CN109188002B - Kit for measuring 25-hydroxyvitamin D - Google Patents

Kit for measuring 25-hydroxyvitamin D Download PDF

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CN109188002B
CN109188002B CN201811070237.1A CN201811070237A CN109188002B CN 109188002 B CN109188002 B CN 109188002B CN 201811070237 A CN201811070237 A CN 201811070237A CN 109188002 B CN109188002 B CN 109188002B
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CN109188002A (en
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李洪伟
吴国平
田君喜
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Maccura Biotechnology Co ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

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Abstract

The application discloses 25-hydroxyvitamin D detect reagent box, 25-hydroxyvitamin D detect reagent box includes: a first reagent comprising a first magnetic particle and a second magnetic particle in an acidic buffer solution, wherein the first magnetic particle is a streptavidin-coated magnetic particle and the second magnetic particle is a non-specific magnetic particle different from the first magnetic particle; a second reagent comprising a 25-hydroxyvitamin D antibody label; and a third reagent comprising biotinylated 25-hydroxyvitamin D. The kit of the present invention is also suitable for the detection of 25-hydroxyvitamin D in serum and plasma samples without the need for additional sample processing reagents and without additional sample processing steps.

Description

Kit for measuring 25-hydroxyvitamin D
Technical Field
The invention relates to a method and a kit for detecting 25-hydroxyvitamin D in a blood sample.
Background
Vitamin D is a fat-soluble vitamin, and belongs to sterol derivatives. Vitamin D has the primary functions of regulating the metabolism of calcium and phosphorus in the body and maintaining the temperature of plasma calcium and phosphorus levels, and is involved in skeletal development. The vitamin D family mainly comprises vitamins D1-D5, of which the most important are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol).
25-hydroxyvitamin D is formed in the liver by the action of the monooxygenase system of stem cell microsomes and is the main storage form of vitamin D in the body. The concentration of 25-hydroxyvitamin D in blood can reflect the level of vitamin D in human body, and is the best biochemical index for detecting the level of vitamin D of individual.
The traditional detection method of 25-hydroxy vitamin D comprises a colloidal gold immunochromatography method, an enzyme-linked immunosorbent assay, a chemiluminescence method and the like. The chemiluminescence method can be used for detection by utilizing automatic equipment, and is relatively simple and convenient to operate.
Chinese patent application CN106199016A proposes a magnetic particle chemiluminescence immunoassay method for detecting 25-hydroxy vitamin D in serum. The method specifically combines carboxylated magnetic particles coated by a 25-hydroxyvitamin D monoclonal antibody with 25-hydroxyvitamin D in a sample, further adds an acridinium ester chemiluminescent marker labeled by a vitamin D derivative for competitive combination, and finally quantitatively detects the content of the 25-hydroxyvitamin D by measuring the luminous intensity. The method uses a standard 25-hydroxyvitamin D sample to test and draw a standard curve in advance, so that a full-automatic chemiluminescence immunoassay system can be used for detection, and the method has high detection sensitivity and accuracy.
However, since 25-hydroxyvitamin D in blood has high affinity for vitamin D-binding protein, it is necessary to dissociate it from the binding protein for the purpose of measuring the total 25-hydroxyvitamin D concentration in human blood, and the dissociation agent used is generally acidic in pH. The plasma sample has higher protein content than the serum sample, can be used for isoelectric precipitation of a large amount of protein substances, particularly plasma fibrin in a pH acidic environment, and the precipitated protein substances can be attached to the surfaces of the magnetic particles, so that the detection method adopting the magnetic particles is interfered, and the measurement results of the serum and the plasma are greatly different. Therefore, most of the current methods for detecting 25-hydroxyvitamin D by using magnetic particles can only detect serum samples, but cannot detect plasma samples.
The Yapei (Abbott) 25-hydroxyvitamin D assay kit is also a kit for measuring 25-hydroxyvitamin D by magnetic microparticle-chemiluminescence. The sample amount was only 10. mu.L, and the samples were incubated in a mixture using two different pretreatment solutions, and a portion thereof was taken out for detection. After the method carries out pretreatment solution treatment and dilution on the sample, the concentration of the interferent is greatly reduced, thereby basically eliminating the influence of the interferent of the plasma sample on the measurement result, and the method is a kit which can be simultaneously used for serum and plasma samples to detect 25-hydroxyvitamin D at present.
