CN117825698A - Dissociating agent for dissociating vitamin D, detection method and application - Google Patents
Dissociating agent for dissociating vitamin D, detection method and application Download PDFInfo
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- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 59
- 238000001514 detection method Methods 0.000 title claims abstract description 46
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- 229930003316 Vitamin D Natural products 0.000 title claims abstract description 23
- 235000019166 vitamin D Nutrition 0.000 title claims abstract description 23
- 239000011710 vitamin D Substances 0.000 title claims abstract description 23
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- JWUBBDSIWDLEOM-NQZHSCJISA-N 25-hydroxy-3 epi cholecalciferol Chemical compound C1([C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=CC=C1C[C@H](O)CCC1=C JWUBBDSIWDLEOM-NQZHSCJISA-N 0.000 description 4
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 4
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of in-vitro diagnosis, and discloses a dissociation agent for dissociating vitamin D, which comprises the following components: tris-HCl buffer, sodium salicylate, polysorbate-20, sodium dodecyl sulfate, proclin950, pH7.0-8.0. Compared with the prior art, the invention has at least the following beneficial effects: (1) The invention has no toxicity and corrosiveness to human body, and no direct or indirect toxicity hazard and risk to producers and users; (2) The dissociating agent component belongs to a degradable component, can not accumulate in the nature or the environment, and has no pollution to the environment; (3) The dissociation agent has good dissociation effect on 25 hydroxy vitamin D in the blood sample, can simultaneously meet the automatic detection of the sample, obtain accurate detection results, and reduce the risk of excessive intake of 25 hydroxy vitamin D2.
Description
Technical Field
The invention relates to an in-vitro diagnosis technology, in particular to a dissociation agent for dissociating vitamin D, a detection method and application.
Background
Vitamin D is a steroid hormone, of two main types: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), which are structurally very similar, but differ in side chains. Vitamin D in humans is mainly derived from 7-dehydrocholesterol in the skin, which is converted into vitamin D3 by uv irradiation, and further includes vitamin D3 taken from foods such as fish, meat, etc., and vitamin D2 in dietary supplements. Either form of vitamin D is bound to the binding protein in the blood and transported to the liver where it is converted to 25 hydroxy vitamin D and then to 1,25- (OH) in the kidney 2 VD, which is a biologically active ingredient that vitamin D plays a role, but is 1,25- (OH) in the human body's internal circulation 2 VD was very low and the half-life was only 4 hours. While the 25 hydroxy vitamin D content in the cycle is 1,25- (OH) 2 VD is 1000 times, is the main existence form of vitamin D in circulation, has a half-life period of 3 weeks, can reflect the real level of vitamin D in human body, and is the best index for reflecting the state of vitamin D in individuals.
25 hydroxy vitamin D is the main storage form of vitamin D in organism, and accounts for more than 95% of the total amount. The half-life is long (2-3 weeks), and is not affected by the level of blood calcium and parathyroid hormone, and therefore is recognized as an optimal index for objectively evaluating the nutritional status of vitamin D.
The detection of the 25-hydroxy vitamin D can reflect the condition of the vitamin D of the human body, and has important significance in the aspects of diagnosis and monitoring of rickets, osteoporosis and the like.
Due to the tight binding of 25 hydroxy vitamin D to the binding protein in the blood circulation (affinity constants up to 1X 10 7 ～1×10 9 ) Accurate quantification of 25 hydroxy vitamin D must be accomplished by first thoroughly separating it from the binding protein.
The method for measuring the concentration of the serum 25 hydroxy vitamin D comprises a plurality of methods such as high performance liquid chromatography, competitive protein binding method, radioimmunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, enzyme-linked immunoassay, fluorescence immunoassay and the like. These methods can be categorized into two broad categories, chromatographic methods and immunological methods.
The most outstanding advantage of the chromatographic method for measuring the 25-hydroxy vitamin D in serum is that the respective concentrations of the 25-hydroxy vitamin D2 and the 25-hydroxy vitamin D3 can be accurately distinguished, but the chromatographic method requires a large sample size, the sample pretreatment is complicated and takes a long time, the instrument operation is complicated, the requirement on operators is high, and the instrument price is high.
