CN115774112A - 25-hydroxy vitamin D dissociation liquid, detection method, application and kit - Google Patents
25-hydroxy vitamin D dissociation liquid, detection method, application and kit Download PDFInfo
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Abstract
The invention relates to the technical field of immunochromatography detection, in particular to 25-hydroxy vitamin D dissociation liquid, a detection method, application and a kit. The 25-hydroxy vitamin D dissociation liquid comprises 4-8 parts of guanidine hydrochloride and 2-4 parts of propylene carbonate, and the pH value is 7.0-9.0. In the 25-hydroxyvitamin D dissociation process, guanidine hydrochloride enables the VDBP protein to be randomly coiled through forming and dissociating peptide bonds in the protein structure, so that the protein loses the active structure and is reversibly denatured. And the polar structure of the propylene carbonate ensures that amino acid residues of all parts after the peptide bond is dissociated are loosened, so that the dissociation effect of the guanidine hydrochloride is more sufficient, and the time required for dissociation is shorter. The two have synergistic effect to improve dissociation efficiency. The detection kit provided by the invention is simple to operate, has lower price, and has higher sensitivity and specificity compared with an enzyme-linked immunosorbent assay.
Description
Technical Field
The invention relates to the technical field of immunochromatography detection, in particular to 25-hydroxy vitamin D dissociation liquid, a detection method, application and a kit.
Background
It is widely believed that the high or low level of serum 25-hydroxyvitamin D reflects the storage level of vitamin D and is associated with clinical symptoms of vitamin D deficiency, and therefore 25-hydroxyvitamin D is a major basis for early diagnosis of clinical symptoms of vitamin D deficiency. Whereas 25-hydroxyvitamin D in serum binds primarily to 25-hydroxyvitamin D binding protein (VDBP), the first requirement for detection of 25-hydroxyvitamin D is dissociation. The 25-hydroxyvitamin D is dissociated from the VDPB.
The dissociation liquid firstly dissociates 25-hydroxyvitamin D from 25-hydroxyvitamin D binding protein, and the current VD dissociation methods have the following several but all have corresponding technical defects.
(1) The strong acid and strong alkali method denatures or changes the conformation of the vitamin D binding protein through too high or too low pH value, so that the binding capacity with the vitamin D is lost and the vitamin D is released, 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, namely adding protease into the sample to hydrolyze the vitamin D binding protein and release the vitamin D. However, the method has the defect that the hydrolysis time is too long, which violates the original purpose of developing a rapid detection kit.
(3) The organic solvent dissolution method uses the principle of similar phase dissolution, 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 cannot be completed automatically and has larger detection error.
(4) VD-binding protein hydrolysis, which involves adding protease to the sample to hydrolyze the vitamin D-binding protein, thereby releasing vitamin D. The drawback of this method is that the enzymatic hydrolysis time is too long, resulting in a long overall detection process.
(5) The combination method, which is currently more common, is to use a reagent of pH3.8-4.8 and 5-30% DMSO, a liquid organic amide, 0.5-5% short chain alcohol Zhong Diqu vitamin D. The method contains DMSO as highly toxic substance, and has biological safety risk.
The prior art CN 109738252B discloses a 25-hydroxy-vitamin D dissociation liquid, which comprises: phosphate, beta-mercaptoethanol, guanidinium isothiocyanate, EDTA-2Na, SDS and Tween-20 as a buffer. The dissociation liquid can efficiently dissociate vitamin D in a combined state in a sample, and has the advantages of short reaction time, complete release, reaction under a neutral pH condition, small influence on subsequent reaction and the like. However, guanidine isothiocyanate and EDTA, which are main components in the dissociation solution, are expensive, the cost of reagents is high, the mass production is not facilitated, and the dissociation solution mainly acts on a chemiluminescence method and has influence on a fluorescence immunochromatography method; on the other hand the dissociation time is relatively long.
