CN108089015B - Release agent for detecting 25 hydroxy vitamin D content in human serum sample and detection method thereof - Google Patents
Release agent for detecting 25 hydroxy vitamin D content in human serum sample and detection method thereof Download PDFInfo
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Abstract
The invention discloses a releasing agent for detecting 25-hydroxy vitamin D content in a human serum sample, which comprises 0.05-0.3% of sodium dodecyl sulfate, 1-20% of dimethyl sulfoxide and 1-10% of ethanol in mass-volume ratio; during preparation, firstly, sodium dodecyl sulfate is weighed and dissolved in a small amount of 0.05M Tris-NaCl buffer solution, then dimethyl sulfoxide and ethanol are added, the volume is determined by 0.05M Tris-NaCl buffer solution, and the pH value is adjusted to 7.0 by concentrated hydrochloric acid, so that a finished releasing agent is obtained. The invention has the advantages that the preparation method is simple, and the used components have less harm to the environment and organisms; during detection, 25-hydroxy vitamin D and metabolites thereof can be effectively released from the binding protein, so that the binding protein can be fully detected; the sample is not required to be pretreated, and can be added with other reagents at the same time, so that the automatic operation is convenient.
Description
Technical Field
The invention relates to an in vitro diagnosis technology, in particular to a releasing agent for quantitatively detecting the content of 25 hydroxyvitamin D in a human serum sample, and also relates to a method for detecting the content of 25 hydroxyvitamin D in the human serum sample by using the releasing agent.
Background
Vitamin D is a steroid hormone, of which there are two main types: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) which are structurally similar but differ in side chain. Vitamin D in human body is mainly derived from 7-dehydrocholesterol in skin, and is converted into vitamin D3 by ultraviolet irradiation, and also comprises vitamin D3 ingested from food such as fish and meat, and vitamin D2 in dietary supplement. Either form of vitamin D, which binds to binding proteins in the blood and is transported to the liver, is converted to 25(OH) VD in the liver and then to 1,25- (OH) in the kidney2VD, which is the bioactive component of vitamin D function, but 1,25- (OH) in the circulation in humans2The VD content is very small, and the half-life period is only 4 h. And the 25(OH) VD content in the cycle is 1,25- (OH)2The VD is 1000 times of the VD, is the main existing form of vitamin D in circulation, has a half-life period of 3 weeks, can reflect the real level of the vitamin D of a human body, and is the optimal index for reflecting the state of the vitamin D of an individual.
In human serum, almost all 25 hydroxyvitamin D binds to binding proteins, of which about 88% binds to vitamin D binding protein and about 12% binds to albumin the concentration of binding protein in serum is about 250-400 mg/L and the binding constant of binding protein to 25 hydroxyvitamin D is as high as Ka =6 × 105M-1，
The concentration of 25 hydroxyvitamin D in serum can only be accurately measured if 25 hydroxyvitamin D is effectively released from the binding protein.
There are various methods for releasing 25 hydroxyvitamin D from binding protein, and the method used in PCT International application WO99/67211 is to precipitate protein with ethanol, remove the precipitate, and recover the supernatant containing 25 hydroxyvitamin D to be detected for detection, but this method cannot be automated, and the detection result has large errors. US7482162 proposes a method of competitively substituting 25 hydroxyvitamin D from a binding protein using ANS, which applicant has verified to be incapable of dissociating the substance to be tested from its binding protein. PCT international application WO2008/092917a1 discloses the use of proteolytic digestion to dissociate 25 hydroxyvitamin D from its binding protein, but this procedure is cumbersome and time consuming. Other methods such as WO2003/023391 disclose the use of direct measurement of 25 hydroxy vitamin D by substitution of hydroxylated aromatic carboxylic acids; the release of 25 hydroxy vitamin D from vitamin D binding proteins using one or more amphoteric reagents with ph3.8 to 4.8 and 5-30% DMSO, liquid organic amides and optionally 0.5-5% short chain alcohols is proposed in EP2007/140962, the amphoteric compounds used in this patent being toxic hazardous substances. WO2012/091569 proposes the use of perfluoroalkyl acids or salts thereof to release 25 hydroxy vitamin D from vitamin D binding proteins, wherein perfluoroalkyl acids may pose a hazard to the environment and organisms.
Disclosure of Invention
The invention aims to provide a releasing agent for detecting the content of 25 hydroxyvitamin D in a human serum sample, aiming at the defects of the prior art, the releasing agent is easy to implement, and the components do not contain components harmful to the environment and organisms, so that the accurate detection of the concentration of 25 hydroxyvitamin D in the serum is ensured.