In the immunological method, the competitive protein binding method and the radioimmunomethod have the defects of higher sensitivity, use of radioisotope labels, short half-life of reagents, requirement of professional equipment such as Gamma counter and the like, difficult treatment of test wastes, larger required sample size and the like, and influence the application of the method. The detection of the ELISA, the chemiluminescent immunoassay, the electrochemiluminescent immunoassay and the fluorescent immunoassay requires smaller sample quantity, and in clinical practical application, many users prefer the ELISA, the chemiluminescent immunoassay, the electrochemiluminescent immunoassay and the fluorescent immunoassay without radioactive pollution.
Although more than 95% of 25 hydroxy vitamin D in human blood is 25 hydroxy vitamin D3, small amounts of 25 hydroxy vitamin D2 are still present and high concentrations of 25 hydroxy vitamin D2 may be measured in patients who are artificially supplemented with 25 hydroxy vitamin D2. Therefore, the cross-reactivity of the detection method to 25 hydroxy vitamin D3 and 25 hydroxy vitamin D2 is 100% when the concentration of 25 hydroxy vitamin D in human blood is measured. Only radioimmunoassay and chemiluminescent immunoassay kits from DiaSorin corporation and fluoroimmunoassay kits from Tosoh Bioscience have been available to achieve 100% cross-reactivity for both 25 hydroxy vitamin D2 and 25 hydroxy vitamin D3. The cross-reactivity of the kit produced by other companies to 25 hydroxy vitamin D2 is not up to 100%. This may lead to an underestimation of the 25 hydroxy vitamin D concentration of blood samples of those patients who are supplemented with 25 hydroxy vitamin D2, thereby exposing them to the risk of excessive intake of 25 hydroxy vitamin D2.
The current commercial immunological methods have various methods for separating vitamin D and binding protein and releasing 25 hydroxy vitamin D from binding protein, and the method used in PCT International application WO99/67211 is to precipitate protein by ethanol, remove precipitate and recover supernatant containing 25 hydroxy vitamin D to be detected for detection, but the method cannot be completed by automation and has larger error in detection result. PCT international application WO2008/092917A1 discloses the use of proteolytic digestion to dissociate 25 hydroxy vitamin D from its binding protein, but this method is cumbersome and time consuming. EP2007/140962 proposes the use of one or more amphoteric agents having a pH of 3.8 to 4.8 and 5-30% DMSO, a liquid organic amide and optionally 0.5-5% short chain alcohol to release 25 hydroxy vitamin D from vitamin D binding protein, 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 the perfluoroalkyl acids may be harmful to the environment and organisms.
Disclosure of Invention
The present invention first provides a dissociating agent for dissociating vitamin D, the dissociating agent comprising: tris-HCl buffer, sodium salicylate, polysorbate-20, sodium dodecyl sulfate, proclin950, pH7.0-8.0; the dissociating agent is used to dissociate vitamin D from the vitamin D binding protein.
Preferably, the dissociating agent comprises 20-100 mM Tris-HCl buffer, 0.005-0.05 g/mL sodium salicylate, 0.05% -0.2% polysorbate-20, 0.001-0.01 g/mL sodium dodecyl sulfate, 0.1% -0.5% Proclin950, pH7.0-8.0.
Preferably, the dissociating agent is 50mM Tris-HCl buffer, 0.01 g/mL sodium salicylate, 0.1% polysorbate-20, 0.005 g/mL sodium lauryl sulfate, 0.3% Proclin950, pH7.5.
The invention also provides a preparation method of the dissociation agent, which comprises the following steps:
(1) Preparing Tris-HCl buffer solution, and sequentially adding sodium salicylate, polysorbate-20, sodium dodecyl sulfate and Proclin950 while stirring to obtain a stable system;
(2) Then hydrochloric acid is used for adjusting the pH value to 7.0-8.0, the mixture is fully and uniformly mixed, and a 0.45 mu m filter membrane is used for filtering, so that the 25-hydroxy vitamin D dissociating agent is obtained.
The invention also provides a 25-hydroxy vitamin D detection kit which comprises the dissociation agent.
Preferably, the kit further comprises a reagent R1, a reagent R2, a product calibrator, a start reagent and a washing liquid.
Preferably, the reaction reagent R1 is a solid phase conjugate with a certain concentration;
optionally the reactant R1 is a 25 hydroxy vitamin D in combination with magnetic beads;
the reactant R2 is a conjugate with a certain concentration; optionally, the reagent R2 is an acridinium ester-labeled anti-25 hydroxyvitamin D-specific antibody;
the product calibrator comprises S1 and S2;
the starting reagent: comprises a solution A and a solution B;
optionally, the liquid a is a pre-excitation liquid; the liquid B is excitation liquid.
