WO2021170053A1 - 25-hydroxyvitamin d analysis method based on immunopurification in combination with mass spectrometric detection - Google Patents

Abstract

A 25-hydroxyvitamin D (25-(OH)D) analysis method based on immunopurification in combination with mass spectrometric detection, mainly comprising the following steps: preparing a biotin-labelled antibody; preparing a magnetic bead that is coated with the biotin-labelled antibody; enriching and purifying a sample; and performing mass spectrometric detection. In the analysis method, by using a high specificity of antibody affinity, 25-(OH)D can be specifically enriched and purified from complex biological samples, and the accurate detection of 25-(OH)D2 and 25-(OH)D3 is separately achieved; according to an antibody-coupled magnetic bead enrichment method in combination with mass spectrometric detection, the use efficiency of a liquid phase mass spectrometry system is greatly improved while high throughput, sensitivity, and accurate quantification are achieved.

Description

An analytical method of 25-hydroxyvitamin D based on immunopurification combined with mass spectrometry detection Technical field

The invention relates to the technical field of biological detection, in particular to a 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection.

Background technique

25-hydroxyvitamin D (25-(OH)D) includes 25-hydroxyvitamin D2 (25-(OH)D2) and 25-hydroxyvitamin D3 (25-(OH)D3), which is the main presence of vitamin D in the body form. The level of serum 25-(OH)D can reflect the body's vitamin D storage level, and is related to the clinical symptoms of vitamin D deficiency. The detection methods of 25-(OH)D2 and 25-(OH)D3 in biological samples (such as whole blood, serum, urine or saliva) mainly include 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 biological samples. Traditional sample processing methods mainly include precipitation, liquid-liquid extraction and solid-phase extraction. According to the method, the substances in the obtained samples are separated by liquid chromatography according to their different properties and then analyzed by mass spectrometry according to the different mass-nucleus ratios. Liquid chromatography mass spectrometry can realize the simultaneous detection of 25-(OH)D2 and 25-(OH)D3.

However, the pre-treatment of liquid chromatography-mass spectrometry samples are all non-specific enrichment methods, and the components of the enriched samples are more complicated. The enriched samples contain 25-(OH)D2 and 25-(OH)D3. It also contains a large amount of matrix materials with properties similar to 25-(OH)D. During chromatographic separation, some non-target analytes are prone to denaturation and precipitation on the chromatographic column or difficult to elute from the chromatographic column, resulting in higher matrix effects, causing column pressure to rise, and affecting the effectiveness of subsequent separation and analysis of target substances Performance and stability, increase the difficulty and burden of liquid mass spectrometry maintenance. Using chromatography to completely separate the target analyte from its isomers or structural analogs is extremely difficult and time-consuming. Incompletely separated matrix materials may also interfere with the mass spectrometric quantification of the analyte.

Immunoassay analysis methods include radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA) and fully automated biochemical methods. The basic principle is to use 25-(OH)D antibodies to specifically capture 25-(OH)D in biological samples, and then detect the corresponding radiation, light or electrochemical signals to achieve the analysis of 25-(OH)D Purpose. But generally 25-(OH)D2 and 25-(OH)D3 will be captured by antibodies, so the immunoassay method detects the total 25-(OH)D level, and it is also vulnerable to other 25-(OH)D structures. Cross interference of similar analytes affects quantification and limits clinical applications.

Therefore, the present invention intends to provide a 25-hydroxyvitamin D analysis method that can accurately detect 25-(OH)D2 and 25-(OH)D3, respectively, and has high specificity.

Summary of the invention

Aiming at the deficiencies of the prior art, the present invention provides a 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

The present invention provides a 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection, which includes the following steps:

Step one, prepare biotin-labeled antibody:

(1) Dialyze the 25-(OH)D antibody with pH 8.1 phosphate buffer solution for 16-24 hours, add the dialyzed antibody to biotin, and then further add phosphate buffer solution to dilute the antibody, and mix to obtain a mixture liquid;

(2) Place the mixed solution in a constant temperature incubator and let it stand for 120±10 minutes to obtain a biotin-labeled antibody;

(3) Dialysis the biotinylated antibody with phosphate buffer at pH 7.4 for 16-24 hours;

