CN113884687B - Dissociation agent for detecting content of 25-hydroxy vitamin D and preparation method thereof - Google Patents
Dissociation agent for detecting content of 25-hydroxy vitamin D and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a dissociating agent for detecting the content of 25-hydroxyvitamin D in a human blood sample, which consists of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidine isothiocyanate, perfluorooctanoic acid and buffer solution; the buffer solution is phosphate buffer solution. The dissociation agent of the vitamin D for the blood sample detection in the technical scheme has full dissociation, solves the problem that the sensitivity of a domestic 25-hydroxyvitamin D detection kit is lower than that of an imported kit, has accurate detection result, small measured value CV, simple operation, short reaction time and thorough release, can be dissociated within 1-2 minutes after the sample is added, and neutral buffer solution does not influence immunoreaction after dissociation and can be matched with the application of a rapid detection kit; ensures the accurate detection of the concentration of 25-hydroxy vitamin D in serum.
Description
Technical Field
The invention relates to the technical field of detection kits, in particular to a dissociation agent for detecting the content of 25-hydroxy vitamin D in a human blood sample and a preparation method thereof.
Background
In clinical practice, serum levels of 25-hydroxyvitamin D are considered to be a major indicator of vitamin D status, an important substance involved in many biological processes in humans and animals. Deficiency of vitamin D can lead to serious diseases such as osteoporosis and osteomalacia. Excessive vitamin D has toxicity, affects intestinal calcium absorption, causes hypercalcemia, and also has hypertension, gastrointestinal problems, etc. By measuring or quantifying vitamin D, i.e. determining its concentration, potential defects or excesses can be identified, making disease control good.
At present, the VD dissociating agents have the following several types, but all have corresponding technical defects.
(1) The strong acid and strong alkali method leads vitamin D binding protein to be denatured or to be changed in conformation through overhigh or overlow pH value, thereby losing the binding capacity with vitamin D and releasing the vitamin D, but the vitamin D buffer solution obtained by the method is not beneficial to the next immunoreaction with VD antibody due to the pH problem;
(2) and (3) a protease hydrolysis method, wherein protease is added into the sample to hydrolyze the vitamin D binding protein and release the vitamin D. However, the method has the defects that the hydrolysis time is too long, and the original purpose of developing a rapid detection kit is violated;
(3) an organic solvent dissolving method, which utilizes the principle of similar intermiscibility, uses ethanol or acetonitrile to extract precipitated protein, removes precipitates, and recovers supernatant containing 25-hydroxy vitamin D to be detected for detection, but the method can not be automatically completed and has larger detection error;
therefore, there is a need to develop a dissociation agent for detecting 25-hydroxyvitamin D content in human blood samples, which has simple operation, sufficient dissociation and good effect.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a dissociation agent for detecting the content of 25-hydroxyvitamin D in a human blood sample, which can realize short reaction time and complete release when dissociating the 25-hydroxyvitamin D and can react under the condition of neutral pH.
In order to solve the technical problems, the technical scheme adopted by the invention is that the dissociation agent for detecting the content of 25-hydroxy vitamin D in a human blood sample consists of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate, perfluorooctanoic acid and buffer solution;
the buffer solution is phosphate buffer solution.
The dissociation agent of the vitamin D for the blood sample detection in the technical scheme has full dissociation, solves the problem that the sensitivity of a domestic 25-hydroxyvitamin D detection kit is lower than that of an imported kit, has accurate detection result, small measured value CV, simple operation, short reaction time and thorough release, can be dissociated within 1-2 minutes after the sample is added, and neutral buffer solution does not influence immunoreaction after dissociation and can be matched with the application of a rapid detection kit; ensures the accurate detection of the concentration of 25-hydroxy vitamin D in blood.
Preferably, the concentration of the phosphate buffer solution is 20mM, wherein the mass volume ratios of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate and perfluorooctanoic acid dissolved in the phosphate buffer solution are 0.5% -1%, 0.8% -1%, 0.5% -1% and 0.5% -1%, respectively.
Preferably, the mass to volume ratios of disodium EDTA, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate and perfluorooctanoic acid dissolved in said phosphate buffer are 0.5%, 0.8%, 0.5% and 0.5%, respectively.