The invention also provides a detection method of the dissociating agent for dissociating the vitamin D, which comprises the following steps:
(1) Sample adding: firstly, sucking a sample by an instrument, adding the dissociating agent according to any one of claims 1-3, and reacting the reaction reagent R2 in a reaction cup;
(2) Incubating;
(3) Sample adding;
(4) Incubating;
(5) Cleaning magnetic beads;
(6) Detecting signals;
the dissociating agent is used to dissociate vitamin D from the vitamin D binding protein.
Preferably, the incubation is at 37 ℃ for 10min; the cleaning is carried out for 3 times, and the liquid injection amount of each cleaning liquid is 500 mu L; the signal detection is that the solution A (pre-excitation solution) is injected firstly, then the reaction cup is moved to the photometry module, the solution B (excitation solution) is injected, and then photometry is carried out.
The invention also provides an application of the dissociation agent or the kit or the detection method, wherein the application is to detect the content of 25 hydroxy vitamin D in a sample.
Preferably, the method is used for non-diagnostic or non-therapeutic purposes.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The invention has no toxicity and corrosiveness to human body, and no direct or indirect toxicity hazard and risk to producers and users;
(2) The dissociating agent component belongs to a degradable component, can not accumulate in the nature or the environment, and has no pollution to the environment;
(3) The dissociation agent has good dissociation effect on 25 hydroxy vitamin D in the blood sample, can simultaneously meet the automatic detection of the sample, obtain accurate detection results, and reduce the risk of excessive intake of 25 hydroxy vitamin D2.
Drawings
FIG. 1 shows the results of the invention [ Y ] and DiaSorin results [ X ].
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
Example 1
Formula of 25 hydroxy vitamin D dissociating agent:
20-100 mM Tris-HCl buffer, 0.005-0.05 g/mL sodium salicylate, 0.05-0.2% polysorbate-20, 0.001-0.01 g/mL sodium lauryl sulfate, 0.1-0.5% Proclin950, pH7.0-8.0.
Dissociating agent # 1: 20 mM Tris-HCl buffer, 0.005 g/mL sodium salicylate, 0.05% polysorbate-20, 0.001 g/mL sodium dodecyl sulfate, 0.1% Proclin950, pH7.0.
Dissociating agent # 2: 50mM Tris-HCl buffer, 0.01 g/mL sodium salicylate, 0.1% polysorbate-20, 0.005 g/mL sodium dodecyl sulfate, 0.3% Proclin950, pH7.5.
Dissociating agent 3#:100 mM Tris-HCl buffer, 0.05 g/mL sodium salicylate, 0.2% polysorbate-20, 0.01 g/mL sodium dodecyl sulfate, 0.5% Proclin950, pH8.0.
Example 2
The preparation method of the 25-hydroxy vitamin D dissociating agent comprises the following steps:
the method comprises the following steps:
the Tris-HCl buffer solution is prepared by weighing Tris and hydrochloric acid, sodium salicylate, polysorbate-20, sodium dodecyl sulfate and Proclin950 are sequentially weighed while stirring continuously, the sodium salicylate, polysorbate-20, sodium dodecyl sulfate and Proclin950 are sequentially added into the Tris-HCl buffer solution, one component is fully dissolved, the other component is added to obtain a stable system, and then the pH is regulated to 7.0-8.0 by hydrochloric acid. Fully and uniformly mixing, and filtering by a 0.45 mu m filter membrane to obtain the 25-hydroxy vitamin D dissociating agent.
Example 3
Detection flow and principle of 25 hydroxy vitamin D detection kit (chemiluminescence method)
1. Reagents and instrumentation required for the reaction:
reactant R1: solid phase conjugate (25 hydroxy vitamin D and magnetic bead conjugate) with certain concentration, wherein the diluent is Tris-HCl buffer solution containing protein protectant, and the pH value is 7.0-8.0;
reaction reagent R2: a certain concentration of conjugate (acridinium ester marked anti-25 hydroxy vitamin D specific antibody), wherein the diluent is Tris-HCl buffer solution containing protein protectant, and the pH value is 7.0-8.0;
reactant R3: a dissociating agent;
and products calibrator S1 and S2 (50 mM Tris-HCl buffer system of calibrator S1 containing about 10ng/mL 25 hydroxy vitamin D antigen, 0.1% preservative, pH 6.8-7.8; 50mM Tris-HCl buffer system of calibrator S2 containing about 40ng/mL 25 hydroxy vitamin D antigen, 0.1% preservative, pH 6.8-7.8.)