The prior art CN 105556313B relates to the use of at least one fluoroalkyl surfactant and at least one alcohol having 1 to 4 carbon atoms for the dissociation of vitamin D and/or vitamin D metabolites from vitamin D binding proteins and also to solutions comprising at least one fluoroalkyl surfactant and at least one alcohol having 1 to 4 carbon atoms. The invention also provides a method for the in vitro detection and quantification of vitamin D and/or at least one vitamin D metabolite in a biological sample, which method comprises the use of at least one fluoroalkyl surfactant and the use of at least one alcohol having 1 to 4 carbon atoms, in order to dissociate the vitamin D and/or B to be detected or one or more metabolites thereof from the vitamin D binding protein. However, the fluoroalkyl surfactants are expensive and the cost of the reagents is high; the fluoroalkyl surfactant is difficult to store at normal temperature, needs to be transported by ice bags, and is difficult to transport and high in cost; on the other hand, some fluoroalkyl groups are classified as hazardous chemicals, with biosafety risks.
The prior art CN 110749664A discloses a dissociation method of 25-hydroxy vitamin D binding protein, comprising the following steps: preparing a mother solution, taking a serum sample, adding the mother solution, uniformly mixing by swirling for 1min, standing the mixed solution for 5min, and then putting the mixed solution into a centrifuge for centrifugation; and adding ethanol into the supernatant of the obtained solution, uniformly mixing by vortex, standing the mixed solution, putting the obtained solution into a centrifuge, after centrifugation is finished, putting the supernatant into a new centrifuge tube, drying by using a nitrogen blow-drying instrument, adding a separation mobile phase, redissolving, uniformly mixing, and performing LC-MS/MS detection. The solution of the invention uses dithiothreitol with a certain concentration, so that the 25-hydroxyvitamin D to be analyzed can be well separated, the detection result of the 25-hydroxyvitamin D can be improved, the whole content condition of the vitamin D can be further determined, and meanwhile, the dithiothreitol has strong reducibility, so that the stability of the 25-hydroxyvitamin D in the purification and extraction processes is improved. However, the method is complex to operate and long in time consumption, and various professional instruments are needed during dissociation, so that the method is difficult to deal with various scenes; and is mainly applied to LC-MS/MS detection, and dissociation liquid has influence on fluorescence immunochromatography.
The prior art CN 113884687B discloses a dissociation agent for detecting 25-hydroxy vitamin D content in a human blood sample, which 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 serum. However, the perfluoro caprylic acid is a fluoroalkyl surfactant, so the price is high, and the cost of the reagent is high; and the ice bag is difficult to store at normal temperature, needs to be transported by ice bags, and has high transportation difficulty and cost.
Disclosure of Invention
Aiming at the problems of complex operation and overlong detection time in the prior art, the invention aims to provide a 25-hydroxy vitamin D dissociation liquid.
In order to achieve the purpose, the invention provides the following technical scheme:
a25-hydroxy vitamin D dissociation liquid comprises, by weight, 4-8 parts of guanidine hydrochloride and 2-4 parts of propylene carbonate, and the pH value of the dissociation liquid is 7.0-9.0.
In the 25-hydroxyvitamin D dissociation process, guanidine hydrochloride enables the VDBP protein to be randomly coiled through forming and dissociating peptide bonds in the protein structure, so that the protein loses the active structure and is reversibly denatured. And the polar structure of the propylene carbonate enables all parts of amino acid residues after peptide bond dissociation to be loosened and further separated, and the hydrophobic bond is weakened, the hydrogen bond polymer is formed to reduce the required dissociation energy of the VDBP protein, so that the N-D (natural-denaturation) reaction is greatly deviated to the direction of dissociation, the dissociation effect of the guanidine hydrochloride is more sufficient, and the time required for dissociation is shorter. The two have synergistic effect to improve dissociation efficiency.
Preferably, the kit also comprises 0.24 to 0.73 part of Tris buffer, 1 to 3 parts of nonylphenol polyoxyethylene ether (NP-40), 0.1 to 2 parts of Tris (2-carboxyethyl) phosphine (TCEP) and 0.1 to 2 parts of diethylene triamine pentaacetic acid pentasodium (DTPA-5 Na).