Step two, prepare magnetic beads coated with biotin-labeled antibody:

(1) Wash the avidin magnetic beads with magnetic bead cleaning solution, and then reconstitute it with binding buffer;

(2) Mix the dissolved avidin magnetic beads with the biotin-labeled antibody prepared in step 1, and then place the biotin-labeled antibody in a constant temperature shaking box to oscillate to obtain biotin-labeled antibody-coated magnetic beads;

(3) After the reaction, wash the magnetic beads with a magnetic bead cleaning solution, dilute, and store at 2-8°C until use;

Step three, enrich and purify the sample: add the sample to the isotope internal standard solution, the magnetic beads coated with the biotin-labeled antibody, stir and incubate at room temperature, and repeat the washing with ultrapure water for at least 3 times; the washed magnetic beads use an organic solvent Perform two elutions and collect the eluate;

Step four, mass spectrometry detection: the eluent obtained in step three is dried and reconstituted or directly transferred to a microplate, and tested by a liquid-phase mass spectrometer.

Further, the dialysis in step one is performed at 4°C or room temperature for 16-24 hours.

Further, the temperature of the thermostat in step one is set to 25±1°C.

Further, the weight ratio of antibody and biotin in step one is 10-20:1.

Further, the preparation method of the magnetic bead cleaning solution in step two is: at room temperature, add 1.212g of Tris, 9.0g of sodium chloride, 0.4mL of HCl, 0.2mL of Tween-20, to a certain volume of purified water. 5.0mL of 20% NaN _3, 0.5mL of Brij-35, pH of the mixed detection 8.3 ± 0.2 qualified, purified water volume to 1L.

Further, the method for preparing the binding buffer in step two is: add 6.06 g of Tris, 9.0 g of sodium chloride and 3.84 mL of HCl to a certain volume of purified water at room temperature, stir for at least 5 minutes until it is completely dissolved, and then add 1.0mL of Tween-20 and 5mL of 20% NaN ₃ , and finally slowly add 10.0g of bovine serum albumin (BSA), and stir for at least 10 minutes to completely dissolve; after all the ingredients are completely dissolved, use a pH meter to measure the pH at 7.2± 0.1 is qualified, and the volume of purified water is set to 1L.

Further, during the cleaning step 2, 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-hydroxy vitamin D2 or ² H ₃ -25-hydroxy vitamin D3.

Further, the incubation conditions in step three are: room temperature, 30 min.

Further, the organic phase used for elution in step 3 is 75% acetonitrile, which contains 0.1% formic acid.

Further, the parameters of the shaking reaction in step 3 are: reacting for 16-24 hours at a temperature of 37±2° C. and an shaking speed of 100 rpm/min.

Compared with the prior art, the present invention adopts the above technical solutions and has the following technical effects:

The invention relates to a 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection, which utilizes the high specificity of antibody affinity, and can specifically enrich and purify 25-(OH)D from complex biological samples, and simultaneously achieve The precise detection of 25-(OH)D2 and 25-(OH)D3 respectively; antibody-coupled magnetic bead enrichment method combined with mass spectrometry detection achieves high-throughput, sensitive and accurate quantification while greatly improving the liquid-phase mass spectrometry system The efficiency of use. In addition, the invention can be used to develop small molecule detection and diagnostic kits, a combination of multiple antibody-coupled magnetic beads, which can simultaneously enrich and detect multiple biomarkers by mass spectrometry at one time. Compared with the traditional immunoassay single-index detection, the throughput Higher, more specific and selective.

Description of the drawings

FIG. 1 is an operation flowchart of an analysis method in an embodiment of the present invention;

Among them, 1 is antibody-coupled magnetic beads; 2 is the enrichment and purification of markers; 3 is the multi-index and Suzuki-specific detection of mass spectrometry.

Detailed ways

The present invention provides a 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection. The present invention will be described in detail and concretely through specific examples below, so as to better understand the present invention, but the following examples do not It does not limit the scope of the present invention.