However, this method requires pretreatment of the sample, not only increasing the detection step, but also extending the detection time. In addition, due to the fact that two pretreatment solutions are added, samples are diluted, the amount of detected samples is small, and the kit is low in sensitivity, linear and the like. Furthermore, despite the prior processing, the final test results still differ somewhat between serum and plasma. Therefore, there is still a need for an improved method for the detection of 25-hydroxyvitamin D that can be used for both serum and plasma samples.
Disclosure of Invention
The object of the present invention is to solve at least one of the above technical problems. Therefore, it is an object of the present invention to provide a kit capable of detecting 25-hydroxyvitamin D in either of serum and plasma samples using a conventional fully automated chemiluminescent immunoassay analyzer, wherein the detection results obtained are substantially free from significant differences depending on whether the sample is serum or plasma.
Specifically, the invention provides a 25-hydroxyvitamin D detection kit, wherein the 25-hydroxyvitamin D detection kit comprises:
a first reagent comprising a first magnetic particle and a second magnetic particle in an acidic buffer solution, wherein the first magnetic particle is a streptavidin-coated magnetic particle and the second magnetic particle is a non-specific magnetic particle different from the first magnetic particle;
a second reagent comprising a 25-hydroxyvitamin D antibody label; and
a third reagent comprising biotinylated 25-hydroxyvitamin D.
In the first reagent, the Streptavidin-coated magnetic particles are not particularly limited, and may be those commercially available, for example, MS300/Streptavidin magnetic particles from JSR life sciences, but are not limited thereto.
The concentration of the first magnetic particles may be 0.1-1mg/mL, preferably 0.1-0.4mg/mL, and most preferably 0.2 mg/mL.
The second magnetic particles may be surface-coated with-COOH, -Tosyl, -NH2or-OH modified magnetic particles. Magnetic fine particles whose surfaces are modified with carboxyl groups are preferable.
The concentration of the second magnetic particles is 0.4-1mg/mL, preferably 0.4-0.8mg/mL, and most preferably 0.6 mg/mL.
The mass ratio of the second magnetic particles to the first magnetic particles is 2:1-8:1, and the mass ratio is preferably 2:1-4:1, and most preferably 3:1 in consideration of cost.
The first and second magnetic particles generally have a particle size in the range of 1.0 to 3.0. mu.m, and may be the same or different. In particular, the particle size of the second magnetic particles in the above range does not significantly affect the detection accuracy.
The acidic buffer solution can be any suitable buffer solution having a pH of 3.00 to 6.00. Specific examples are: 0.2M acetic acid-sodium acetate buffer (. about.pH4.5), but is not limited thereto.
According to a preferred embodiment, the first reagent may consist of a buffer of pH3.00-6.00, 0.1-0.4mg/mL of said first magnetic particles, 0.4-0.8mg/mL of said second magnetic particles. According to a more preferred embodiment, the first reagent may be composed of 0.2M acetic acid-sodium acetate buffer having a pH of 4.5, 0.2mg/mL streptavidin-coated first magnetic particles having a particle size of 1.5 μ M, and 0.6mg/mL carboxylic acid-modified second magnetic particles having a particle size of 1.5 μ M. The 25-hydroxyvitamin D antibody marker in the second reagent is a substance which can be used for quantitatively detecting 25-hydroxyvitamin D, wherein the marker can be any one of horseradish peroxidase, alkaline phosphatase, acridinium ester and ruthenium terpyridyl, and is most preferably horseradish peroxidase.
The concentration of the 25-hydroxyvitamin D antibody marker in the second reagent is 50-200ng/mL, preferably 80-130ng/mL, most preferably 100 ng/mL.
The second agent may conventionally contain necessary additives including, but not limited to, a buffer, sodium chloride, sucrose, a surfactant, a preservative, and the like. These additives may be added according to conventional requirements without particular limitation.
The second reagent in the present invention is a reagent containing a 25-hydroxyvitamin D antibody label for detection in a conventional manner, and is not particularly limited.
According to an exemplary embodiment, the second reagent consists of 0.1mol/L Tris-HCl buffer, 8.5g/L sodium chloride, 20g/L sucrose, 20g/L polyethylene glycol 20000, 1mL/L Procline950, 0.5mL/L Tween-20, 100ng/mL horseradish peroxidase-labeled 25-hydroxyvitamin D antibody.
