CN109188003B - Method for measuring 25-hydroxy vitamin D - Google Patents
Method for measuring 25-hydroxy vitamin D Download PDFInfo
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- CN109188003B CN109188003B CN201811070658.4A CN201811070658A CN109188003B CN 109188003 B CN109188003 B CN 109188003B CN 201811070658 A CN201811070658 A CN 201811070658A CN 109188003 B CN109188003 B CN 109188003B
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- JWUBBDSIWDLEOM-XHQRYOPUSA-N (3e)-3-[(2e)-2-[1-(6-hydroxy-6-methylheptan-2-yl)-7a-methyl-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol Chemical compound C1CCC2(C)C(C(CCCC(C)(C)O)C)CCC2\C1=C\C=C1/CC(O)CCC1=C JWUBBDSIWDLEOM-XHQRYOPUSA-N 0.000 title claims abstract description 87
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 16
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- 239000011780 sodium chloride Substances 0.000 claims description 16
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 14
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 12
- 239000007974 sodium acetate buffer Substances 0.000 claims description 9
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 9
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims description 4
- 229910052707 ruthenium Inorganic materials 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
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- 238000001514 detection method Methods 0.000 abstract description 19
- 238000012545 processing Methods 0.000 abstract description 5
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- 238000003018 immunoassay Methods 0.000 description 12
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- 150000003710 vitamin D derivatives Chemical class 0.000 description 7
- 229930003316 Vitamin D Natural products 0.000 description 6
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
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- 239000011710 vitamin D Substances 0.000 description 6
- 229940046008 vitamin d Drugs 0.000 description 6
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- 239000000126 substance Substances 0.000 description 3
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 229940088594 vitamin Drugs 0.000 description 2
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- 102000014914 Carrier Proteins Human genes 0.000 description 1
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- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 102000050760 Vitamin D-binding protein Human genes 0.000 description 1
- 101710179590 Vitamin D-binding protein Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
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- 150000001732 carboxylic acid derivatives Chemical group 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 238000010494 dissociation reaction Methods 0.000 description 1
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- 229950003499 fibrin Drugs 0.000 description 1
- 238000013100 final test Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
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- 238000001556 precipitation Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- 210000000130 stem cell Anatomy 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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- 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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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- 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 a method for detecting 25-hydroxyvitamin D, which comprises the following steps: incubating a biological sample with a first reagent and a second reagent, wherein the first reagent comprises 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 different magnetic particle than the first magnetic particle; the second reagent comprises a 25-hydroxyvitamin D antibody label; further adding a third reagent comprising biotinylated 25-hydroxyvitamin D for co-incubation; separating the magnetic particles; and adding a luminescent substrate and detecting the intensity of the luminescence. The method 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
Technical Field
The invention relates to a method 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 method for detecting 25-hydroxyvitamin D in any one 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 present invention provides a method for detecting 25-hydroxyvitamin D, the method comprising:
incubating a biological sample with a first reagent and a second reagent, wherein the first reagent comprises 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 different magnetic particle than the first magnetic particle; the second reagent comprises a 25-hydroxyvitamin D antibody label;
further adding a third reagent comprising biotinylated 25-hydroxyvitamin D for co-incubation; and
separating the first and second magnetic particles;
a luminescent substrate is added and the intensity of the luminescence is measured.
The Streptavidin-coated magnetic particle is not particularly limited, and may be those commercially available, for example, MS300/Streptavidin magnetic particle from JSR life sciences, Inc., but is not limited thereto. The concentration of the first magnetic particles in the first reagent may vary from product to product and may conveniently be in the range 0.1-1mg/mL, preferably 0.1-0.4mg/mL, 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 in the first reagent is 0.4-1mg/mL, preferably 0.4-0.8mg/mL, and most preferably 0.6 mg/mL.
In the first reagent, 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 in the first reagent may be any suitable buffer solution having a pH of 3.00-6.00. 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 having a pH of 3.00-6.00, 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 label in the second reagent is 50-200ng/mL, preferably 80-130ng/mL, and 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.
The co-incubation of the biological sample with the first and second reagents is performed at about 37 ℃ for 10-30 minutes.
The biological sample may be a serum or plasma sample. In a specific embodiment, the biological sample is taken from a mammal, in particular from a human.
The biotinylated 25-hydroxy vitamin D in the third reagent may be any commercially available product, such as the cat # of the company fenpeng bio-inc: 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.
The co-incubation with the biotinylated 25-hydroxyvitamin D is carried out at about 37 ℃ for 10-30 minutes, preferably 20 minutes.
According to a preferred embodiment, the method of the present invention for detecting 25-hydroxyvitamin D comprises the steps of:
co-incubating the biological sample with a first reagent and a second reagent, wherein
The first reagent comprises a second magnetic particle and a first magnetic particle in an acidic buffer solution with the pH value of 3.00-6.00, wherein the mass ratio of the second magnetic particle to the first magnetic particle is 2:1-4:1, the first magnetic particle is a magnetic particle coated with streptavidin, and the second magnetic particle is a non-specific magnetic particle with the surface modified with carboxyl, tosyl, amino or hydroxyl; and
the second reagent contains a 25-hydroxyvitamin D antibody labeled by horseradish peroxidase, alkaline phosphatase, acridinium ester or ruthenium terpyridyl;
further adding a third reagent comprising biotinylated 25-hydroxyvitamin D for co-incubation;
separating the first and second magnetic particles; and
a luminescent substrate is added and the intensity of the luminescence is measured.