The invention aims to solve another technical problem of providing a preparation method of a dissociation agent for detecting the content of 25-hydroxy vitamin D in a human blood sample, which comprises the following steps:
s1: preparing 20mM phosphate buffer solution with the pH value of 7.4;
s2: dissolving EDTA disodium and guanidinium isothiocyanate in phosphate buffer solution, and adjusting pH to 7.4;
s3: dissolving dimethylethanolamine and dimethylformamide into the phosphate buffer solution obtained in the step S2, and putting the dimethylethanolamine and dimethylformamide into an ultrasonic cleaning machine for ultrasonic dissolution;
s4: and (4) dissolving perfluorooctanoic acid in the phosphate buffer solution obtained in the step S3, putting the solution into an ultrasonic cleaning machine for ultrasonic dissolution, and adjusting the pH value to 7.4 after ultrasonic dissolution.
Preferably, in step S2, 0.5% by weight/volume of disodium EDTA and 0.5% by weight/volume of guanidine isothiocyanate are weighed.
Preferably, in step S3, 0.8% by weight/volume of dimethylethanolamine and 0.8% by weight/volume of dimethylformamide are weighed.
Preferably, in step S4, 0.5% by mass/volume of perfluorooctanoic acid is weighed.
Drawings
The following further detailed description of embodiments of the invention is made with reference to the accompanying drawings:
FIG. 1 is a graph showing the correlation analysis of the results of the detection in the first embodiment;
FIG. 2 is a graph showing the correlation analysis of the results of the detection in the second embodiment;
FIG. 3 is a graph showing the correlation analysis of the results of the detection in the third embodiment;
FIG. 4 is a graph showing the correlation analysis of the results of the detection in the fourth embodiment.
Detailed Description
The dissociation agent for detecting the content of 25-hydroxy vitamin D in a human blood sample consists of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidine isothiocyanate, perfluorooctanoic acid and buffer solution; the buffer solution is a phosphate buffer solution, the concentration of the phosphate buffer solution is 20mM, wherein the mass volume ratios of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate and perfluorooctanoic acid dissolved in the phosphate buffer solution are respectively 0.5-1%, 0.8-1%, 0.5-1% and 0.5-1%; the optimum content of the components is 0.5%, 0.8%, 0.5% and 0.5% by mass/volume of disodium EDTA, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate and perfluorooctanoic acid, respectively, dissolved in a 20mM phosphate buffer.
Selecting 20 blood samples, respectively dissociating the 20 blood samples by using the first to fourth schemes, determining the dissociated blood samples by using a Roche kit (25-hydroxyvitamin D detection kit: cat 07464215), wherein a dissociating agent in the Roche kit is not used, comparing the determination result with the data of the blood samples which are completely determined by using the Roche kit, and performing deviation comparison and correlation analysis on the two.
Wherein:
the first embodiment is as follows: the content of the phosphate buffer solution in this example was 20mM, and the content of disodium EDTA added was 0.5%; the content of dimethylethanolamine is 0.8%, the content of dimethylformamide is 0.8%, and the pH is 7.4; the component content percentages in this example are mass to volume ratios.
The specific measurement data table is shown in the following table 1:
TABLE 1
The correlation analysis of the measurement results in the first embodiment is shown in fig. 1, and it can be seen from the table data and fig. 1 that the first embodiment has dissociation effect but does not completely dissociate.
The second embodiment is: the content of the phosphate buffer solution in this example was 20mM, and the content of disodium EDTA added was 0.5%; the content of dimethylethanolamine is 0.8 percent, the content of dimethylformamide is 0.8 percent, the content of perfluorooctanoic acid is 0.5 percent, and the pH value is 7.4; the component content percentages in this example are mass to volume ratios.
The specific measurement data table is shown in the following table 2:
TABLE 2
The correlation analysis of the measurement results in the second embodiment is shown in fig. 2, and it can be seen from the table data and fig. 2 that the dissociation effect is enhanced and the correlation trend is good as compared with the first embodiment.