Starting the reagent: comprises solution A (pre-excitation solution containing 0.01 mol/L nitric acid and 1.3% hydrogen peroxide) and solution B (excitation solution containing 0.35 mol/L sodium hydroxide)
Cleaning solution (Tris-HCl buffer system containing polysorbate-20 in proper amount)
Full-automatic chemiluminescence immunoassay instrument
2. Sample reaction scheme:
(1) sample adding: the instrument firstly absorbs 10 mu L of sample, 80 mu L of dissociation agent and 50 mu L of reaction reagent R2 (conjugate) into the reaction cup for reaction;
(2) incubation: incubation time at 37 ℃ is 10min;
(3) sample adding: adding 50 μl of the solid phase conjugate;
(4) incubation: incubation time at 37 ℃ is 10min;
(5) and (3) cleaning magnetic beads: cleaning for 3 times, wherein the liquid injection amount of each cleaning liquid is 500 mu L;
(6) and (3) signal detection: liquid A (pre-excitation liquid) is injected first, then the reaction cup is moved to the photometry module, liquid B (excitation liquid) is injected, and then photometry is performed.
All the above steps are automatically completed by a full-automatic chemiluminescence immunoassay analyzer.
3. Reaction principle:
the detection principle of the 25-hydroxy vitamin D detection kit (chemiluminescence method) is a direct competition method, and the 25-hydroxy vitamin D in serum is quantitatively detected by adopting a chemiluminescence technology. On the first incubation, 25 hydroxyvitamin D is separated from the binding protein and bound to an anti-25 hydroxyvitamin D specific antibody on the conjugate. After 10 minutes incubation, the solid phase conjugate (25 hydroxyvitamin D combined with magnetic beads) was added, followed by 10 minutes incubation and washing to remove unbound material. Then, a starter reagent is added to produce a transient chemiluminescent reaction. The resulting light signal (RLU) is measured with a photomultiplier tube and is inversely proportional to the concentration of 25 hydroxy vitamin D in the calibrator, sample or quality control.
Example 4
(1) 31 clinical serum samples are selected, the 25 hydroxy vitamin D detection kit containing the dissociating agent (prepared according to a dissociating agent No. 2 formula) is used for detection on a matched full-automatic chemiluminescence immunoassay analyzer, and the detection result is compared with the detection result of a chemiluminescence immunoassay system of DiaSorin company, and the correlation of the detection result is shown in Table 1.
TABLE 1 correlation comparison data of DiaSorin results with the results of the present invention
From the data, the correlation between the detection result of the invention and the detection result of the DiaSorin reagent is better (figure 1), and the correlation coefficient r is more than 0.99, which indicates that the dissociation effect of the dissociation agent of the invention is better, and the clinical detection requirement of the 25 hydroxy vitamin D at present can be met.
(2) The 25-hydroxy vitamin D detection kit containing the dissociating agent (prepared according to the formula of dissociating agent 1#, dissociating agent 2#, and dissociating agent 3# respectively) is used for detection on a complete set of full-automatic chemiluminescence immunoassay analyzer, the detection is repeated 10 times, the average value and the imprecision (CV) of the 10 results are calculated, and the repeatability of the detection result is determined.
TABLE 2 repeatability test data for 25 hydroxy vitamin D test kit containing dissociating agent # 1
TABLE 3 repeatability test data for 25 hydroxy vitamin D test kit containing dissociating agent # 2
TABLE 4 repeatability test data for 25 hydroxy vitamin D test kit containing dissociating agent 3#
As can be seen from the data in tables 2 to 4, the detection was performed on a complete set of full-automatic chemiluminescence immunoassay using the 25 hydroxy vitamin D detection kit containing the dissociating agent of the present invention, and the inaccuracy (CV) was controlled to be within 10%, and the reproducibility of the results was good.