NP-40 is a surfactant, lyses the cell membrane in the whole blood sample, makes vitamin D binding protein mix with reactant completely, makes VDBP denaturate at the same time, and can be preserved at normal temperature, reduce dissociation time, increase accuracy.
DTPA-5Na is used as a complexing agent, so that denatured VDBP can aggregate and precipitate, the dissociation efficiency of VDBP and the release amount of vitamin D are increased, dissociated vitamin D is prevented from being combined with VDBP again, the amount of dissociated vitamin D is increased, dissociation time is shortened, and accuracy is improved.
TCEP is used as a reducing agent, and the function of the TCEP is to dissociate disulfide bonds in a protein structure, so that the guanidine hydrochloride-propylene carbonate system can further influence the internal structure of the protein and improve dissociation efficiency.
A diluent comprises, by weight, 0.24-0.73 parts of Tris buffer, 0.5-3 parts of C13-15 alcohol polyoxyethylene ether, 0.1-2 parts of Tetronic1307 (S9), 0.02-1 parts of Proclin300, 0.2-2 parts of TX-100, and the pH is 7.4-8.8.
In the diluent system, C13-15 alcohol polyoxyethylene ether is used as a macromolecular surfactant, so that the surface tension of water can be reduced, and due to the structure, 25-hydroxyvitamin D is gathered, the stability of 25-hydroxyvitamin D is protected, the combination of 25-hydroxyvitamin D and an antibody on a chromatographic membrane can be promoted, and the accuracy of a reagent card is improved.
Tetronic1307 (S9) can be combined with other substances in blood through a special structure, so that the matrix effect in blood is reduced, the possibility of false positive is reduced, and the accuracy of the reagent card is improved.
The second purpose of the invention is to provide a method for detecting 25-hydroxy vitamin D.
A method for detecting 25-hydroxyvitamin D comprising the steps of:
a. uniformly mixing the dissociation solution and the whole blood sample, shaking for 1-3min, adding the diluent, and uniformly mixing to obtain a sample;
b. dripping the sample on a 25-hydroxy vitamin D detection reagent card, and reacting for 10-15min;
c. the reagent card is put into a fluorescence immunoassay analyzer for detection, data is read, and the content of the 25-hydroxyvitamin D is obtained through fluorescence intensity (a calculated T/C value, namely the ratio of a signal value measured by a T line to a signal value measured by a C line).
Preferably, the 25-hydroxyvitamin D detection reagent card comprises a PVC lining plate, a nitrocellulose membrane, a combination pad, a sample pad and a water absorption pad.
Preferably, the sample pad is treated with a sample pad treatment solution including Tris buffer, trehalose, BSA, and TX-100 before being added to the reagent card. The Tris buffer solution provides a stable ionic environment for the combination of the antibody protein and the vitamin D, and prevents the accumulation of the antibody protein by the interaction of ions. The combination of trehalose and BSA prevents the antibody protein from being hydrolytically inactivated and protects the antibody protein from being affected by components in the dissociation solution, thereby losing the detection activity and affecting the experimental result, and TX-100 is faster to move on the reagent card by reducing the surface tension, and is more fully combined with vitamin D.
The nitrocellulose membrane contains a T line and a C line and is prepared by a gold spraying and membrane scribing instrument. The T line is processed by spraying a T line coating on the nitrocellulose membrane. The C wire is processed by spraying a coating of the C wire on a nitrocellulose membrane.
Preferably, the coating for treating the C line and the T line includes: the T coat comprises Tris buffer, trehalose, BSA and anti-25-hydroxy vitamin D antibody protein, and the C coat comprises Tris buffer, trehalose, BSA and goat anti-mouse IgG antibody protein.
The anti-25-hydroxyvitamin D antibody protein on the T line is combined with the anti-25-hydroxyvitamin D antibody protein marked by the time-resolved fluorescent microspheres through being combined with the vitamin D, so that the anti-25-hydroxyvitamin D antibody protein stays on the T line of the nitrocellulose membrane. The more vitamin D is, the more time-resolved fluorescent microspheres stay in the T line, the higher the fluorescence intensity is, and the content of the vitamin D can be detected by detecting a T/C value (the ratio of a signal value measured by the T line to a signal value measured by the C line) calculated by fluorescence intensity.