In the third reagent, the biotinylated 25-hydroxyvitamin D is not particularly limited, and may be any commercially available product, such as a product number of philippine bio-corporation: VD-BIO, but is not so limited.
The concentration of the biotinylated 25-hydroxyvitamin D in the third reagent may be generally 0.5-2ng/mL, preferably 1 ng/mL.
The same third agent may conveniently contain additives such as: buffers, sodium chloride, sucrose, surfactants, preservatives, and the like, but are not limited thereto. These additives may be added according to conventional requirements without particular limitation.
The third reagent of the present invention is also a conventional reagent, and is not particularly limited
According to an exemplary embodiment, the third reagent consists of 0.1mol/L Tris-HCl buffer, 8.5g/L NaCl, 20g/L sucrose, 1mL/L Procline950, 0.5mL/L Tween-20, 1ng/mL biotinylated 25-hydroxyvitamin D.
According to a preferred embodiment, the kit for detecting 25-hydroxyvitamin D of the present invention comprises:
the first reagent comprises second magnetic particles and first magnetic particles in an acidic buffer solution with the pH value of 3.00-6.00 at the mass ratio of 2:1-4:1, wherein the first magnetic particles are magnetic particles coated with streptavidin, and the second magnetic particles are nonspecific magnetic particles with the surfaces modified with carboxyl, tosyl, amino or hydroxyl;
a second reagent comprising a 25-hydroxyvitamin D antibody labeled with horseradish peroxidase, alkaline phosphatase, acridinium ester, or ruthenium terpyridyl;
a third reagent comprising biotinylated 25-hydroxyvitamin D.
According to a most preferred embodiment, the kit for detecting 25-hydroxyvitamin D of the present invention comprises:
the reagent comprises a first reagent, a second reagent and a third reagent, wherein the first reagent consists of 0.2M acetic acid-sodium acetate buffer solution with the pH value of 4.5, 0.2mg/mL first magnetic particles coated with streptavidin and 0.6mg/mL second magnetic particles with the surfaces modified by carboxylic acid;
the second reagent consists of 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 20g/L polyethylene glycol 20000, 1mL/L Procline950, 0.5mL/L Tween-20 and 100ng/mL horseradish peroxidase-labeled 25-hydroxy vitamin D antibody;
the third reagent consists of 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 1mL/L Procline950, 0.5mL/L Tween-20 and 1ng/mL biotinylated 25-hydroxy vitamin D.
The kit of the invention is used for detecting 25-hydroxyvitamin D in serum or plasma samples. In a specific embodiment, the subject is a mammal, in particular a human.
The detection and use method of the kit comprises the following steps:
incubating a biological sample with a first reagent and a second reagent;
further adding a third reagent comprising biotinylated 25-hydroxyvitamin D for co-incubation;
separating the magnetic particles; and
a luminescent substrate is added and the intensity of the luminescence is measured.
Wherein the two incubations may be performed, for example, at about 37 ℃ for 10 to 30 minutes, preferably for 20 minutes.
The kit of the present invention is also suitable for the detection of 25-hydroxyvitamin D in serum and plasma samples without the need for additional sample processing reagents and without additional sample processing steps. The kit of the invention can use conventional automatic detection equipment, namely, the detection can be carried out according to the original steps of the conventional magnetic particle detection kit. The standard deviation of the detection results of the serum sample and the plasma sample from the same subject is within ± 15%, and in a preferred embodiment, the standard deviation can be within ± 10%, which completely meets the requirement of the detection accuracy of the detection kit.
Detailed Description
The invention is further illustrated by the following specific examples.
Example 1 comparison of results of determination of serum and plasma samples of the same origin
Materials and reagents
Serum/plasma (EDTA-K2) pairs from 20 subjects were taken,
25-hydroxyvitamin D Main calibrator of Mike biological products Ltd 1 set,
McEtBioGmbH 25-hydroxyvitamin D assay kit (chemiluminescence method) 2 boxes (lot No. 0417011, Specification: 100 test/box),
McBioSicGreens Ltd washing buffer (Cat: IM4202458, lot: 0417141)
Substrate solution (enzymatic chemiluminescent substrate solution (HRP)) for full-automatic immunoassay system of Mike biological products Ltd (cat # IM4202459, lot # 0417031)
IS1200 full-automatic chemiluminescence immunoassay analyzer (equipment number: 1015-.