(3) And respectively selecting a sufficient amount of each one of the 25 hydroxy vitamin D clinical serum samples with low, medium and high concentration levels, subpackaging, storing in an ultralow temperature refrigerator below minus 20 ℃ for standby, and respectively detecting one tube of the 25 hydroxy vitamin D clinical serum samples with low, medium and high concentration levels after subpackaging when the 25 hydroxy vitamin D detection kit containing the dissociation agent (prepared according to the dissociation agent 2# formula) stored at 2-8 ℃ is detected for 0, 3, 6, 9, 12, 15 and 18 months, wherein 0 month is the time for starting stability monitoring, calculating the result range by using +/-15% of the detection result deviation of 0 month, and if the later detection result exceeds the range, indicating that the detection result is inaccurate, namely the 25 hydroxy vitamin D detection kit containing the dissociation agent is invalid. The detection results are shown in Table 5.
TABLE 5 monitoring results of the long-term stability of 25 hydroxy vitamin D detection kit containing the dissociating agent of the present invention
As can be seen from the data in Table 5, the 25 hydroxy vitamin D detection kit containing the dissociating agent of the invention has accurate detection results and stable dissociating effect when being stored for 18 months at the temperature of 2-8 ℃.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A dissociating agent for dissociating vitamin D, the dissociating agent comprising: tris-HCl buffer, sodium salicylate, polysorbate-20, sodium dodecyl sulfate, proclin950, pH7.0-8.0, and dissociating agent for dissociating vitamin D from vitamin D binding protein.
2. The debonding agent of claim 1, wherein said debonding agent comprises 20-100 mM Tris-HCl buffer, 0.005-0.05 g/mL sodium salicylate, 0.05% -0.2% polysorbate-20, 0.001-0.01 g/mL sodium lauryl sulfate, 0.1% -0.5% Proclin950, pH7.0-8.0.
3. The dissociating agent of claim 1, wherein said dissociating agent is 50mM Tris-Hcl buffer, 0.01 g/mL sodium salicylate, 0.1% polysorbate-20, 0.005 g/mL sodium dodecyl sulfate, 0.3% Proclin950, ph7.5.
4. A method of preparing a dissociating agent as claimed in any one of claims 1 to 3, comprising the steps of:
(1) Preparing Tris-HCl buffer solution, and sequentially adding sodium salicylate, polysorbate-20, sodium dodecyl sulfate and Proclin950 while stirring to obtain a stable system;
(2) Then hydrochloric acid is used for adjusting the pH value to 7.0-8.0, the mixture is fully and uniformly mixed, and a 0.45 mu m filter membrane is used for filtering, so that the 25-hydroxy vitamin D dissociating agent is obtained.
5. A 25 hydroxy vitamin D assay kit comprising the dissociating agent of any one of claims 1-3;
optionally, the kit further comprises a reagent R1, a reagent R2, a product calibrator, a start reagent, and a wash.
6. The kit according to claim 5, wherein the reaction reagent R1 is a concentration of a solid phase conjugate;
optionally the reactant R1 is a 25 hydroxy vitamin D in combination with magnetic beads;
the reactant R2 is a conjugate with a certain concentration; optionally, the reagent R2 is an acridinium ester-labeled anti-25 hydroxyvitamin D-specific antibody;
the product calibrator comprises S1 and S2;
the starting reagent: comprises a solution A and a solution B;
optionally, the liquid a is a pre-excitation liquid; the liquid B is excitation liquid.
7. A method for detecting a dissociating agent for dissociating vitamin D, comprising the steps of:
(1) Sample adding: firstly, sucking a sample by an instrument, adding the dissociating agent according to any one of claims 1-3, and reacting the reaction reagent R2 in a reaction cup;
(2) Incubating;
(3) Sample adding;
(4) Incubating;
(5) Cleaning magnetic beads;
(6) Detecting signals;
the dissociating agent is used to dissociate vitamin D from the vitamin D binding protein.
8. The method according to claim 7, wherein the incubation is at 37 ℃ for a period of 10min; the cleaning is carried out for 3 times, and the liquid injection amount of each cleaning liquid is 500 mu L; the signal detection is that the solution A (pre-excitation solution) is injected firstly, then the reaction cup is moved to the photometry module, the solution B (excitation solution) is injected, and then photometry is carried out.
9. Use of a dissociating agent according to any one of claims 1-3 or a kit according to any one of claims 5-6 or a detection method according to any one of claims 7-8, for detecting 25 hydroxy vitamin D content in a sample.
10. The use according to claim 9, wherein the method is for non-diagnostic or non-therapeutic purposes.
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