CN111366646B - 25-hydroxyvitamin D analysis method based on immune purification and mass spectrometry detection - Google Patents
25-hydroxyvitamin D analysis method based on immune purification and mass spectrometry detection Download PDFInfo
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Abstract
The invention discloses a 25-hydroxyvitamin D analysis method based on immune purification combined with mass spectrometry detection, which mainly comprises the following steps: preparing a biotin-labeled antibody, preparing a magnetic bead coated by the biotin-labeled antibody, enriching and purifying a sample and detecting mass spectrum. The analysis method utilizes the high specificity of antibody affinity, can specifically enrich and purify 25- (OH) D from a complex biological sample, and simultaneously realizes the respective accurate detection of 25- (OH) D2 and 25- (OH) D3; the antibody coupling magnetic bead enrichment method is combined with mass spectrum detection, so that the use efficiency of a liquid phase mass spectrum system is greatly improved while high flux, sensitivity and accurate quantification are realized.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a 25-hydroxyvitamin D analysis method based on immune purification and mass spectrometry detection.
Background
25-hydroxyvitamin D (25- (OH) D), including 25-hydroxyvitamin D2(25- (OH) D2) and 25-hydroxyvitamin D3(25- (OH) D3), is the predominant form of vitamin D present in the body. The high or low serum 25- (OH) D level may reflect the storage level of vitamin D in humans and is associated with clinical symptoms of vitamin D deficiency. The detection method of 25- (OH) D2 and 25- (OH) D3 in biological samples (such as whole blood, serum, urine or saliva) mainly comprises liquid chromatography-mass spectrometry and immunoassay.
Before detection by liquid chromatography-mass spectrometry, 25- (OH) D2 and 25- (OH) D3 need to be enriched and separated from a biological sample, the traditional sample processing method mainly comprises a precipitation method, a liquid-liquid extraction method and a solid-phase extraction method, substances in the obtained sample are separated by liquid chromatography according to different properties, and then are detected and analyzed by mass spectrometry according to different mass-to-nuclear ratios. Liquid chromatography mass spectrometry can achieve simultaneous detection of 25- (OH) D2 and 25- (OH) D3.
However, the pretreatment of the sample by the liquid chromatography-mass spectrometry is a non-specific enrichment method, the enriched sample has more complicated components, and the enriched sample contains a large amount of matrix substances with similar properties to 25- (OH) D besides 25- (OH) D2 and 25- (OH) D3. During chromatographic separation, some non-target analyte substances are easy to denature and precipitate on a chromatographic column or are difficult to elute from the chromatographic column, so that a higher matrix effect is caused, the column pressure is increased, the effectiveness and stability of subsequent separation and analysis of target substances are influenced, and the difficulty and burden of liquid mass spectrum maintenance are increased. The difficulty of using chromatography to completely separate the target analyte and its isomers or structural analogues is extremely time consuming and the incompletely separated matrix material may also interfere with mass spectrometric quantification of the analyte.
Immunoassay methods include Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), and fully automated biochemical methods. The basic principle is that 25- (OH) D in a biological sample is captured specifically by using an antibody of 25- (OH) D, and then the purpose of analyzing the 25- (OH) D is achieved by detecting corresponding radioactive quantity, light or electrochemical signals. However, both 25- (OH) D2 and 25- (OH) D3 are typically captured by antibodies, and thus the immunoassay detects levels of total 25- (OH) D, and is also susceptible to cross-interference with other analytes with similar 25- (OH) D structures, affecting quantitation and limiting clinical use.