The 25-hydroxy vitamin D determination kit comprises the following reagents:
first reagent (R1): 0.2mol/L acetic acid-sodium acetate buffer (pH4.5), 0.2mg/mL streptavidin magnetic particles as the first magnetic particles;
third agent (R2): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 9501 mL/L Procline, 200.5 mL/L tween-L and biotinylated 25-hydroxy vitamin D1 ng/mL;
second reagent (R3): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 2000020 g/L polyethylene glycol, 9501 Procline 9501 mL/L Tween-200.5 mL/L, and 100ng/mL horseradish peroxidase-labeled 25-hydroxy vitamin D antibody.
Additionally, preparing second magnetic particles: carboxylic magnetic particles (manufacturer: JSR life sciences, cat # MS160/Carboxyl, particle size: 1.5 μm).
Firstly, detecting a 25-hydroxyvitamin D main calibrator and 20 pairs of serum/plasma samples by using a 25-hydroxyvitamin D determination kit without adding second magnetic particles, and calculating the deviation between a plasma measured value and a serum measured value;
the 25-hydroxyvitamin D master calibrator and the same 20 pairs of serum/plasma samples were then tested using a 25-hydroxyvitamin D assay kit with second magnetic microparticles added to a final concentration of 0.6mg/mL, and the deviation between the plasma and serum measurements was calculated.
The specific method comprises mixing 15 μ L sample with 100 μ L first reagent and 100 μ L second reagent, reacting at 37 deg.C for 20 min, adding 100 μ L third reagent, mixing, and reacting at 37 deg.C for 20 min. Magnetic separation to obtain magnetic particles, washing with washing buffer solution for 3 times, adding luminous substrate solution, and detecting signal value.
Finally, the above assay was repeated as required by the instructions using the Yapei (Abbott) 25-hydroxyvitamin D assay kit.
The results of the two measurements are shown in table 1 below:
TABLE 1
Figure BDA0001799342270000071
From the above table, it can be seen that the relative deviation of 20 to the serum/plasma sample measured by the kit without the second magnetic particles is between 30.25% and 106.12%, and the difference is very large. After the second magnetic particles are added, the relative deviation is between 2.29 and 9.89 percent, and the effect is obvious. The 20 pairs were tested using the commercial Abbott kit, which is capable of testing serum plasma samples, with relative deviations between-4.96% and 10.52%. The kit added with the magnetic particles meets the requirement of the YY/T1585-2017 total 25-hydroxyvitamin D determination kit (the relative deviation is within the range of +/-15%) on the accuracy of the kit as the Yapei kit, can be used for measuring serum samples and plasma samples, and has the effect equivalent to that of the Yapei kit.
Example 2 different addition of second magnetic beads;
materials and reagents
The same serum/plasma (EDTA-K2) pairs from 20 subjects as in example 1,
25-hydroxyvitamin D Main calibrator of Mike biological products Ltd 1 set,
McEvTokyi Ltd 25-hydroxyvitamin D assay kit (chemiluminescence method) 3 boxes (lot No. 0417011, specification: 100 test/box),
McBioSicGreens Ltd washing buffer (Cat: IM4202458, lot: 0417141)
Substrate solution (enzymatic chemiluminescent substrate solution (HRP)) for full-automatic immunoassay system of Mike biological products Ltd (cat # IM4202459, lot # 0417031)
McBioSiteOfficial IS1200 full-automatic chemiluminescence immunoassay analyzer (Equipment number: 1015-
The 25-hydroxy vitamin D determination kit comprises the following reagents:
first reagent (R1): 0.2mol/L acetic acid-sodium acetate buffer (pH4.5), 0.2mg/mL streptavidin magnetic particles as first magnetic particles;
third agent (R2): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 9501 mL/L Procline, 200.5 mL/L tween-L and biotinylated 25-hydroxy vitamin D1 ng/mL;
second reagent (R3): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 2000020 g/L polyethylene glycol, 9501 Procline 9501 mL/L Tween-200.5 mL/L, and 100ng/mL horseradish peroxidase-labeled 25-hydroxy vitamin D antibody.
Additionally, preparing second magnetic particles: carboxylic magnetic particles (manufacturer: JSR life sciences, cat # MS160/Carboxyl, particle size: 1.5 μm).
The second magnetic particles with final concentrations of 0.2mg/mL, 0.4mg/mL and 0.8mg/mL were added to the first reagent in 3 cartridges, and the 25-hydroxyvitamin D assay kit with the second magnetic particles in 3 cartridges was used to detect the 25-hydroxyvitamin D master calibrator and 20 pairs of serum/plasma samples, and the deviation between the plasma measurement value and the serum measurement value was calculated.