According to a most preferred embodiment, the method of detecting 25-hydroxyvitamin D of the present invention comprises:
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 first and second magnetic particles;
adding a luminescent substrate and detecting the intensity of the luminescence,
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 modified with carboxylic acid 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.
The method 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 method of the present invention can use conventional magnetic particle detection kit and automatic detection equipment, and can detect the magnetic particle in the original steps only by adding the second magnetic particle of the present invention in a certain proportion to the original reagent containing the magnetic particle. 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 (pH 4.5), 0.2mg/mL streptavidin magnetic particle as the first magnetic particle;
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
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 (pH 4.5), 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
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 (PH 4.5), 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
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 (PH 4.5), 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
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 (21)
1. A method of detecting 25-hydroxyvitamin D, the method comprising:
incubating a biological sample with a first reagent and a second reagent, wherein,
the first reagent comprises a first magnetic particle and a second magnetic particle in an acid buffer solution, wherein the first magnetic particle is a streptavidin-coated magnetic particle, and the second magnetic particle is coated with-COOH, -Tosyl, -NH2or-OH-modified magnetic particles different from the first magnetic particles, and the second reagentThe agent comprises a 25-hydroxyvitamin D antibody label;
further adding a third reagent comprising biotinylated 25-hydroxyvitamin D for co-incubation;
separating the first and second magnetic particles; and
adding a luminescent substrate and detecting the intensity of the luminescence,
in the first reagent, the mass ratio of the second magnetic particles to the first magnetic particles is 2:1-8: 1.
2. The method of claim 1, wherein the first magnetic particle has a concentration of 0.1-1mg/mL in the first reagent.
3. The method of claim 2, wherein the concentration of the first magnetic particle in the first reagent is 0.1-0.4 mg/mL.
4. The method of claim 2, wherein the first magnetic particle has a concentration of 0.2mg/mL in the first reagent.
5. The method of claim 1, wherein the second magnetic particle concentration in the first reagent is 0.4-1 mg/mL.
6. The method of claim 5, wherein the second magnetic particle concentration in the first reagent is 0.4-0.8 mg/mL.
7. The method of claim 5, wherein the second magnetic particle concentration is 0.6mg/mL in the first reagent.
8. The method of claim 1, wherein the mass ratio of the second magnetic particles to the first magnetic particles in the first reagent is 2:1-4: 1.
9. The method of claim 1, wherein the first reagent has a mass ratio of the second magnetic particles to the first magnetic particles of 3: 1.
10. The method of claim 1, wherein the pH of the acidic buffer solution in the first reagent is between 3.00 and 6.00.
11. The method of claim 1, wherein the first reagent consists of a buffer having a pH of 3.00-6.00, 0.1-0.4mg/mL of the first magnetic particle, 0.4-0.8mg/mL of the second magnetic particle.
12. The method of claim 11, wherein the first reagent consists of 0.2M acetate-sodium acetate buffer at pH4.5, 0.2mg/mL streptavidin-coated first magnetic particles at a particle size of 1.5 μ ι η, and 0.6mg/mL carboxyl-modified second magnetic particles at a particle size of 1.5 μ ι η.
13. The method of 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.
14. The method of claim 13, wherein the concentration of the 25-hydroxyvitamin D antibody label in the second reagent is 50-200 ng/mL.
15. The method of claim 13, wherein the concentration of the 25-hydroxyvitamin D antibody label in the second reagent is 80-130 ng/mL.
16. The method of claim 13, wherein the concentration of the 25-hydroxyvitamin D antibody label in the second reagent is 100 ng/mL.
17. The method of claim 1, wherein the concentration of biotinylated 25-hydroxyvitamin D in the third reagent is 0.5-2 ng/mL.
18. The method of claim 17, wherein the concentration of biotinylated 25-hydroxyvitamin D in the third reagent is1 ng/mL.
19. The method of claim 1, wherein the co-incubation with the biological sample with the first reagent and the second reagent and further addition of a third reagent comprising biotinylated 25-hydroxyvitamin D are each performed at 37 ℃ for 10-30 minutes.
20. The method of claim 1 comprising the steps of:
co-incubating the biological sample with a first reagent and a second reagent, wherein
The first reagent comprises a second magnetic particle and a first magnetic particle in an acidic buffer solution with the pH value of 3.00-6.00, wherein the mass ratio of the second magnetic particle to the first magnetic particle is 2:1-4:1, the first magnetic particle is a magnetic particle coated with streptavidin, and the second magnetic particle is a non-specific magnetic particle with the surface modified with carboxyl, tosyl, amino or hydroxyl; and
the second reagent contains a 25-hydroxyvitamin D antibody labeled by horseradish peroxidase, alkaline phosphatase, acridinium ester or ruthenium terpyridyl;
further adding a third reagent comprising biotinylated 25-hydroxyvitamin D for co-incubation;
separating the first and second magnetic particles; and
a luminescent substrate is added and the intensity of the luminescence is measured.
21. The method of claim 20, comprising the steps of:
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 first and second magnetic particles;
adding a luminescent substrate and detecting the intensity of the luminescence,
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 modified with carboxyl on the surface;
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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