A 25-hydroxyvitamin d dissociation agent, and methods and kits for making same

Abstract

The embodiment of the application belongs to the technical field of biomedical detection, and relates to a 25-hydroxyvitamin D dissociation agent and a preparation method thereof. The 25-hydroxyvitamin D dissociation agent comprises a surfactant, a reducing agent, a stabilizer, a protective agent and a buffer. The content of the surfactant is 0.5%-4%(w / v); the content of the reducing agent is 0.1%-1.5%(w / v); the content of the stabilizer is 0.2-1.0%(w / v); the concentration of the protective agent is 1-20mM; and the concentration of the buffer is 20-50mM. The technical scheme provided in the application can fully release 25-hydroxyvitamin D from vitamin D binding protein, and the stability is good, so that the sensitivity and accuracy of detection are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, more particularly, to a 25-hydroxy vitamin D dissociation agent, a preparation method thereof and a kit. BACKGROUND

[0002] Vitamin D is an essential fat-soluble vitamin for the human body, which contains a cyclopentane polyhydrogenophenanthrene skeleton in the molecular structure, belongs to a sterol derivative, and has important physiological functions such as regulating calcium and phosphorus metabolism, promoting intestinal calcium and phosphorus absorption and bone calcification, and maintaining blood calcium and blood phosphorus balance. Vitamin D mainly has two forms of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), and the structural differences between the two are that the side chains are different. Vitamin D2 cannot be synthesized by the human body and can only be obtained through daily diet. Vitamin D3 is mainly derived from the conversion of 7-dehydrocholesterol in the skin under ultraviolet irradiation, and can also be obtained through diet, accounting for 90%-95% of the total content in the human body.

[0003] Vitamin D itself has no biological activity, needs to be combined with vitamin D binding protein, and is transported to the liver and kidney to undergo hydroxylation transformation to generate 25-hydroxy vitamin D and 1,25-hydroxy vitamin D before it can exert biological activity. Among them, 25-hydroxy vitamin D circulating in the human body is the main form of vitamin D, with a half-life of up to 3 weeks, suitable for reflecting the true content of vitamin D as a measured substance, so its blood level is considered in clinical practice as the best indicator reflecting the vitamin D status. Vitamin D binding protein is abundant in blood and firmly binds to vitamin D with high affinity, so how to make 25-hydroxy vitamin D dissociate from vitamin D efficiently and stably is the premise and basis for accurately measuring 25-hydroxy vitamin D in the sample.

[0004] The conventional several dissociation agents for dissociating vitamin D all have corresponding technical defects, such as: using the principle of similar dissolves similar to dissolve vitamin D in an organic solvent, but involving subsequent centrifugation and other steps, which is not convenient for automatic operation (organic solvent method); relying on too high or too low pH value to make vitamin D binding protein denature or change its conformation, thereby losing the ability to bind with vitamin D, but the system environment is not conducive to the subsequent immune reaction (strong acid and strong base method); using protease to hydrolyze vitamin D binding protein to release vitamin D, but the required enzymolysis time is too long (enzyme hydrolysis method); using vitamin D analogues to competitively replace vitamin D from vitamin D binding protein, but the added vitamin D analogues will interfere with the detection.

[0005] Based on the technical defects existing in the above several methods, there are also some reports on the optimization of vitamin D dissociation system at present, but there is still a lack of a simple operation, ideal dissociation effect, strong stability and high sensitivity dissociation system. For example: the preparation method of vitamin D dissociation agent is disclosed in the invention patent CN112557675A, which realizes the efficient dissociation of 25-hydroxyvitamin D and its metabolites by using composite surfactant (8-aniline-1-naphthalenesulfonic acid and fluorinated alkyl), but the solvent system is composed of dimethylformamide and dimethyl sulfoxide amine which has poor biological friendliness; the invention patent CN109738252A provides a 25-hydroxyvitamin D dissociation agent, which includes 10-50mM phosphate buffer, 0.1%-2% β-mercaptoethanol, 0.1%-2% guanidine isothiocyanate, 0.1%-2% disodium ethylenediaminetetraacetate, 0.1%-2% sodium dodecyl sulfonate and 0.1%-5% Tween-20, the dissociation reaction can be carried out under neutral pH conditions, but the extremely toxic β-mercaptoethanol is used as a reducing agent, and the preparation of the whole solution system is also relatively complicated; the invention patent CN111521832A discloses a 25-hydroxyvitamin D determination kit, the dissociation agent used is sodium acetate, which can improve the specificity and sensitivity of the measured substance in a low pH environment, but in a relatively mild neutral environment, the stability of the dissociation agent is poor, and the dissociation efficiency is also reduced. SUMMARY

[0006] The technical problem to be solved by the embodiments of the present application is that the dissociation of 25-hydroxyvitamin D in the prior art is complicated, the dissociation stability is poor, and the dissociation efficiency is low.