The specific method comprises mixing 15 μ L sample with 100 μ L first reagent and 100 μ L second reagent, reacting at 37 deg.C for 20 min, adding 100 μ L third reagent, mixing, and reacting at 37 deg.C for 20 min. Magnetic separation to obtain magnetic particles, washing with washing buffer solution for 3 times, adding luminous substrate solution, and detecting signal value.
The results of the two measurements are shown in table 2 below:
TABLE 2
Figure BDA0001799342270000091
From the above table, it can be seen that the relative deviation of the kit measurement 20, in which 0.2mg/mL of the second magnetic particles are added at a ratio of 1:1 (the second magnetic particles are compared with the first magnetic particles), to the serum/plasma sample is between 5.01% and 48.33%, and the accuracy of the kit (the relative deviation is within a range of +/-15%) exceeding the accuracy requirement of the YY/T1585-2017 total 25-hydroxyvitamin D measurement kit (the labeled immunoassay method) is judged to be incapable of measuring the serum/plasma; the kit with 0.4mg/mL second magnetic particles added in a ratio of 2:1 has a relative deviation of 5.18-14.77%, can meet the requirement of the accuracy of the kit, and can be judged as measurable serum and plasma, but the difference is large; the kit added with 0.8mg/mL second magnetic particles in a ratio of 4:1 has a relative deviation of 3.81-8.82%, meets the requirement of the accuracy of the kit, and is judged to be capable of determining serum and plasma.
Example 3
Materials and reagents
The same serum/plasma (EDTA-K2) pairs from 20 subjects as in example 1,
25-hydroxyvitamin D Main calibrator of Mike biological products Ltd 1 set,
McEvTokyi Ltd 25-hydroxyvitamin D assay kit (chemiluminescence method) 3 boxes (lot No. 0417011, specification: 100 test/box),
McBioSicGreens Ltd washing buffer (Cat: IM4202458, lot: 0417141)
Substrate solution (enzymatic chemiluminescent substrate solution (HRP)) for full-automatic immunoassay system of Mike biological products Ltd (cat # IM4202459, lot # 0417031)
McBioSiteOfficial IS1200 full-automatic chemiluminescence immunoassay analyzer (Equipment number: 1015-
The 25-hydroxy vitamin D determination kit comprises the following reagents:
first reagent (R1): 0.2mol/L acetic acid-sodium acetate buffer (PH4.5), 0.2mg/mL streptavidin magnetic particles as the first magnetic particles;
third agent (R2): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 9501 mL/L Procline, 200.5 mL/L tween-L and biotinylated 25-hydroxy vitamin D1 ng/mL;
second reagent (R3): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 2000020 g/L polyethylene glycol, 9501 Procline 9501 mL/L Tween-200.5 mL/L, and 100ng/mL horseradish peroxidase-labeled 25-hydroxy vitamin D antibody.
Second magnetic particles: tosyl magnetic particles (manufacturer: JSR life sciences, cat. No.: MS160/Tosyl, particle diameter: 1.5 μm).
The second magnetic particles with final concentrations of 0.4mg/mL, 0.6mg/mL and 0.8mg/mL were added to the first reagent in 3 cartridges, and the 25-hydroxyvitamin D assay kit with the second magnetic particles in 3 cartridges was used to detect the 25-hydroxyvitamin D master calibrator and 20 pairs of serum/plasma samples, and the deviation between the plasma measurement value and the serum measurement value was calculated.
The specific method comprises mixing 15 μ L sample with 100 μ L first reagent and 100 μ L second reagent, reacting at 37 deg.C for 20 min, adding 100 μ L third reagent, mixing, and reacting at 37 deg.C for 20 min. Magnetic separation to obtain magnetic particles, washing with washing buffer solution for 3 times, adding luminous substrate solution, and detecting signal value.
The results of the two measurements are shown in table 3 below:
TABLE 3
Figure BDA0001799342270000111
The above table shows that the relative deviation of the kit added with 0.4mg/mL Tosyl magnetic particles for determining 20 to the serum and plasma samples is between 0.60% and 14.52%, and the requirement of the kit on accuracy is met; the kit added with 0.6mg/ml of oligosyl magnetic particles has the relative deviation of 1.82-11.56% and meets the requirement of the accuracy of the kit; the kit added with 0.8mg/ml of oligosyl magnetic particles has the relative deviation of 2.04-9.96%, and meets the requirement of accuracy of the kit.
