CN111721944A - 25-hydroxy vitamin D detection kit, preparation method and detection method - Google Patents
25-hydroxy vitamin D detection kit, preparation method and detection method Download PDFInfo
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- CN111721944A CN111721944A CN202010560055.3A CN202010560055A CN111721944A CN 111721944 A CN111721944 A CN 111721944A CN 202010560055 A CN202010560055 A CN 202010560055A CN 111721944 A CN111721944 A CN 111721944A
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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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention relates to the technical field of in-vitro diagnosis and detection, in particular to a 25-hydroxy vitamin D detection kit, a preparation method and a detection method. A 25-hydroxyvitamin D detection kit comprising: the reagent kit comprises an R1 reagent, an R2 reagent, an R3 reagent and a sample processing reagent, wherein the R1 reagent comprises a streptavidin magnetic particle solution; the R2 reagent comprises an alkaline phosphatase-labeled 25-hydroxy vitamin D monoclonal antibody solution; the R3 reagent comprises a biotin-labeled 25-hydroxyvitamin D antigen solution; the sample processing reagent comprises a weakly acidic buffer, a reducing agent and a lower alcohol solvent. The kit has higher sensitivity, specificity and wider detection range, can detect only 40 mu L of serum or plasma, has good patient experience, can be used together with a full-automatic chemiluminescence analyzer, and realizes automatic monitoring.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis and detection, in particular to a detection kit, a preparation method and a detection method of 25-hydroxyvitamin D.
Background
Vitamin D is a fat-soluble steroid derivative, and mainly comprises two forms of vitamin D2 and D3. Vitamin D2 is produced by ultraviolet irradiation of ergosterol in plants; vitamin D3 is prepared by converting 7-dehydrocholesterol contained in human epidermis and dermis by ultraviolet irradiation in sunlight. In humans, vitamins D2 and D3 bind to vitamin D binding proteins in plasma and are transported to the liver where they are acted upon by the monooxygenase system (25-hydroxylase) in the hepatocyte microsomes to form 25-hydroxyvitamin D (including 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3). The 25-hydroxy vitamin D is converted into 1, 25-dihydroxy vitamin D under the action of alpha hydroxylase system in mitochondria of renal proximal tubular epithelial cells, which is the most biologically active form of vitamin D and can promote the synthesis of intestinal calcium binding protein. And 25-hydroxyvitamin D is a main storage form in the metabolism of vitamin D in the human body and can reflect the vitamin D level of the body, so the vitamin D level of the human body is evaluated by detecting 25-hydroxyvitamin D.
Vitamin D is a main element for maintaining the health of bones, promotes the growth of the bones in a human body by regulating the bone calcium metabolism and the blood phosphorus metabolism in the human body, participates in the differentiation of cells of the human body and the regulation of immunologic function, and is directly related to the balance of the metabolism of the human body so as to influence the health condition of the human body. Vitamin D deficiency will lead to muscle weakness, and severe vitamin D deficiency in childhood will lead to skeletal deformity, i.e. rickets; if the disease occurs in adults, mature skeletal calcification insufficiency occurs, and osteomalacia is caused. Studies have shown that vitamin D deficiency may increase the incidence of certain cardiovascular, autoimmune and infectious diseases, and prolonged intake of excessive vitamin D causes hypercalcemia and hypercalcemia, and severe cases die due to renal calcification, calcification of the heart and aorta. More and more medical professionals and patients have realized that the lack or excess of vitamin D causes health risks, and the test amount of medical institutions is increased rapidly, so that a more stable and accurate detection reagent is needed to help doctors obtain accurate detection results. Of course, the test results of the 25-hydroxyvitamin D assay reagent are only used for clinical reference and cannot be used alone as a basis for diagnosis or elimination of cases.
However, the chemiluminescence immunoassay method adopted at present for detecting 25-hydroxyvitamin D still has the problems of low sensitivity, poor specificity, poor reproducibility, narrow detection range and the like. The development of a 25-hydroxyvitamin D detection reagent with high detection sensitivity, strong specificity, wide detection range and good reproducibility is still needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a 25-hydroxyvitamin D detection kit, a preparation method and a detection method, wherein the kit has the advantages of high detection sensitivity, strong specificity, wide detection range and good reproducibility.