Therefore, the invention aims to provide a 25-hydroxyvitamin D analysis method which can accurately detect 25- (OH) D2 and 25- (OH) D3 respectively and has high specificity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a 25-hydroxyvitamin D analysis method based on immune purification combined with mass spectrometry detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a 25-hydroxyvitamin D analysis method based on immune purification combined with mass spectrometry detection, which comprises the following steps:
step one, preparing a biotin labeled antibody:
(1) dialyzing 25- (OH) D antibody for 16-24h by using phosphate buffer solution with pH of 8.1, adding biotin into the dialyzed antibody, further adding phosphate buffer solution to dilute the antibody, and uniformly mixing to obtain mixed solution;
(2) placing the mixed solution in a constant temperature incubator, standing and reacting for 120 +/-10 min to obtain a biotin labeled antibody;
(3) dialyzing the biotinylated antibody for 16-24h with phosphate buffer solution with pH of 7.4;
step two, preparing a biotin-labeled antibody coated magnetic bead:
(1) washing the avidin magnetic beads with a magnetic bead cleaning solution, and then re-dissolving with a binding buffer solution;
(2) mixing the dissolved avidin magnetic beads with the biotin-labeled antibody prepared in the first step, and then placing the mixture in a constant-temperature oscillation box for oscillation reaction to obtain biotin-labeled antibody-coated magnetic beads;
(3) after reaction, cleaning the magnetic beads by using a magnetic bead cleaning solution, diluting, and storing at 2-8 ℃ for later use;
step three, enriching and purifying samples: adding an isotope internal standard solution and the biotin-labeled antibody coated magnetic beads into a sample, stirring and incubating at room temperature, and repeatedly washing with ultrapure water for at least 3 times; eluting the washed magnetic beads for 2 times by using an organic solvent, and collecting eluent;
step four, mass spectrum detection: and drying the eluent obtained in the third step, redissolving the eluent or directly transferring the eluent into a micropore plate, and detecting the eluent by a liquid phase mass spectrometer.
Further, the dialysis in step one is performed at 4 ℃ or room temperature for 16-24 h.
Further, the temperature of the oven in the first step was set to 25 ± 1 ℃.
Further, the weight ratio of the antibody to the biotin in the first step is 10-20: 1.
further, the preparation method of the magnetic bead cleaning solution in the second step comprises the following steps: at room temperature, a volume of purified water was added with 1.212g Tris, 9.0g NaCl, 0.4mL HCl, 0.2mL Tween-20, 5.0mL 20% NaN 3 And 0.5mL of Brij-35, the pH value is detected to be 8.3 +/-0.2 after the uniform mixing is carried out, the quality is qualified, and the volume of the purified water is fixed to 1L.
Further, the preparation method of the binding buffer solution in the step two comprises the following steps: to a volume of purified water at room temperature was added 6.06g of Tris, 9.0g of NaCl and 3.84mL of HCl, stirring for at least 5min to dissolve completely, then adding 1.0mL of Tween-20 and 5mL of 20% NaN 3 Finally, 10.0g of Bovine Serum Albumin (BSA) is slowly added and stirred for at least 10min until complete dissolution; after all the components are completely dissolved, the pH value is measured to be qualified when the pH value is 7.2 +/-0.1 by using a pH meter, and the volume of purified water is fixed to 1L.
Further, during the cleaning in the second step, the volume ratio of the magnetic beads to the magnetic bead cleaning solution is less than or equal to 1: 2.
further, the isotope internal standard solution is 2 H 3 -25-hydroxyvitamin D2 or 2 H 3 -25-hydroxyvitamin D3.
Further, the incubation conditions in step three are: room temperature, 30 min.
Further, the organic phase used for elution in step three was 75% acetonitrile, which contained 0.1% formic acid.