In order to achieve the purpose, the invention can adopt the following technical scheme:
the releasing agent for detecting the content of 25-hydroxy vitamin D in a human serum sample comprises 0.05-0.3% of sodium dodecyl sulfate, 1-20% of dimethyl sulfoxide and 1-10% of ethanol by mass volume ratio;
during preparation, firstly, sodium dodecyl sulfate is weighed and dissolved in a small amount of 0.05M Tris-NaCl buffer solution, then dimethyl sulfoxide and ethanol are added, the volume is determined by 0.05M Tris-NaCl buffer solution, and the pH value is adjusted to 7.0 by concentrated hydrochloric acid, so that a finished releasing agent is obtained.
Furthermore, the components comprise, by mass volume, 0.1-0.2% of sodium dodecyl sulfate, 5-15% of dimethyl sulfoxide and 3-8% of ethanol.
More preferably: in the components, the content of sodium dodecyl sulfate is 0.15%, the content of dimethyl sulfoxide is 10%, and the content of ethanol is 5%.
The method for detecting the content of 25-hydroxy vitamin D in a human serum sample by using the release agent prepared by the invention comprises the following steps:
firstly, respectively adding a calibrator and a specimen into a reaction container, sequentially adding a release agent, magnetic microspheres coupled with a 25-hydroxy vitamin D antibody and horseradish peroxidase-labeled 25-hydroxy vitamin D, oscillating and incubating;
secondly, adding a cleaning solution into the reaction container for washing five times;
and thirdly, adding a luminescent substrate into the reaction vessel, measuring the luminous intensity, drawing a standard curve according to the luminous intensity of the calibrator, and calculating the concentration of 25 hydroxyvitamin D in the sample to be tested.
The invention has the advantages that the preparation method is simple, and the used components have less harm to the environment and organisms; during detection, 25-hydroxy vitamin D and metabolites thereof can be effectively released from the binding protein, so that the binding protein can be fully detected; the sample is not required to be pretreated, and can be added with other reagents at the same time, so that the automatic operation is convenient.
Specifically, the innovation of the invention is mainly as follows:
1. sodium Dodecyl Sulfate (SDS) is applied to the dissociation process of 25-hydroxy vitamin D and binding protein for the first time, and can effectively prevent the secondary binding with the binding protein after releasing the 25-hydroxy vitamin D, thereby ensuring that all 25-hydroxy vitamin D in human serum treated by a releasing agent participates in the subsequent detection process;
2. all components in the release agent are the optimal formula under the system, and powerful guarantee is provided for effective dissociation of the release agent;
3. the releasing agent taking the sodium dodecyl sulfate as the main component is used for detecting the 25-hydroxy vitamin D in the serum sample, a sample pretreatment step is not needed during detection, and the final detection result has better consistency with the detection result of the Roche reagent kit, thereby having very good application prospect.
Drawings
FIG. 1 is a graph showing a standard substance obtained by the detection method of the present invention.
FIG. 2 is a graph showing the comparison between the detection results of the present invention and those of the Roche reagent kit.
Detailed Description
Firstly, the preparation of the releasing agent
The components are as follows: 0.15 percent (w/v) of sodium dodecyl sulfate, 10 percent (w/v) of dimethyl sulfoxide and 5 percent (w/v) of ethanol;
the preparation method comprises the following steps: firstly, dissolving sodium dodecyl sulfate in a small amount of 0.05M Tris-NaCl buffer solution, adding dimethyl sulfoxide and ethanol, then diluting to constant volume with 0.05M Tris-NaCl buffer solution, and finally adjusting the pH value to 7.0 with concentrated hydrochloric acid to obtain a finished releasing agent.
