pH 7.4 PB buffer to obtain the acridine ester labeled labeled antibody; then adding an equal volume of glycerol, and storing the labeled antibody at -20 DEG C for standby; and adding the labeled antibody into a treatment solution, and uniformly mixing to obtain the luminescent reagent.

7. Use of a 25-hydroxy vitamin D dissociation solution as defined in any one of claims 1 or a 25-hydroxy vitamin D kit as defined in any one of claims 2 to 6, characterized in that, The application is used for detecting the content of 25-hydroxyvitamin D in a sample.

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