The third embodiment is: the content of the phosphate buffer solution in this example was 20mM, and the content of disodium EDTA added was 0.5%; the content of dimethylethanolamine is 0.8%, the content of dimethylformamide is 0.8%, the content of guanidinium isothiocyanate is 0.5%, and the pH is 7.4; the component content percentages in this example are mass to volume ratios.
The specific measurement data table is shown in the following table 3:
TABLE 3
The correlation analysis of the measurement results in the third embodiment is shown in fig. 3, and it can be seen from the table data and fig. 3 that the dissociation effect is enhanced and the correlation trend is good as compared with the second embodiment.
The fourth embodiment is: the content of the phosphate buffer solution in this example was 20mM, and the content of disodium EDTA added was 0.5%; the content of dimethylethanolamine is 0.8%, the content of dimethylformamide is 0.8%, the content of guanidinium isothiocyanate is 0.5%, the content of perfluorooctanoic acid is 0.5%, and the pH is 7.4; the component content percentages in this example are mass to volume ratios.
The specific measurement data table is shown in table 4 below:
TABLE 4
The correlation analysis of the measurement results in the fourth embodiment is shown in fig. 4, and it can be seen from the table data and fig. 4 that the dissociation effect is significantly enhanced compared to the first, second, and third embodiments, and the measured values are very consistent with those of the roche kit and the deviation is not large; the fourth scheme is the best scheme.
According to the fourth preferred embodiment compared with the first to fourth embodiments, 20 blood samples were dissociated for 1-8min and then measured with a Roche kit once per minute to confirm the dissociation effect (the effect of dissociation time on subsequent reactions).
The specific measurement data table is shown in table 5 below:
TABLE 5
From the results in the table, it can be seen that the sample can be completely dissociated after the dissociating agent is added, the result is close to that in 1-8min, the dissociation effect is stable, the dissociation time is short, and the sample can be fully dissociated without long time.
According to the best scheme IV compared with the first to fourth embodiments, three groups of samples are selected and used for respectively measuring TT3 and TT4, the samples are measured by using a Roche kit after being dissociated, the same sample is repeatedly measured for 20 times, and the influence of the dissociating agent on the measured value CV is observed; three groups of samples were assayed simultaneously and completely using the Roche kit.
The specific measurement data table is shown in table 6 below:
TABLE 6
As can be seen from the results in the table above, the measured value CV after dissociation of the dissociation agent for detecting the content of 25-hydroxyvitamin D in a human blood sample is relatively stable, and the problem of insufficient dissociation does not exist.
It should be noted that the preparation method of the dissociation agent for detecting the content of 25-hydroxy vitamin D in the human blood sample of the best embodiment comprises the following steps:
s1: preparing 20mM phosphate buffer solution with the pH value of 7.4;
s2: dissolving EDTA disodium and guanidinium isothiocyanate in phosphate buffer solution, and adjusting pH to 7.4;
s3: dissolving dimethylethanolamine and dimethylformamide into the phosphate buffer solution obtained in the step S2, and putting the dimethylethanolamine and dimethylformamide into an ultrasonic cleaning machine for ultrasonic dissolution;
s4: dissolving perfluorooctanoic acid in the phosphate buffer solution obtained in the step S3, placing the solution into an ultrasonic cleaning machine for ultrasonic dissolution, and adjusting the pH value to 7.4 after ultrasonic dissolution;
in the step S2, 0.5% by mass volume of disodium EDTA and 0.5% by mass volume of guanidine isothiocyanate are weighed;
in the step S3, dimethylethanolamine and dimethylformamide are weighed according to the mass volume ratio of 0.8%; putting the mixture into an ultrasonic cleaning machine for ultrasonic treatment for 15 minutes;
in the step S4, the perfluorooctanoic acid with a mass volume ratio of 0.5% is weighed; putting the mixture into an ultrasonic cleaning machine for ultrasonic treatment for 15 minutes.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A dissociating agent for detecting the content of 25-hydroxy vitamin D in a human blood sample is characterized by consisting of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidine isothiocyanate, perfluorooctanoic acid and buffer solution;
the buffer solution is phosphate buffer solution;
the concentration of the phosphate buffer solution is 20mM, wherein the mass volume ratios of EDTA disodium, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate and perfluorooctanoic acid dissolved in the phosphate buffer solution are 0.5% -1%, 0.8% -1%, 0.5% -1% and 0.5% -1%, respectively.