Example 4
Materials and reagents
The same serum/plasma (EDTA-K2) pairs from 20 subjects as in example 1,
25-hydroxyvitamin D Main calibrator of Mike biological products Ltd 1 set,
McEtBioGmbH 25-hydroxyvitamin D assay kit (chemiluminescence method) 2 boxes (lot No. 0417011, Specification: 100 test/box),
McBioSicGreens Ltd washing buffer (Cat: IM4202458, lot: 0417141)
Substrate solution (enzymatic chemiluminescent substrate solution (HRP)) for full-automatic immunoassay system of Mike biological products Ltd (cat # IM4202459, lot # 0417031)
McBioSiteOfficial IS1200 full-automatic chemiluminescence immunoassay analyzer (Equipment number: 1015-
The 25-hydroxy vitamin D determination kit comprises the following reagents:
first reagent (R1): 0.2mol/L acetic acid-sodium acetate buffer (PH4.5), 0.2mg/mL streptavidin magnetic particles as the first magnetic particles;
third agent (R2): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 9501 mL/L Procline, 200.5 mL/L tween-L and biotinylated 25-hydroxy vitamin D1 ng/mL;
second reagent (R3): 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 2000020 g/L polyethylene glycol, 9501 Procline 9501 mL/L Tween-200.5 mL/L, and 100ng/mL horseradish peroxidase-labeled 25-hydroxy vitamin D antibody.
Two additional second magnetic particles were prepared: tosyl magnetic particles (manufacturer: JSR life sciences, cat # MS300/Tosyl, particle size: 3.0 μm) and Carboxyl magnetic particles (manufacturer: JSR life sciences, cat # MX200/Carboxyl, particle size: 2.1. mu.m).
Two-pack kit the Tosyl magnetic particles were added to the first reagent of the first pack at a final concentration of 0.6mg/mL, the carboxyl magnetic particles were added to the first reagent of the second pack at a final concentration of 0.6mg/mL, and the 25-hydroxyvitamin D master calibrator and 20 pairs of serum/plasma samples were tested using the 2-pack 25-hydroxyvitamin D assay kit with the second magnetic particles added, and the deviation between the plasma measurement value and the serum measurement value was calculated.
The specific method comprises mixing 15 μ L sample with 100 μ L first reagent and 100 μ L second reagent, reacting at 37 deg.C for 20 min, adding 100 μ L third reagent, mixing, and reacting at 37 deg.C for 20 min. Magnetic separation to obtain magnetic particles, washing with washing buffer solution for 3 times, adding luminous substrate solution, and detecting signal value.
The results of the two measurements are shown in table 4 below:
TABLE 4
Figure BDA0001799342270000131
The above table shows that the relative deviation of the kit 20 added with the Tosyl magnetic particles with the particle size of 0.6mg/mL being 3.0 mu m to the serum and plasma sample is between 1.89 and 11.40 percent, and the accuracy requirement of the kit is met; after the carboxyl magnetic particles with the particle size of 2.1 mu m of 0.6mg/mL are added, the relative deviation is measured to be between 0.48 and 10.46 percent, and the requirement of the accuracy of the kit is also met.

Claims (23)

1. A 25-hydroxyvitamin D detection kit, the 25-hydroxyvitamin D detection kit comprising:
a first reagent comprising a first magnetic particle and a second magnetic particle in an acidic buffer solution, wherein the first magnetic particle is a streptavidin-coated magnetic particle and the second magnetic particle is a non-specific magnetic particle different from the first magnetic particle;
a second reagent comprising a 25-hydroxyvitamin D antibody label; and
a third reagent comprising biotinylated 25-hydroxyvitamin D;
in the first reagent, the mass ratio of the second magnetic particles to the first magnetic particles is 2:1-8: 1.
2. The kit of claim 1, wherein the first magnetic particle is present in the first reagent at a concentration of 0.1-1 mg/mL.
3. The kit of claim 2, wherein the first magnetic particle is present in the first reagent at a concentration of 0.1-0.4 mg/mL.
4. The kit of claim 3, wherein the first reagent has a concentration of the first magnetic particle of 0.2 mg/mL.
5. The kit according to claim 1, wherein in the first reagent, the second magnetic particles are surface-coated with-COOH, -Tosyl, -NH2or-OH modified magnetic particles.