[0007] In order to solve the above technical problems, the embodiments of the present application provide a 25-hydroxyvitamin D dissociation agent, which adopts the following technical scheme:

[0008] The surface active agent, the reducing agent, the stabilizer, the protective agent and the buffer solution;

[0009] The content of the surface active agent is 0.5%-4%(w / v); the content of the reducing agent is 0.1%-1.5%(w / v); the content of the stabilizer is 0.2-1.0%(w / v); the concentration of the protective agent is 1-20mM; and the concentration of the buffer solution is 20-50mM.

[0010] Further, the surface active agent is at least one of sodium dodecyl sulfonate, perfluorooctanoic acid, Tween-20, Tween-40 and Triton X-100.

[0011] Further, the reducing agent is at least one of dithiothreitol, β-mercaptoethanol and tris(2-carboxyethyl) phosphine.

[0012] Further, the stabilizer is at least one of disodium ethylenediaminetetraacetate, 7-dehydrocholesterol, cholesterol, bile acid and the like.

[0013] Further, the protective agent is at least one of anthocyanins, coumarins, cinnamoyl esters, flavonoids, chromones.

[0014] Further, the buffer is a phosphate buffer.

[0015] Further, the buffer is used to provide a buffer environment, and the buffer environment pH value of the phosphate buffer is 6.8-7.4.

[0016] Further, the surfactant is sodium dodecyl sulfonate, the reducing agent is dithiothreitol, the stabilizer is disodium ethylenediaminetetraacetate, and the protective agent is anthocyanins.

[0017] To solve the above technical problems, the embodiment of the present application also provides a preparation method of 25-hydroxyvitamin D dissociation agent, which adopts the technical scheme as follows:

[0018] The surfactant, the reducing agent, the stabilizer, the protective agent and the buffer are respectively prepared;

[0019] The surfactant, the reducing agent, the stabilizer, the protective agent and the buffer are respectively prepared;

[0020] The content of the surfactant is 0.5%-4%(w / v); the content of the reducing agent is 0.1%-1.5%(w / v); the content of the stabilizer is 0.2-1.0%(w / v); the concentration of the protective agent is 1-20mM; and the concentration of the buffer is 20-50mM.

[0021] To solve the above technical problems, the embodiment of the present application also provides a 25-hydroxyvitamin D detection kit, which comprises the 25-hydroxyvitamin D dissociation agent as described above or prepared by the preparation method as described above.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The 25-hydroxyvitamin D dissociation agent provided by the present application has high dissociation efficiency for 25-hydroxyvitamin D, can fully release it from vitamin D binding protein, and has good stability, thereby improving the sensitivity and accuracy of detection. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the solutions in the application, the following will briefly introduce the drawings needed to be used in the embodiments, and obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0025] Figure 1 is a linear correlation result diagram according to the embodiment 1 of the application;

[0026] Figure 2 is a linear correlation result diagram according to the embodiment 2 of the application;

[0027] Figure 3 is a linear correlation result diagram according to the embodiment 3 of the application. DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application; the description and claims of this application as well as the above discussion of the drawings merely provide illustrative examples of the application, and one skilled in the art will recognize further variations of the application. It is therefore contemplated to cover any and all modifications, variations or equivalents that fall within the spirit and scope of the present application. As used herein, the terms "including", "containing", "having" and the like are intended to be open-ended terms that are to be interpreted in the context of the entire specification, and are not intended to be limiting. The terms "first", "second", and the like, as used herein do not denote any order, quantity, or importance, but are used to distinguish one element from another.

[0029] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily mutually exclusive of one another. As will be apparent to those of ordinary skill in the art, embodiments described herein can be combined with other embodiments.

[0030] In order to make the technical personnel in the art better understand the solutions of the application, the technical solutions in the embodiments of the application will be clearly and completely described below in conjunction with the drawings.

[0031] The 25-hydroxyvitamin D dissociation agent provided by the embodiments of the application comprises a surfactant, a reducing agent, a stabilizer, a protective agent and a buffer.

[0032] The content of the surfactant is 0.5%-4%(w / v); the content of the reducing agent is 0.1%-1.5%(w / v); the content of the stabilizer is 0.2-1.0%(w / v); the concentration of the protective agent is 1-20mM; and the concentration of the buffer is 20-50mM.

[0033] The application adds a reducing agent and a protective agent in the dissociation agent, which can efficiently destroy the protein conformation, protect the dissociated 25-hydroxy vitamin D, and make it exist stably, which is very beneficial to the subsequent detection.

[0034] In some embodiments, the surfactant is at least one of sodium dodecyl sulfonate, perfluorooctanoic acid, Tween-20, Tween-40, and Triton X-100.