2. The dissociation agent for detecting 25-hydroxyvitamin D content in a human blood sample according to claim 1, wherein the mass-to-volume ratios of disodium EDTA, dimethylethanolamine, dimethylformamide, guanidinium isothiocyanate and perfluorooctanoic acid dissolved in the phosphate buffer are 0.5%, 0.8%, 0.5% and 0.5%, respectively.
3. A preparation method of a dissociation agent for detecting the content of 25-hydroxy vitamin D in a human blood sample is characterized by comprising the following steps:
s1: preparing 20mM phosphate buffer solution with the pH value of 7.4;
s2: dissolving EDTA disodium and guanidinium isothiocyanate in phosphate buffer solution, and adjusting pH to 7.4;
s3: dissolving dimethylethanolamine and dimethylformamide into the phosphate buffer solution obtained in the step S2, and putting the dimethylethanolamine and dimethylformamide into an ultrasonic cleaning machine for ultrasonic dissolution;
s4: and (4) dissolving perfluorooctanoic acid in the phosphate buffer solution obtained in the step S3, putting the solution into an ultrasonic cleaning machine for ultrasonic dissolution, and adjusting the pH value to 7.4 after ultrasonic dissolution.
4. The method of claim 3, wherein in step S2, disodium EDTA and 0.5% guanidinium isothiocyanate are weighed in a mass/volume ratio of 0.5%.
5. The method as claimed in claim 3, wherein in step S3, the mass volume ratio of dimethylethanolamine and dimethylformamide is 0.8%.
6. The method for preparing a dissociation agent for detecting 25-hydroxy vitamin D content in a human blood sample according to claim 3, wherein in the step S4, perfluorooctanoic acid with a mass-to-volume ratio of 0.5% is weighed.
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| US20040132104A1 (en) * | 2003-01-07 | 2004-07-08 | Sackrison James L. | Vitamin D assay |
| ATE472734T1 (en) * | 2007-02-01 | 2010-07-15 | Immundiagnostik Ag | DIRECT DETERMINATION OF VITAMIN D IN SERUM OR PLASMA |
| US20140370616A1 (en) * | 2011-03-25 | 2014-12-18 | Nanospeed Diagnostics Inc. | Lateral flow immunoassay for detecting vitamins |
| US9140711B2 (en) * | 2011-04-04 | 2015-09-22 | Immundiagnostik Ag | Determination of vitamin D metabolites in dried blood |
| US20140162294A1 (en) * | 2012-12-06 | 2014-06-12 | General Atomics | Methods and compositions for assaying vitamin d |
| US20140273021A1 (en) * | 2013-03-14 | 2014-09-18 | Enzo Biochem, Inc. | Vitamin d assays |
| US20150212099A1 (en) * | 2014-01-30 | 2015-07-30 | General Atomics | Methods and compositions for assaying vitamin d |
| EP3168619B1 (en) * | 2014-07-10 | 2021-10-20 | Shenzhen New Industries Biomedical Engineering Co., Ltd. | Detection agent for detecting 25-hydroxy vitamin d, preparation method and use |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101273272A (en) * | 2005-09-29 | 2008-09-24 | 霍夫曼-拉罗奇有限公司 | Release reagent for vitamin D compounds |
| CN102985826A (en) * | 2010-04-01 | 2013-03-20 | 未来诊断有限公司 | Immunoassay for free vitamin D |
| CN102906570A (en) * | 2010-05-20 | 2013-01-30 | 霍夫曼-拉罗奇有限公司 | Release reagent for vitamin d compounds |
| CN104955841A (en) * | 2013-01-28 | 2015-09-30 | 索灵股份公司 | Method and kit for detecting 1,25-dihydroxyvitamin D and related antibodies |
| CN105556313A (en) * | 2013-09-17 | 2016-05-04 | 生物梅里埃公司 | Solution for dissociating vitamin D from vitamin D-binding protein, associated detection method and use |
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