Example

Referring to Figure 1, this embodiment provides a 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection, which includes the following steps:

Step one, prepare biotin-labeled antibody:

Take 1 mg of 25-(OH)D antibody and dialyze it with phosphate buffer (pH 8.1) at 4°C or room temperature for no less than 16-24 hours; add 0.05 mg of biotin to the dialyzed antibody, and then add phosphate buffer ( pH 8.1) Make the final concentration of the antibody 1mg/mL, mix for 5 minutes; place the mixture in a constant temperature incubator at 25±1°C, let stand for 120±10 minutes, and finally use phosphate buffer (pH 7.4) at 4°C or The biotinylated antibody was dialyzed at room temperature for 16-24h.

Step two, prepare magnetic beads coated with biotin-labeled antibody:

Shake the streptavidin magnetic beads well, then suck 20mg magnetic beads (volume x μL), wash twice with a magnetic bead cleaning solution not less than twice the volume of the magnetic beads, and then reconstitute the magnetic beads to Not less than 5mg/mL; mix the magnetic beads with the above-mentioned biotinylated antibody, the final concentration of the magnetic beads is not less than 10mg/mL, the magnetic beads are placed in a constant temperature shaking box 37±2℃, 100rmp/min shake and react for 16-24h ; Remove the reacted magnetic beads, wash 4 times with a magnetic bead cleaning solution of not less than 2 times the reaction volume, dilute the magnetic beads to 400μg/mL with binding buffer, and store at 2-8°C until use.

Step three, enrich and purify the sample: take 100μL of serum, add 100μL of internal standard solution (containing ² H ₃ -25-hydroxyvitamin D2 (20ng/mL), ² H ₃ -25-hydroxyvitamin D3 (20ng/mL)), 300μL antibody magnetic beads, incubate with stirring at room temperature for 30min, transfer the magnetic beads to 500μL ultrapure water, stir and mix, and repeat washing at least 3 times. The washed magnetic beads were transferred to 100 μL of 75% acetonitrile (containing 0.1% formic acid), mixed well for 5 minutes, and collected the eluate. The magnetic beads were washed once again, and the eluates were collected twice.

Step 4. Mass spectrometry detection: The eluent can be dried, reconstituted with an appropriate solvent, or directly transferred 150μL to a 96-well microtiter plate, and tested by a liquid-phase mass spectrometer.

Among them, the preparation method of the magnetic bead cleaning solution in step two is: at room temperature, add 1.212g of Tris, 9.0g of sodium chloride, 0.4mL of HCl, 0.2mL of Tween-20, 5.0 to a certain volume of purified water mL of 20% NaN ₃ , 0.5 mL of Brij-35, after mixing uniformly, the pH value is 8.3±0.2, it is qualified, and the volume of purified water is adjusted to 1L.

The method for preparing the binding buffer in step two is: add 6.06g of Tris, 9.0g of sodium chloride and 3.84mL of HCl to a certain volume of purified water at room temperature, stir for at least 5min until completely dissolved, and then add 1.0mL Tween-20 and 5mL 20% NaN ₃ , and finally slowly add 10.0g bovine serum albumin (BSA), stirring at least 10 minutes to completely dissolve; after all the ingredients are completely dissolved, use a pH meter to measure the pH value at 7.2 ± 0.1 Qualified, the volume of purified water is set to 1L.

The effect of this evaluation method is analyzed, and the results shown in Table 1-3 are obtained.

Table 1 Calculation results of precision

Table 2 Calculation results of linear range

Table 3 Recovery rate of 25-hydroxyvitamin D

Analyte	average(%)	95% confidence interval
25-hydroxy vitamin D2	94	88-100
25-hydroxyvitamin D3	90	79-100

Comparison

This comparative example uses traditional liquid chromatography mass spectrometry to detect and analyze 25-hydroxyvitamin D. The results are shown in Table 4-6 below:

Table 4 Calculation results of precision

Table 5 Calculation results of linear range

Table 6 Recovery rate of 25-hydroxyvitamin D

Analyte	average(%)	95% confidence interval
25-hydroxy vitamin D2	91	85-100
25-hydroxyvitamin D3	88	74-100

In summary, the method provided by the present invention can specifically enrich and purify 25-(OH)D from complex biological samples, while simultaneously achieving precise detection of 25-(OH)D2 and 25-(OH)D3, respectively. Compared with traditional methods, it has the characteristics of high specificity, high throughput and accurate quantification.