Further, the parameters of the oscillatory reaction in step three are: the reaction is carried out for 16 to 24 hours under the conditions that the temperature is 37 +/-2 ℃ and the oscillation speed is 100 rmp/min.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the invention relates to a 25-hydroxyvitamin D analysis method based on immune purification and mass spectrum detection, which utilizes the high specificity of antibody affinity, can specifically enrich and purify 25- (OH) D from a complex biological sample, and simultaneously realizes the respective accurate detection of 25- (OH) D2 and 25- (OH) D3; the antibody coupling magnetic bead enrichment method is combined with mass spectrum detection, so that the use efficiency of a liquid phase mass spectrum system is greatly improved while high flux, sensitivity and accurate quantification are realized. In addition, the invention can be used for developing small molecule detection and diagnosis kits, various antibodies are coupled with magnetic bead combinations, various biomarkers can be simultaneously enriched and detected by mass spectrometry at one time, and compared with the traditional immunoassay method which is used for single-index detection, the invention has higher flux and stronger specificity and selectivity.
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FIG. 1 is a flow chart illustrating the operation of an analysis method according to an embodiment of the present invention;
wherein, 1 is antibody coupling magnetic beads; 2, enriching and purifying the marker; and 3, detecting the multi-index and Suzuki specificity of the mass spectrum.
Detailed Description
The present invention provides a method of 25-hydroxyvitamin D assay based on immuno-purification coupled with mass spectrometric detection, and is described in detail and specifically by the following examples to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
Examples
Referring to fig. 1, the present example provides a 25-hydroxyvitamin D assay based on immunopurification coupled with mass spectrometry detection, comprising the following steps:
step one, preparing a biotin labeled antibody:
dialyzing 1mg of 25- (OH) D antibody against phosphate buffer (pH 8.1) at 4 deg.C or room temperature for at least 16-24 hr; adding 0.05mg biotin into dialyzed antibody, adding phosphate buffer solution (pH 8.1) to make the final concentration of antibody 1mg/mL, and mixing for 5 min; placing the mixed solution in a constant temperature incubator at 25 +/-1 ℃, standing for reaction for 120 +/-10 min, and finally dialyzing the biotinylated antibody for 16-24h at 4 ℃ or room temperature by using a phosphate buffer solution (pH 7.4).
Step two, preparing a biotin-labeled antibody coated magnetic bead:
shaking up streptavidin magnetic beads, then sucking 20mg magnetic beads (volume x mu L), washing twice with a magnetic bead cleaning solution with the volume not less than twice of the magnetic bead volume, and then re-dissolving the magnetic beads to be not less than 5mg/mL by using a binding buffer solution; mixing magnetic beads with the biotinylated antibody, wherein the final concentration of the magnetic beads is not lower than 10mg/mL, placing the magnetic beads in a constant-temperature oscillation box at 37 +/-2 ℃, and shaking uniformly at 100rmp/min for reacting for 16-24 h; taking down the reacted magnetic beads, washing the magnetic beads for 4 times by using magnetic bead washing liquid with the reaction volume not less than 2 times, diluting the magnetic beads to 400 mu g/mL by using binding buffer solution, and storing the magnetic beads at the temperature of 2-8 ℃ for later use.
Step three, enriching and purifying samples: 100 μ L of serum was taken and 100 μ L of internal standard solution (containing 2 H 3 -25-hydroxyvitamin D2(20ng/mL), 2 H 3 25-hydroxyvitamin D3(20ng/mL)), 300. mu.L of antibody magnetic beads, stirring and incubating at room temperature for 30min, transferring the magnetic beads to 500. mu.L of ultrapure water, stirring and mixing, and repeatedly washingAt least 3 times. The washed magnetic beads were transferred to 100. mu.L of 75% acetonitrile (containing 0.1% formic acid), mixed thoroughly for 5min, the eluate was collected, the magnetic beads were washed once again, and the two eluates were pooled.
Step four, mass spectrum detection: the eluent can be selectively dried by blowing, redissolved by using a proper solvent, or directly transferred into a 96-hole microporous plate by 150 mu L, and detected by a liquid phase mass spectrometer.