[0035] Preferably, the surfactant is selected from sodium dodecyl sulfonate. Sodium dodecyl sulfonate is an anionic surfactant that can destroy the hydrogen bond force within and between protein molecules. Its dodecyl hydrophobic structure can insert into the internal hydrophobic region of the protein, destroy the hydrophobic interaction of the protein, and thus destroy the higher structure of the protein, so that the vitamin D binding protein is unfolded, and thus the 25-hydroxy vitamin D is dissociated.

[0036] The dodecyl sulfonate can be combined with the protein at a mass ratio of 1.4:1, which is the most commonly used high-efficiency surfactant for denatured protein in biochemical laboratories.

[0037] In some embodiments, the reducing agent is at least one of dithiothreitol, β-mercaptoethanol, and tris(2-carboxyethyl) phosphine.

[0038] Preferably, the reducing agent is selected from dithiothreitol.

[0039] Dithiothreitol is a strong reducing agent that can reduce the disulfide bonds formed between the cysteine residues of the protein, change the conformation of the protein, and facilitate the penetration of the protein structure by itself and sodium dodecyl sulfonate, thereby destroying the structure of the protein and releasing more 25-hydroxy vitamin D. Compared with the common reducing agent β-mercaptoethanol, dithiothreitol has lower irritation, toxicity, and action concentration, and has better performance.

[0040] In some embodiments, the stabilizing agent is at least one of disodium ethylenediaminetetraacetate, 7-dehydrocholesterol, cholesterol, bile acid, and the like.

[0041] Among them, the stabilizing agent is selected from disodium ethylenediaminetetraacetate.

[0042] Disodium ethylenediaminetetraacetate is used as a complexing agent here, which can cause the denatured vitamin D binding protein to aggregate and precipitate, so that the 25-hydroxy vitamin D buried in the deep structure of the vitamin D binding protein can be more completely dissociated.

[0043] In some embodiments, the protective agent is at least one of anthocyanins, coumarins, cinnamoyl esters, flavonoids, and chromones.

[0044] Among them, the protective agent is preferably selected from anthocyanins.

[0045] Anthocyanin is a water-soluble natural pigment, widely exists in plant body, belongs to flavonoid compounds, and is used as a protective agent. Anthocyanin is a strong antioxidant, which can protect the body from free radical damage. The addition of anthocyanin in the dissociation solution can effectively remove the free radicals existing in the sample, protect 25-hydroxy vitamin D from free radical attack, and ensure the accurate detection of 25-hydroxy vitamin D.

[0046] In some embodiments, the buffer is a phosphate buffer.

[0047] The phosphate buffer has the advantages of simple preparation, less influence of pH by temperature and dilution, etc., and is used herein to provide a buffer environment, so that the dissociation is carried out in a neutral environment with a pH of 6.8-7.4, which can provide a suitable environment for the binding of 25-hydroxy vitamin D and antibodies after dissociation.

[0048] In some embodiments, in the 25-hydroxy vitamin D dissociation agent, the surfactant is sodium dodecyl sulfate, the reducing agent is dithiothreitol, the stabilizer is disodium ethylenediaminetetraacetate, and the protective agent is anthocyanin.

[0049] The 25-hydroxy vitamin D dissociation agent of the present application can efficiently dissociate 25-hydroxy vitamin D from vitamin D binding protein, and has the advantages of complete dissociation, good stability, and little influence on subsequent reactions.

[0050] Based on the above-mentioned 25-hydroxy vitamin D dissociation agent, the present application further provides a preparation method of a 25-hydroxy vitamin D dissociation agent, comprising the following steps:

[0051] The surfactant, reducing agent, stabilizer, protective agent and buffer are prepared respectively;

[0052] The surfactant, reducing agent, stabilizer, protective agent and buffer are combined to form a 25-hydroxy vitamin D dissociation agent.

[0053] The present application also provides a 25-hydroxy vitamin D detection kit, which comprises the 25-hydroxy vitamin D dissociation agent as described above or prepared by the preparation method as described above.

[0054] The content of the present application will be described more specifically and further illustrated by the following specific examples, but these examples are by no means limiting to the present application.

[0055] Example 1

[0056] The composition and concentration of the dissociation agent of the present embodiment are shown in Table 1.

[0057] Table 1

[0058] Component Concentration Surfactant Sodium dodecylsulfate 1.5% (w / v) Reducing agent Dithiothreitol 0.8% (w / v) Stabilizer Disodium ethylenediaminetetraacetate 0.5% (w / v) Protectant Anthocyanin 3 mM Buffer Phosphate buffer 30 mM

[0059] The serum samples for 25-hydroxy vitamin D project which have been measured by the imported dissociation reagent (control) were collected, totally 60 cases (including low, medium and high values). A certain amount of each sample was taken into 3 sample reaction cups, respectively, a certain amount of dissociation reagent of Example 1 was added for dissociation, and the concentration (ng / mL) of 25-hydroxy vitamin D was determined by chemiluminescence method. Then the values measured by Example 1 and the control reagent were linearly fitted to obtain the correlation, and the results are shown in Table 2.