Second, method for detecting 25 hydroxy vitamin D content in human serum sample by using releasing agent prepared by the invention
First, preparation of relevant reagents
1. Preparation of magnetic microparticle suspension coated with anti-25-hydroxyvitamin D antibody: taking appropriate amount of carboxyl magnetic particles according to the use amount, activating with excessive EDC and NHS under acidic condition (pH 4.5-pH 5.5) with 0.05-0.1M MES (2- (N-morpholino) ethanesulfonic acid) buffer solution for 30 min; after activation, adding a magnetic field, and standing for 5min to separate the magnetic particles from the liquid; the supernatant was discarded and washed twice with 0.01M PBS buffer, pH7.6, to wash off excess activator; adding appropriate amount of anti-25-hydroxyvitamin D antibody to make its concentration be 22 μ g/mg magnetic particle, shaking and reacting in 0.05M-0.1M MES buffer (pH4.5-pH5.5) for 1 h; after the reaction, adding a magnetic field, standing for 5min to separate the magnetic particles from the liquid, discarding the supernatant, blocking with 0.01M PBS buffer (pH7.6) containing 1% Bovine Serum Albumin (BSA), and repeatedly blocking for 5 times, each for 10 min; after the sealing is finished, adding a proper amount of sealing liquid to preserve the magnetic particles, so that the final concentration of the magnetic particles is 0.5 mg/ml; storing the magnetic particle suspension at 2-8 deg.C for use;
2. preparation of 25-hydroxy vitamin D series of calibrators: preparing a series of calibration products with labeled concentrations of 0ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml and 140ng/ml by using a Tris-NaCl buffer solution containing 1-3% BSA and 0.1-0.3% PC300, wherein the colors of a bottle cover are white, yellow, green, blue, red, purple and black in sequence;
3. preparation of horseradish peroxidase-labeled 25-hydroxy vitamin D: condensing carboxyl on the 25-hydroxy vitamin D derivative and amino of horseradish peroxidase (HRP) molecules into an amide compound under the action of EDC, and dialyzing to obtain the enzyme-labeled 25-hydroxy vitamin D; adding glycerol into the marked solution at equal ratio and preserving at-20 ℃ for later use; adding the marked enzyme-labeled 25-hydroxy vitamin D into a Tris-NaCl buffer solution with the pH of 7.4 and containing 20 to 50 percent of calf serum and 0.15 to 0.25 percent of PC according to the proportion of 1:2000 to 1:5000, and uniformly mixing to obtain an enzyme conjugate used when the release agent is applied;
4. the human serum 25 hydroxy vitamin D releasing agent is prepared according to the method of the invention;
5. the luminescence substrate A solution is prepared from 0.2M Tris-HCl, 0.15mM L mininol, 0.59mM hydroxycoumarin and 0.35mM gallic acid, the luminescence substrate B solution is prepared from 0.85mM amino acid oxidase, 0.8% Tween 20, 0.5mM DTPA and 0.12mM vitamin C, and the concentrated washing solution is prepared from NaH2PO4·2H2O 4.06g, Na2HPO4·12H2O62.32 g, Tween 202-10 ml and distilled water 1000 ml.
Second, detecting
1. Taking out a certain amount of reaction containers (reaction holes) and numbering, and respectively adding 50 mul of calibrator, 50 mul of releasing agent prepared by the invention and 50 mul of clinical serum specimen into each reaction hole according to the experimental requirements;
2. shaking up the magnetic particle suspension, and adding 20 mul into each hole;
3. adding 50 mul of enzyme label into each hole;
4. mixing the solution in the reaction hole uniformly, and incubating for 15 minutes at 37 ℃;
5. washing the magnetic particles in the reaction holes with a washing solution for 5 times by using magnetic separation and washing equipment;
6. fully oscillating the washed reaction hole to disperse the magnetic particles;
7. adding 50 mul of luminescent substrate A and luminescent substrate B into each hole, shaking, uniformly mixing, and keeping out of the sun to react for 5 minutes;
8. detecting the luminescence intensity by using a chemiluminescence detection instrument;
9. and establishing a calibration curve by adopting a four-parameter fitting mode and taking the concentration value of the calibrator as an X axis and the logarithm value of the luminous intensity of the calibrator as a Y axis. And calculating back the corresponding concentration value according to the luminous intensity value of the sample to be detected.
By adopting the method, the detection of the calibrator is carried out, the linear R of the standard curve is more than 0.999, as shown in figure 1, the abscissa is the concentration of the calibrator, and the ordinate is the signal value corresponding to each calibrator.
Third, comparative experiment
177 clinical serum samples are taken, detection is carried out according to the steps of the method for detecting the content of 25 hydroxyvitamin D in the human serum sample, and meanwhile, a 25 hydroxyvitamin D detection kit of Roche company is adopted for detection. The data of the detection results are compared in table 1 below.
TABLE 1 comparison of results of Roche assay with the assay of the present invention
The result shows that the linear regression equation of the detection result (y) by adopting the method and the detection result (x) of the 25-hydroxyvitamin D kit of Roche company is y = 0.9763x-0.849, and the related coefficient can reach R2=0.9544, as shown in fig. 2, the abscissa (x axis) is the detection result of the kit of roche company, the ordinate (y axis) is the detection result of the detection method of the present invention, the fitting linear regression equation is y = 0.9786x-0.8874, and the correlation coefficient can reach R2= 0.9572. The data show that the detection method has better consistency with the detection result of the Roche kit due to the adoption of the 25-hydroxy vitamin D releasing agent, can be better applied to the detection of 25-hydroxy vitamin D in a human serum sample, and has better application prospect.