The preparation method of the magnetic bead cleaning solution in the second step comprises the following steps: at room temperature, a volume of purified water was added with 1.212g Tris, 9.0g NaCl, 0.4mL HCl, 0.2mL Tween-20, 5.0mL 20% NaN 3 And 0.5mL of Brij-35, the pH value is detected to be 8.3 +/-0.2 after the uniform mixing is carried out, the quality is qualified, and the volume of the purified water is fixed to 1L.
The preparation method of the combined buffer solution in the second step comprises the following steps: adding 6.06g Tris, 9.0g sodium chloride and 3.84mL HCl into a certain volume of purified water at room temperature, stirring for at least 5min until completely dissolving, and adding 1.0mL Tween-20 and 5mL 20% NaN 3 Finally, 10.0g of Bovine Serum Albumin (BSA) is slowly added, and the mixture is stirred for at least 10min until the mixture is completely dissolved; after all the components are completely dissolved, the pH value is measured to be qualified at 7.2 +/-0.1 by a pH meter, and the volume of purified water is determined to be 1L.
The results of this evaluation method were analyzed to obtain the results shown in tables 1 to 3.
TABLE 1 results of calculation of precision
TABLE 2 calculation of the Linear Range
TABLE 325 recovery of hydroxyvitamin D
| Analyte | Average (%) | 95% confidence interval |
| 25-hydroxyvitamin D2 | 94 | 88-100 |
| 25-hydroxyvitamin D3 | 90 | 79-100 |
Comparative example
This comparative example uses conventional liquid chromatography mass spectrometry to detect and analyze 25-hydroxyvitamin D, and the results are shown in tables 4-6 below:
TABLE 4 results of calculation of precision
TABLE 5 calculation of Linear Range
TABLE 625 recovery of hydroxyvitamin D
| Analyte | Average (%) | 95% confidence interval |
| 25-hydroxyvitamin D2 | 91 | 85-100 |
| 25-hydroxyvitamin D3 | 88 | 74-100 |
In conclusion, the method provided by the invention can specifically enrich and purify 25- (OH) D from a complex biological sample, simultaneously realize respective accurate detection of 25- (OH) D2 and 25- (OH) D3, and has the characteristics of high specificity, high flux, accurate quantification and the like compared with the traditional method.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (10)
1. A 25-hydroxyvitamin D assay based on immuno-purification coupled with mass spectrometric detection, wherein the 25-hydroxyvitamin D comprises 25- (OH) D2 and/or 25- (OH) D3, the 25-hydroxyvitamin D assay comprising the steps of:
step one, preparing a biotin labeled antibody:
(1) dialyzing the 25-hydroxyvitamin D antibody for 16-24h by using a phosphate buffer solution with the pH value of 8.1, adding biotin into the dialyzed antibody, further adding the phosphate buffer solution into the antibody to dilute the antibody, and uniformly mixing to obtain a mixed solution;
(2) placing the mixed solution in a constant temperature incubator, standing and reacting for 120 +/-10 min to obtain a biotin labeled antibody;
(3) dialyzing the biotinylated antibody against phosphate buffer pH 7.4 for 16-24 h;
step two, preparing a biotin-labeled antibody coated magnetic bead:
(1) washing the avidin magnetic beads with a magnetic bead cleaning solution, and then re-dissolving with a binding buffer solution;
(2) mixing the dissolved avidin magnetic beads with the biotin-labeled antibody prepared in the first step, and then placing the mixture in a constant-temperature oscillation box for oscillation reaction to obtain magnetic beads coated with the biotin-labeled antibody;
(3) after reaction, cleaning the magnetic beads by using a magnetic bead cleaning solution, diluting, and storing at 2-8 ℃ for later use;
step three, enriching and purifying samples: adding a sample into an isotope internal standard solution and the magnetic beads coated by the biotin-labeled antibody, stirring and incubating at room temperature, and repeatedly washing with ultrapure water for at least 3 times; eluting the washed magnetic beads for 2 times by using an organic solvent, and collecting eluent;
step four, mass spectrum detection: and drying the eluent obtained in the step three, redissolving the eluent or directly transferring the eluent into a microporous plate, and detecting the eluent by a liquid-phase mass spectrometer.