[0060] Table 2

[0061]

[0062]

[0063] Example 2

[0064] The composition and concentration of the dissociation reagent of this example are shown in Table 3.

[0065] Table 3

[0066] Component Concentration Surfactant Sodium dodecylsulfate 2% (w / v) Reducing agent Dithiothreitol 1.5% (w / v) Stabilizer Disodium ethylenediaminetetraacetate 0.5% (w / v) Protectant Anthocyanin 4 mM Buffer Phosphate buffer 20 mM

[0067] The serum samples for 25-hydroxy vitamin D project which have been measured by the imported dissociation reagent (control) were collected, totally 60 cases (including low, medium and high values). A certain amount of each sample was taken into 3 sample reaction cups, respectively, a certain amount of dissociation reagent of Example 1 was added for dissociation, and the concentration (ng / mL) of 25-hydroxy vitamin D was determined by chemiluminescence method. Then the values measured by Example 1 and the control reagent were linearly fitted to obtain the correlation, and the results are shown in Table 2.

[0068] Table 4

[0069]

[0070]

[0071] Example 3

[0072] The composition and concentration of the dissociation reagent of this example are shown in Table 5.

[0073] Table 5

[0074] Component Concentration Surfactant Sodium dodecylsulfate 1.5% (w / v) Reducing agent Dithiothreitol 0.8% (w / v) Stabilizer Disodium ethylenediaminetetraacetate 0.6% (w / v) Protectant Anthocyanin 2 mM Buffer Phosphate buffer 40 mM

[0075] Collect 60 serum samples (including low, medium and high values) for 25-hydroxy vitamin D project which have been measured by the imported dissociation agent (control). Take a certain amount of volume of each sample into 3 sample reaction cups, respectively, add a certain amount of dissociation agent of Example 3 for dissociation, and determine the concentration (ng / mL) of 25-hydroxy vitamin D by chemiluminescence method. Then, linear fitting is performed on the measured values of Example 3 and the control reagent to obtain the correlation, and the results are shown in Table 6.

[0076] Table 6

[0077]

[0078]

[0079] The results of Table 2, Table 4 and Table 6 show that the dissociation performance of Examples 1-3 is excellent, and the measured values are basically consistent with the results measured by the imported dissociation agent. In combination with Figures 1-3 It can also be seen that the linear correlation of Examples 1-3 is good, and the R2 value is above 0.96, indicating that the correlation is good.

[0080] Therefore, the novel dissociation agent for 25-hydroxy vitamin D combined with vitamin D binding protein provided in the present application can efficiently dissociate 25-hydroxy vitamin D in the sample.

[0081] Obviously, the above-described embodiments are only a part of the embodiments of the present application, and are not all the embodiments. The preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application.

[0082] The present application can be implemented in many different forms, and conversely, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacements to some technical features therein. Any equivalent structure made by using the content of the specification and drawings, directly or indirectly applied to other related technical fields, is also within the scope of the patent protection of the present application.

Claims

1. A 25-hydroxy vitamin D dissociating agent, characterized by, The 25-hydroxyvitamin D dissociation agent comprises: a surfactant, a reducing agent, a stabilizer, a protective agent and a buffer; wherein the content of the surfactant is 1.5%-2%(w / v); the content of the reducing agent is 0.8%-1.5%(w / v); the content of the stabilizer is 0.5-0.6%(w / v); the concentration of the protective agent is 2-4mM; and the concentration of the buffer is 20-50mM. The surfactant is sodium dodecyl sulfate, the reducing agent is dithiothreitol, the stabilizer is disodium ethylenediaminetetraacetate, and the protective agent is anthocyanin.

2. The 25-hydroxy vitamin D dissociating agent of claim 1, wherein, The buffer is a phosphate buffer.

3. The 25-hydroxy vitamin D dissociating agent of claim 2, wherein, The buffer is used to provide a buffer environment, and the buffer environment pH value of the phosphate buffer is 6.8-7.

4.

4. A process for the preparation of a 25-hydroxy vitamin D dissociating agent according to claim 1, characterized in that, The method comprises the following steps: preparing the surfactant, the reducing agent, the stabilizer, the protective agent and the buffer respectively; composing the surfactant, the reducing agent, the stabilizer, the protective agent and the buffer into the 25-hydroxyvitamin D dissociation agent.

5. A 25-hydroxyvitamin D test kit characterized in that, The 25-hydroxyvitamin D dissociation agent prepared by the preparation method of claim 4 is also included.

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