Claims (2)
1. A releasing agent for detecting 25 hydroxyvitamin D content in a human serum sample, comprising: according to the mass volume ratio, the components contain 0.1-0.2% of sodium dodecyl sulfate, 5-15% of dimethyl sulfoxide and 3-8% of ethanol;
during preparation, firstly, weighing sodium dodecyl sulfate, dissolving the sodium dodecyl sulfate in a small amount of 0.05M Tris-NaCl buffer solution, adding dimethyl sulfoxide and ethanol, fixing the volume by using 0.05M Tris-NaCl buffer solution, and adjusting the pH value to 7.0 by using concentrated hydrochloric acid to obtain a finished releasing agent;
the method for detecting the content of 25-hydroxy vitamin D in a human serum sample by using the releasing agent comprises the following steps:
firstly, respectively adding a calibrator and a specimen into a reaction container, sequentially adding a release agent, magnetic microspheres coupled with a 25-hydroxy vitamin D antibody and horseradish peroxidase-labeled 25-hydroxy vitamin D, oscillating and incubating;
secondly, adding a cleaning solution into the reaction container for washing five times;
and thirdly, adding a luminescent substrate into the reaction vessel, measuring the luminous intensity, drawing a standard curve according to the luminous intensity of the calibrator, and calculating the concentration of 25 hydroxyvitamin D in the sample to be tested.
2. The releasing agent for detecting 25 hydroxyvitamin D content in human serum samples according to claim 1, wherein: in the components, sodium dodecyl sulfate is 0.15%, dimethyl sulfoxide is 10%, and ethanol is 5%.
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| CN109632444A (en) * | 2018-12-24 | 2019-04-16 | 郑州安图生物工程股份有限公司 | Liquid 25-hydroxy-vitamin D calibration object dilution |
| CN109813592A (en) * | 2019-03-26 | 2019-05-28 | 济南维瑞医药科技开发有限公司 | The identification pre-treating method of vitamine D3 in one vegetable oil |
| CN110749664A (en) * | 2019-08-29 | 2020-02-04 | 重庆同怡生物技术研究院有限公司 | Dissociation method of 25-hydroxy vitamin D binding protein |
| CN113495159A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | System reaction liquid, 25-hydroxy vitamin D quantitative detection kit and use method thereof |
| CN112557675B (en) * | 2020-12-11 | 2024-08-02 | 郑州安图生物工程股份有限公司 | Dissociation agent for 25-hydroxy vitamin D and metabolite thereof, and preparation method and application thereof |
| CN117825698B (en) * | 2024-03-05 | 2024-06-07 | 江苏奥雅生物科技有限公司 | Dissociating agent for dissociating vitamin D, detection method and application |
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| WO2012136720A1 (en) * | 2011-04-04 | 2012-10-11 | Immundiagnostik Ag | Analysing vitamin d metabolites in dried blood |
| CN106199015A (en) * | 2016-06-27 | 2016-12-07 | 盐城拜明生物技术有限公司 | Vitamin D releasing agent for serum sample detection, preparation method and application |
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| US7087395B1 (en) * | 2001-01-16 | 2006-08-08 | Quest Diagnostics Investments Incorporated | Vitamin D assay |
| JP4852614B2 (en) * | 2005-09-29 | 2012-01-11 | エフ．ホフマン−ラ ロシュ アーゲー | Release reagents for vitamin D compounds |
| CN102906570B (en) * | 2010-05-20 | 2014-11-05 | 霍夫曼-拉罗奇有限公司 | Release reagent for vitamin D compounds |
| PL3358356T3 (en) * | 2013-09-17 | 2021-03-08 | bioMérieux | Solution for dissociating vitamin d from vitamin d-binding protein, associated detection method and use |
| CN106199016A (en) * | 2016-06-30 | 2016-12-07 | 深圳市亚辉龙生物科技股份有限公司 | 25 hydroxy-vitamine D chemiluminescence immune detection reagent kits and preparation method thereof |
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| WO2012136720A1 (en) * | 2011-04-04 | 2012-10-11 | Immundiagnostik Ag | Analysing vitamin d metabolites in dried blood |
| CN106199015A (en) * | 2016-06-27 | 2016-12-07 | 盐城拜明生物技术有限公司 | Vitamin D releasing agent for serum sample detection, preparation method and application |
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