2. The method of claim 1, wherein the dialysis in step one is performed at 4 ℃ or room temperature for 16-24 hours.
3. The 25-hydroxyvitamin D assay method according to claim 1, wherein the constant temperature incubator temperature in step one is set to 25 ± 1 ℃.
4. The method of claim 1, wherein the weight ratio of the antibody to biotin in step one is 10-20: 1.
5. the method of claim 1, wherein the step of analyzing is performed in the presence of a reagent such as a reagentThe preparation method of the magnetic bead cleaning solution in the second step comprises the following steps: at room temperature, a volume of purified water was added with 1.212g Tris, 9.0g NaCl, 0.4mL HCl, 0.2mL Tween-20, 5.0mL 20% NaN 3 And 0.5mL of Brij-35, the pH value is detected to be 8.3 +/-0.2 after the uniform mixing is carried out, the quality is qualified, and the volume of the purified water is fixed to 1L.
6. The method of claim 1, wherein the binding buffer of step two is formulated by: adding 6.06g Tris, 9.0g sodium chloride and 3.84mL HCl into a certain volume of purified water at room temperature, stirring for at least 5min until complete dissolution, and adding 1.0mL Tween-20 and 5mL 20% NaN 3 Finally, slowly adding 10.0g of bovine serum albumin, and stirring for at least 10min until the bovine serum albumin is completely dissolved; after all the components are completely dissolved, the pH value is measured to be qualified at 7.2 +/-0.1 by a pH meter, and the volume of purified water is determined to be 1L.
7. The method of claim 1, wherein in the second washing step, the volume ratio of the magnetic beads to the magnetic bead washing solution is less than or equal to 1: 2.
8. the method of claim 1, wherein the isotopic internal standard solution is 2 H 3 -25-hydroxyvitamin D2 or 2 H 3 -25-hydroxyvitamin D3.
9. The 25-hydroxyvitamin D assay of claim 1, wherein the organic solvent used in step three elution is 75% acetonitrile containing 0.1% formic acid.
10. The method of claim 1, wherein the parameters of the oscillatory reaction in step three are: the reaction is carried out for 16 to 24 hours under the conditions that the temperature is 37 +/-2 ℃ and the oscillation speed is 100 rmp/min.
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| CN111879876A (en) * | 2020-08-07 | 2020-11-03 | 苏州新波生物技术有限公司 | Rapid and sensitive analysis method for 25 hydroxyvitamin D based on immune mass spectrometry technology |
| CN113063871B (en) * | 2021-03-24 | 2022-12-27 | 杭州佰辰医疗器械有限公司 | Metabolic small molecule detection method and detection system |
| CN114371065A (en) * | 2021-12-28 | 2022-04-19 | 上海固容生物科技有限公司 | Method for processing liquid biopsy sample of biological sample (magnetic bead separation method) and application thereof |
| CN114487209A (en) * | 2022-03-08 | 2022-05-13 | 天津国科医工科技发展有限公司 | Liquid chromatography tandem mass spectrometry detection method of fat-soluble vitamins based on magnetic solid phase extraction |
| CN114544836A (en) * | 2022-03-11 | 2022-05-27 | 天津国科医工科技发展有限公司 | Pretreatment method and detection method for detecting trace estrogen, 17-hydroxypregnanolone, aldosterone and dehydroepiandrosterone sulfate |
| CN116203256A (en) * | 2023-03-06 | 2023-06-02 | 深圳泰乐德医疗有限公司 | Kit for detecting vitamin B6 and detection method thereof |
| CN116500281B (en) * | 2023-06-26 | 2023-11-07 | 天津赛飞乐生物技术有限公司 | 1, 25-dihydroxyvitamin D detection material, preparation method thereof and 1, 25-dihydroxyvitamin D detection method |
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