6. The kit of claim 1, wherein the concentration of the second magnetic particles in the first reagent is 0.4-1 mg/mL.
7. The kit of claim 6, wherein the concentration of the second magnetic particles in the first reagent is 0.4-0.8 mg/mL.
8. The kit of claim 7, wherein the concentration of the second magnetic particles in the first reagent is 0.6 mg/mL.
9. The kit of any one of claims 1-8, wherein the mass ratio of the second magnetic particles to the first magnetic particles in the first reagent is 2:1-4: 1.
10. The kit of claim 9, wherein the first reagent has a mass ratio of the second magnetic particles to the first magnetic particles of 3: 1.
11. The kit according to claim 1, wherein the acidic buffer solution has a pH value of 3.00-6.00.
12. The kit of claim 1, wherein the first reagent consists of a buffer at ph3.00-6.00, 0.1-0.4mg/mL of the first magnetic particle, 0.4-0.8mg/mL of the second magnetic particle.
13. The kit according to claim 12, wherein the first reagent consists of 0.2M acetic acid-sodium acetate buffer solution with pH of 4.5, 0.2mg/mL streptavidin-coated first magnetic particles with a particle size of 1.5 μ M, and 0.6mg/mL carboxyl-modified second magnetic particles with a particle size of 1.5 μ M.
14. The kit according to claim 1, wherein the 25-hydroxyvitamin D antibody label in the second reagent is one selected from horseradish peroxidase, alkaline phosphatase, acridinium ester and ruthenium terpyridyl.
15. The kit of claim 14, wherein the concentration of the 25-hydroxyvitamin D antibody marker in the second reagent is 50-200 ng/mL.
16. The kit of claim 15, wherein the concentration of the 25-hydroxyvitamin D antibody marker in the second reagent is 80-130 ng/mL.
17. The kit of claim 16, wherein the concentration of the 25-hydroxyvitamin D antibody marker in the second reagent is 100 ng/mL.
18. The kit of claim 14, wherein the second reagent consists of 0.1mol/L Tris-HCl buffer, 8.5g/L sodium chloride, 20g/L sucrose, 20g/L polyethylene glycol 20000, 1mL/L Procline950, 0.5mL/L tween-20, 100ng/mL horseradish peroxidase-labeled 25-hydroxyvitamin D antibody.
19. The kit of claim 1, wherein the concentration of biotinylated 25-hydroxyvitamin D in the third reagent is 0.5-2 ng/mL.
20. The kit of claim 19, wherein the concentration of biotinylated 25-hydroxyvitamin D in the third reagent is1 ng/mL.
21. The kit of claim 19, wherein the third reagent consists of 0.1mol/L Tris-HCl buffer, 8.5g/L sodium chloride, 20g/L sucrose, 1mL/L Procline950, 0.5mL/L tween-20, 1ng/mL biotinylated 25-hydroxyvitamin D.
22. The kit of claim 1, comprising:
the first reagent comprises second magnetic particles and first magnetic particles in an acidic buffer solution with the pH value of 3.00-6.00 at the mass ratio of 2:1-4:1, wherein the first magnetic particles are magnetic particles coated with streptavidin, and the second magnetic particles are nonspecific magnetic particles with the surfaces modified with carboxyl, tosyl, amino or hydroxyl;
a second reagent comprising a 25-hydroxyvitamin D antibody labeled with horseradish peroxidase, alkaline phosphatase, acridinium ester, or ruthenium terpyridyl;
a third reagent comprising biotinylated 25-hydroxyvitamin D.
23. The kit of claim 22, comprising: the first reagent consists of 0.2M acetic acid-sodium acetate buffer solution with the pH value of 4.5, 0.2mg/mL first magnetic particles coated with streptavidin and 0.6mg/mL second magnetic particles with modified carboxyl on the surfaces;
the second reagent consists of 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 20g/L polyethylene glycol 20000, 1mL/L Procline950, 0.5mL/L Tween-20 and 100ng/mL horseradish peroxidase-labeled 25-hydroxy vitamin D antibody;
the third reagent consists of 0.1mol/L Tris-HCl buffer solution, 8.5g/L sodium chloride, 20g/L sucrose, 1mL/L Procline950, 0.5mL/L Tween-20 and 1ng/mL biotinylated 25-hydroxy vitamin D.
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