The goat anti-mouse IgG antibody protein is combined with the time-resolved fluorescent microsphere labeled mouse IgG protein, so that the goat anti-mouse IgG antibody protein stays on a nitrocellulose membrane C line, the vitamin D content can be detected preferably through a T/C value (the ratio of a signal value measured by the T line to a signal value measured by the C line) calculated by the fluorescence intensity of the C line, the combination pad is sprayed with a fluorescent marker, and the fluorescent marker comprises the time-resolved fluorescent microsphere labeled anti-25-hydroxy vitamin D antibody protein and the time-resolved fluorescent microsphere labeled mouse IgG protein.
The fluorescent marker can emit fluorescence under proper stimulation, and the vitamin D content can be measured by detecting the fluorescence intensity. The anti-25-hydroxyvitamin D antibody protein marked by the time-resolved fluorescent microspheres is combined with vitamin D, and can be combined with the anti-25-hydroxyvitamin D antibody protein on the nitrocellulose membrane T line through the vitamin D. The time-resolved fluorescent microsphere labeled mouse IgG protein can be combined with the goat anti-mouse IgG antibody protein on the nitrocellulose membrane C line.
The invention also aims to provide application of the 25-hydroxy vitamin D dissociation liquid.
The invention claims the application of the 25-hydroxyvitamin D dissociation solution in dissociation or detection of 25-hydroxyvitamin D.
The invention claims the application of the 25-hydroxyvitamin D dissociation solution in preparing a 25-hydroxyvitamin D dissociation or detection reagent.
The invention also claims a 25-hydroxyvitamin D dissociation or detection kit, which comprises the 25-hydroxyvitamin D dissociation solution.
The invention is further explained below:
in the process of VD dissociation, NP-40 acts as a common detergent, and at a sufficiently high concentration, the polar hydrophilic regions of each molecule are oriented toward the polar solute (water), while the hydrophobic regions are brought together to form a thermodynamically stable micelle with a hydrophobic core. The hydrophobic core region of the detergent micelle binds to the hydrophobic surface of the protein to form a soluble protein-detergent complex. In the process, bipolar lipid membranes of cells are damaged, so that cell membranes of the membrane-bound proteins Bai Jieli are released and dissolved, polymers of 25-hydroxy vitamin D protein and VDBP in the cells are released, the probability of reaction of the polymers and guanidine hydrochloride-propylene carbonate is increased, and the dissociation efficiency and the content of dissociated vitamin D are increased. Based on the principle of binding with the hydrophobic surface of protein, NP-40 can also cause the denaturation and inactivation of VDBP to a certain extent, and can also increase the separation efficiency and the content of dissociated vitamin D.
TCEP is used as a reducing agent, and the function of the TCEP is to dissociate disulfide bonds in a protein structure, so that the guanidine hydrochloride-propylene carbonate system can further influence the internal structure of the protein and improve dissociation efficiency. Under the guanidine hydrochloride-propylene carbonate-TCEP protein denaturant system, NP-40 further denatures VDBP, loses the capacity of combining with 25-hydroxy vitamin D, and further releases free 25-hydroxy vitamin D. DTPA-5Na is used as a complexing agent, forms a more thermodynamically stable ionic state with VDBP through ionic bond combination, can enable the denatured VDBP to aggregate and precipitate, and also increases the dissociation efficiency of the VDBP and the release amount of vitamin D. The components accelerate the dissociation efficiency of the 25-hydroxy vitamin D through the synergistic effect.
Secondly, in the process of applying the dissociation liquid to the fluorescence immunochromatography, the invention provides other reagents such as sample pad treatment liquid and coating liquid, wherein trehalose and BSA can protect antibody protein required by detection from being influenced by components in the dissociation liquid, so that the detection activity is lost, and the experimental result is influenced, thereby ensuring the accuracy of the test.
Compared with the prior art, the invention has the following beneficial effects:
1. the dissociation liquid is stored at normal temperature, so that transportation and storage are facilitated, and the detection accuracy is improved.