The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should all fall within the scope of the present invention.

Claims (10)

1. A 25-hydroxyvitamin D analysis method based on immunopurification combined with mass spectrometry detection is characterized in that it comprises the following steps:

   Step one, prepare biotin-labeled antibody:

   (1) Dialyze the 25-hydroxyvitamin D antibody with pH 8.1 phosphate buffer for 16-24 hours, add biotin to the dialyzed antibody, and then further add phosphate buffer to dilute the antibody, and mix to obtain a mixed solution ；

   (2) Place the mixed solution in a constant temperature incubator and let it stand for 120±10 minutes to obtain a biotin-labeled antibody;

   (3) Dialysis the biotinylated antibody with a phosphate buffer with a pH of 7.4 for 16-24 hours;

   Step two, prepare magnetic beads coated with biotin-labeled antibody:

   (1) Wash the avidin magnetic beads with magnetic bead cleaning solution, and then reconstitute it with binding buffer;

   (2) Mix the dissolved avidin magnetic beads with the biotin-labeled antibody prepared in step 1, and then place the avidin-labeled antibody-coated magnetic beads in a constant temperature shaking box for shaking reaction;

   (3) After the reaction, wash the magnetic beads with a magnetic bead cleaning solution, dilute, and store at 2-8°C until use;

   Step three, enrich and purify the sample: add the sample to the isotope internal standard solution, the magnetic beads coated with the biotin-labeled antibody, stir and incubate at room temperature, and repeat the washing with ultrapure water for at least 3 times; the washed magnetic beads use an organic solvent Perform two elutions and collect the eluate;

   Step four, mass spectrometry detection: the eluent obtained in step three is dried and reconstituted or directly transferred to a microplate, and detected by a liquid-phase mass spectrometer.
2. The 25-hydroxyvitamin D analysis method of claim 1, wherein the dialysis in step 1 is performed at 4°C or room temperature for 16-24 hours.
3. The 25-hydroxyvitamin D analysis method according to claim 1, wherein the temperature of the incubator in the step one is set to 25±1°C.
4. The 25-hydroxyvitamin D analysis method of claim 1, wherein the weight ratio of the antibody and biotin in the first step is 10-20:1.
5. The 25-hydroxyvitamin D analysis method according to claim 1, wherein the preparation method of the magnetic bead cleaning solution in step two is: adding 1.212g of Tris, 9.0 to a certain volume of purified water at room temperature. g of sodium chloride, 0.4mL of HCl, 0.2mL of Tween-20, 5.0mL of 20% NaN ₃ , 0.5mL of Brij-35, after mixing evenly, the pH value is 8.3±0.2 as qualified, and the purified water is constant volume To 1L.
6. The 25-hydroxyvitamin D analysis method according to claim 1, wherein the method for preparing the binding buffer in step two is: adding 6.06g of Tris, 9.0g to a certain volume of purified water at room temperature sodium chloride and 3.84mL of HCl, stirred 5min at least until complete dissolution, followed by addition of 1.0mL of Tween-20 and 5mL 20% NaN _3, and finally slowly added 10.0g of bovine serum albumin, at least 10min stirring until completely dissolved; After all the ingredients are completely dissolved, use a pH meter to measure the pH value of 7.2±0.1 as qualified, and dilute the purified water to 1L.
7. The 25-hydroxyvitamin D analysis method according to claim 1, characterized in that, during the cleaning step 2, the volume ratio of the magnetic beads to the magnetic bead cleaning solution is less than or equal to 1:2.
8. The 25-hydroxyvitamin D analysis method of claim 1, wherein the isotope internal standard solution is ² H ₃ -25-hydroxy vitamin D2 or ² H ₃ -25-hydroxy vitamin D3.
9. The 25-hydroxyvitamin D analysis method according to claim 1, wherein the organic solvent used for elution in step 3 is 75% acetonitrile, which contains 0.1% formic acid.
10. The 25-hydroxyvitamin D analysis method according to claim 1, characterized in that the parameters of the shaking reaction in step 3 are: reacting at a temperature of 37±2° C. and an shaking speed of 100 rmp/min for 16-24 hours.

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