2. The method is more convenient and rapid, improves the accuracy and specificity, improves the minimum detection limit of the vitamin D, reduces the false positive probability, and can be used for the in vitro detection of 25-hydroxyvitamin D.
3. The invention greatly shortens the dissociation time and can effectively improve the detection efficiency.
4. Compared with the chemiluminescence method, the detection kit provided by the invention has the advantages of simple operation, lower equipment price compared with LC-MS, and high sensitivity and specificity compared with the enzyme-linked immunosorbent assay.
Drawings
FIG. 1 shows a 25-hydroxyvitamin D detection reagent card;
FIG. 2 shows the results of the linear range experiment of the method of the present invention.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
1. Preparation of the reagent (percentages are percentage mass fraction):
dissociation liquid: mixing Tris buffer solution with pH value of 7.4 and concentration of 40mmol/L with guanidine hydrochloride, NP-40, TCEP, propylene carbonate and DTPA-5Na, wherein the dissociation solution contains 5% of guanidine hydrochloride, 1.5% of NP-40, 0.5% of TCEP, 3% of propylene carbonate and 0.3% of DTPA-5 Na.
Diluting liquid: mixing a Tris buffer solution with the pH value of 7.4 and the concentration of 40mmol/L with Proclin300, tx-100, C13-15 alcohol polyoxyethylene ether and Proclin300, wherein the content of Proclin in the diluent is 0.02%, the content of TX-100 is 0.8%, the content of C13-15 alcohol polyoxyethylene ether is 0.8%, and the content of Tetronic1307 is 1%.
Fluorescent microsphere working solution: mixing Tris buffer solution with pH value of 7.4 and concentration of 40mmol/L with sodium caseinate, trehalose, BSA and fluorescent microsphere labeled anti-25-hydroxy vitamin D antibody protein, and controlling the content of sodium caseinate in the obtained fluorescent microsphere working solution to be 0.2%, the content of trehalose to be 1%, the content of BSA to be 2% and the content of fluorescent microsphere labeled anti-25-hydroxy vitamin D antibody protein to be 1.5ug/ml.
T-line coating liquid: mixing a Tris buffer solution with the pH value of 7.4 and the concentration of 40mmol/L with trehalose, BSA and fluorescent microsphere labeled anti-25-hydroxy vitamin D antibody protein, and controlling the content of the trehalose in the obtained T-line coating solution to be 1.5%, the content of the BSA to be 2% and the content of the fluorescent microsphere labeled anti-25-hydroxy vitamin D antibody protein to be 1.0ug/ml.
C, coating liquid: mixing Tris buffer solution with pH of 7.4 and concentration of 40mmol/L with trehalose, BSA and goat anti-mouse IgG antibody protein, and controlling the content of trehalose in the obtained C-line coating solution to be 1.5%, the content of BSA to be 2% and the content of goat anti-mouse IgG antibody protein to be 0.8ug/ml.
Sample pad treatment solution: and (2) mixing Tris buffer solution with the pH value of 7.4 and the concentration of 40mmol/L with trehalose, BSA and TX-100, and controlling the content of the trehalose, the content of the BSA and the content of the TX-100 in the diluent in the obtained fluorescent microsphere working solution to be 2%, 1% and 0.8%.
2. Determination of 25-hydroxyvitamin D content
As shown in FIG. 1, the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are arranged from left to right, the sample pad covers the combination pad, the combination pad covers the nitrocellulose membrane, and the absorbent paper covers the nitrocellulose membrane.
Spraying the fluorescent microsphere working solution on a gold-labeled binding pad, spraying the T-line coating solution and the C-line coating solution on an NC membrane, and treating the glass fiber membrane by using the sample pad binding solution. And then assembling the obtained gold-labeled combined pad, the NC membrane, the sample pad, the absorbent paper and the PVC plate together to form the 25-hydroxy vitamin D detection reagent card.
Respectively adding 25-hydroxyvitamin D standard substances into a biological sample (specifically a whole blood sample) to obtain calibrator with 25-hydroxyvitamin D concentration of 0, 1, 3, 10, 20, 55 and 125 ng/mL. Adding 15 μ L of calibrator into 60 μ L of dissociation liquid, shaking and mixing for 1-3min. 240ul of diluent was added to the dissociated samples and mixed well.
Adding 80 mu L of the diluent into the sample, adding the sample into the sample adding hole of the detection card, and reacting for 10-15min at room temperature (25 ℃) to obtain the reaction detection card of the calibrator.
And (2) placing the reaction detection card of the calibrator into a fluorescence immunoassay analyzer for detection to obtain the fluorescence intensity of the reaction detection card (namely the fluorescence intensity of the calibrator), calculating to obtain a T/C value (the ratio of the signal value measured by the T line to the signal value measured by the C line), drawing a calibration curve by using the concentration of 25-hydroxyvitamin D in the calibrator and the T/C value, writing the calibration curve into an ID card, and recording the calibration curve into the fluorescence immunoassay analyzer (Jin Haofeng KHF 01).
Taking 15 mu L of biological sample (specifically whole blood sample) as a sample to be detected, mixing with 60 mu L of dissociation liquid, and shaking and uniformly mixing for 1-3min. 240ul of diluent was added to the dissociated samples and mixed well.
Adding 80 mu L of the diluent into the sample, adding the sample into the sample adding hole of the detection card, and reacting for 10-15min at room temperature (25 ℃) to obtain the reaction detection of the sample to be detected.
And (3) placing the reaction detection card of the sample to be detected into a fluorescence immunoassay analyzer for detection, and obtaining the content of the 25-hydroxy vitamin D in the biological sample according to the calibration curve and the fluorescence intensity of the reaction detection card (namely the fluorescence intensity of the sample to be detected).
Example 2
Effect of additional ingredients on dissociation time
10 samples to be tested of 25-hydroxyvitamin D are prepared, a reagent card prepared by using the dissociation liquid formula and the related working solution provided by the invention is used for testing according to the method in the step of measuring the content of 25-hydroxyvitamin D in the embodiment 1, wherein the dissociation time is respectively 30s, 1min and 5min, and the samples to be tested are tested according to the description of a product specification by using a product A (Xiamen is a 25-light-based vitamin D measuring kit of Zhengzhi Biotech Co., ltd.), wherein the dissociation time is set to be 5min, 10min and 15min. In addition, compared with the dissociation solution of the invention, six groups of reagent cards prepared by the formula of the dissociation solution without guanidine hydrochloride, propylene carbonate, NP-40, TCEP and DTPA-5Na are used for testing samples to be tested, wherein the dissociation time is set to be 5min, 10min and 15min. The concentrations (ng/ml) of the samples to be tested in the five cases at different dissociation times are determined in total, so that the influence of the dissociation time on the determination result is verified by each formula. As a supplementary experiment, a reagent card prepared from a commercially available dissociation liquid A and a relevant working solution provided by the present invention was tested according to the method in "determination of 25-hydroxyvitamin D content in example 1", wherein the dissociation time was set to 5min, 10min, and 15min. In summary, this embodiment provides example 1 and comparative example 8, which include product a, and the dissociation liquid of product a and the reagent card combination provided by the present invention.
Dissociation liquid of comparative example 1: mixing Tris buffer solution with pH value of 7.4 and concentration of 40mmol/L with NP-40, TCEP and DTPA-5Na, wherein the dissociation solution contains NP-40 1.5%, TCEP 0.5% and DTPA-5Na 0.3%.
Dissociation liquid of comparative example 2: mixing a Tris buffer solution with the pH value of 7.4 and the concentration of 40mmol/L with NP-40, TCEP, propylene carbonate and DTPA-5Na, wherein the dissociation solution contains 1.5 percent of NP-40, 0.5 percent of TCEP, 3 percent of propylene carbonate and 0.3 percent of DTPA-5 Na.
Dissociation liquid of comparative example 3: mixing Tris buffer solution with pH value of 7.4 and concentration of 40mmol/L with guanidine hydrochloride, NP-40, TCEP and DTPA-5Na, wherein the dissociation solution contains 5% of guanidine hydrochloride, 1.5% of NP-40, 0.5% of TCEP and 0.3% of DTPA-5 Na.
Dissociation liquid of comparative example 4: mixing a Tris buffer solution with the pH value of 7.4 and the concentration of 40mmol/L with guanidine hydrochloride, propylene carbonate, TCEP and DTPA-5Na, wherein the content of guanidine hydrochloride, propylene carbonate, TCEP and DTPA-5Na in the dissociation solution is 5%, 3%, 0.5% and 0.3% respectively.
Dissociation liquid of comparative example 5: mixing a Tris buffer solution with the pH value of 7.4 and the concentration of 40mmol/L with guanidine hydrochloride, NP-40, propylene carbonate and DTPA-5Na, wherein the content of the guanidine hydrochloride, the content of the NP-40, the content of the propylene carbonate and the content of the DTPA-5Na in the dissociation solution are respectively 5%, 1.5% and 3%, respectively 0.3%.
Dissociation liquid of comparative example 6: mixing Tris buffer solution with pH value of 7.4 and concentration of 40mmol/L with guanidine hydrochloride, NP-40, TCEP and propylene carbonate, wherein the dissociation solution contains 5% of guanidine hydrochloride, 1.5% of NP-40, 0.5% of TCEP and 3% of propylene carbonate.
The other reagents were the same as in example 1.
As a supplementary experiment, comparative example 7 was conducted by using a reagent card prepared from a commercially available dissociation liquid of product A and a related working solution provided by the present invention according to the method of "measuring 25-hydroxyvitamin D content in example 1".
As can be seen from tables 1 to 5, the formulations of product a on the market and the comparative example after removing the core component in example 1 were stable after 10min of dissociation for about fifteen minutes, whereas the present invention can achieve the effect of product a10min within 1min, and from the viewpoint of only a small increase in concentration in the 5min result, the dissociation was fully completed at about 1 min. Furthermore, there was a small increase in the concentration of dissociated vitamin D. Therefore, compared with the prior art, the method greatly improves the dissociation efficiency, shortens the dissociation time, and increases the content of the dissociated vitamin D in a small range. The 3-fold ratio with the minor component removed showed that the full dissociation time was still after 10min and that the dissociated vitamin D content was also significantly reduced, but increased compared to the 3-fold ratio with the core component removed. It is demonstrated that the minor ingredients also play an important role in the formulation of the present invention. Therefore, experiments prove that the components of the invention play a synergistic effect to jointly increase the dissociation efficiency and the amount of dissociated vitamin D.
Example 3
Method repeatability test and method accuracy test
And (3) taking the samples with the standard of 10ng/mL and 80ng/mL as samples to be detected, and performing the test according to the method for measuring the content of 25-hydroxyvitamin D in the embodiment 1, wherein each sample is parallelly measured for 10 times, and the variation coefficient of the obtained detection result is calculated.
As can be seen from Table 6, when the sample concentrations are 10ng/mL and 80ng/mL, the variation coefficient of the detection result obtained by the repeatability test is not more than 10%, and the method provided by the invention is proved to be high in precision.
Method accuracy test
The international reference SRM 972a was used as a sample to be tested, and the reagent card prepared from the dissociation solution formulation and the related working solution provided by the present invention was tested according to the method of "determination of 25-hydroxyvitamin D content in example 1" for 5 times in parallel, and the relative deviation was calculated. And testing the sample to be tested by using a product A on the market according to the description of the product specification. In addition, three groups of reagent cards prepared by the dissociation liquid and the related working liquid provided in comparative examples 1 to 3 are used for testing samples to be tested. The concentrations (ng/ml) of the samples to be tested in the five cases at different dissociation times were determined in total, thereby verifying the accuracy of each formulation.
As can be seen from Table 7, the relative deviation between the measured value and the labeled value is not more than +/-7.00 percent and is far less than +/-10 percent, and the kit is proved to have good accuracy. Furthermore, the tendency to deviate more than the original formulation of the selected example 1 from the other formulations or the commercial product a, indicates that the invention provides a significant improvement in the accuracy of the kit.
Example 4
Method linear range experiment and method detection limit verification
The high-value samples were diluted 2 times, 4 times, 8 times, 16 times, 32 times, 64 times and 128 times, respectively, and 3 times of repeated tests were carried out according to the method of "determination of 25-hydroxyvitamin D content in example 1", the average value was calculated, the average value of the determined concentration and the multiple concentration were subjected to straight line fitting by the least square method, and the linear correlation coefficient R was calculated.
As can be seen from Table 8 and FIG. 2, the kit of the present invention has a wide linear range, the linear range reaches [1,120] ng/ml, and the correlation coefficient R >0.995, indicating that the correlation of the fitting data is good.
Method detection limit verification
5 samples to be tested with the concentration close to the detection limit are taken, repeated tests are carried out for 3 times according to the method for measuring the content of 25-hydroxyvitamin D in the embodiment 1, and the undetected probability is counted.
As can be seen from Table 9, the undetected probability is less than 5%, and the detection limit of the kit of the present invention is not higher than 1ng/ml.
The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.
Claims (10)
1. A25-hydroxy vitamin D dissociation liquid is characterized by comprising, by weight, 4-8 parts of guanidine hydrochloride and 2-4 parts of propylene carbonate, wherein the pH value is 7.0-9.0.
2. The 25-hydroxyvitamin D dissociation solution according to claim 1, further comprising 0.24 to 0.73 parts of Tris buffer, 1 to 3 parts of nonylphenol polyoxyethylene ether, 0.1 to 2 parts of Tris (2-carboxyethyl) phosphine, and 0.1 to 2 parts of pentasodium diethylenetriaminepentaacetate.
3. The method for detecting 25-hydroxyvitamin D is characterized by comprising the following steps:
a. uniformly mixing the 25-hydroxyvitamin D dissociation solution as defined in claim 1 or 2 with a whole blood sample, shaking for 1-3min, adding a diluent, and uniformly mixing to obtain a sample;
b. dripping the sample on a 25-hydroxy vitamin D detection reagent card, and reacting for 10-15min;
c. and (3) placing the reagent card into a fluorescence immunoassay analyzer for detection, reading data, and obtaining the content of 25-hydroxy vitamin D according to fluorescence intensity.
4. The detection method according to claim 3, wherein the diluent comprises, by weight, 0.24 to 0.73 part of Tris buffer, 0.5 to 3 parts of C13-15 alcohol polyoxyethylene ether, 0.1 to 2 parts of Tetronic1307, 0.02 to 1 part of Proclin300, 0.2 to 2 parts of TX-100, and the pH is 7.4 to 8.8.
5. The assay of claim 3, wherein the 25-hydroxyvitamin D assay reagent card comprises a nitrocellulose membrane and a conjugate pad; the nitrocellulose membrane contains a T line and a C line; the T line is formed by spraying a T line coating object on a nitrocellulose membrane, and the C line is formed by spraying a C line coating object on the nitrocellulose membrane.
6. The assay of claim 5, wherein the T-line coating comprises Tris buffer, trehalose, BSA, and anti-25-hydroxyvitamin D antibody protein; the C-line coating comprises Tris buffer, trehalose, BSA and goat anti-mouse IgG antibody protein.
7. The detection method according to claim 5, wherein the binding pad is sprayed with a fluorescent marker, and the fluorescent marker comprises anti-25-hydroxyvitamin D antibody protein labeled with time-resolved fluorescent microspheres and mouse IgG protein labeled with time-resolved fluorescent microspheres.
8. Use of a 25-hydroxyvitamin D dissociation solution according to claim 1 or 2 for dissociation or detection of 25-hydroxyvitamin D.
9. Use of a 25-hydroxyvitamin D dissociation solution according to claim 1 or 2 in the preparation of a reagent for dissociation or detection of 25-hydroxyvitamin D.
10. A kit for dissociation or detection of 25-hydroxyvitamin D, comprising the 25-hydroxyvitamin D dissociation solution according to claim 1 or 2.
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