In order to realize the purpose, the invention provides a 25-hydroxy vitamin D detection kit, which adopts the following technical scheme:
a25-hydroxyvitamin D detection kit, comprising: r1 reagent, R2 reagent, R3 reagent and sample processing reagent,
the R1 reagent comprises a streptavidin-coated magnetic particle solution, the concentration of the streptavidin-coated magnetic particles is 0.05-1 mg/ml, and the particle size of the magnetic particles is 1-4 μm;
the R2 reagent comprises an alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody solution, the concentration of the alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody is 0.2-1.0 ng/mL, and the labeling ratio of the anti-25-hydroxyvitamin D monoclonal antibody to alkaline phosphatase is 1: 1-10: 1;
the R3 reagent comprises a biotin-labeled 25-hydroxyvitamin D antigen, and the concentration of the biotin-labeled 25-hydroxyvitamin D antigen is 0.05-0.2%;
the sample processing reagent comprises a weakly acidic buffer, a reducing agent and a lower alcohol solvent, and is used for dissociating 25-hydroxyvitamin D from conjugated protein.
After a large number of research and experimental researches, the inventor discovers that 25-hydroxyvitamin D in a sample to be detected in the traditional technology is often insufficiently combined with a 25-hydroxyvitamin D monoclonal antibody marked by alkaline phosphatase, so that the detection result is inaccurate, and the sensitivity of the kit is low. One reason is that the sample to be detected is usually dissociated from 25-hydroxyvitamin D by using a strong alkaline solution, but the activity of alkaline phosphatase is reduced to a certain extent by using the strong alkaline solution, so that the dissociated 25-hydroxyvitamin D is not fully combined with the alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody, and the detection sensitivity is low and the detection result is inaccurate; the other reason is that 25-hydroxyvitamin D belongs to a small molecular hydrophobic substance and is not easy to be combined with the 25-hydroxyvitamin D monoclonal antibody marked by alkaline phosphatase, so that the detection sensitivity is low and the detection result is inaccurate.
On the basis of the research findings, the invention improves the traditional technology, and uses a sample processing reagent with a weak acid buffer solution to effectively dissociate 25-hydroxyvitamin D from a sample to be detected and simultaneously keep the activity of the 25-hydroxyvitamin D monoclonal antibody marked by alkaline phosphatase; a reducing agent is adopted to avoid the conversion of 25-hydroxyvitamin D, improve the antigen activity of the 25-hydroxyvitamin D and promote the combination of the 25-hydroxyvitamin D and the 25-hydroxyvitamin D monoclonal antibody marked by alkaline phosphatase; the dissolution of 25-hydroxy vitamin D can be promoted by adopting a lower alcohol solvent. The synergistic effect of the components of the sample treatment reagent can effectively dissociate 25-hydroxyvitamin D from a sample to be detected, maintain the activity of the 25-hydroxyvitamin D and the 25-hydroxyvitamin D monoclonal antibody marked by alkaline phosphatase, and fully combine the 25-hydroxyvitamin D and the alkaline phosphatase, thereby achieving the effects of accurate detection and high sensitivity of the detection kit.
In one embodiment, the pH value of the weak acid buffer solution is 5.5-6.9; the weak acidic buffer solution is selected from one of a citric acid buffer solution, an acetic acid buffer solution or a phosphoric acid buffer solution; the weak acid buffer is preferably a citric acid buffer; the concentration of the citric acid buffer solution is 0.05-2.5%, and the concentration of the citric acid buffer solution is preferably 1.5%.
In one embodiment, the reducing agent is selected from dithiothreitol, tris (2-carboxyethyl) phosphine hydrochloride, reduced glutathione, cysteine, or β -mercaptoethanol; the reducing agent is preferably beta-mercaptoethanol, the concentration of the beta-mercaptoethanol is 0.05% -5%, and the concentration of the beta-mercaptoethanol is preferably 0.1% -2.5%; more preferably, the concentration of beta-mercaptoethanol is 0.25 to 1 percent, and most preferably, the concentration of beta-mercaptoethanol is 0.5 percent;
in one embodiment, the concentration of the lower alcohol solvent is 10% to 20%, preferably the concentration of the lower alcohol solvent is 10% to 15%, and more preferably the concentration of the lower alcohol solvent is 15%; the lower alcohol solvent is selected from one of methanol or ethanol.
In one embodiment, the sample processing reagent further comprises 0.02-0.1% of a preservative, and the preservative is one of sodium azide or Procline 300.
In one embodiment, the R1 reagent further includes 0.02 to 0.1mol/L of 2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02 to 0.1% of preservative, wherein the preservative is one of sodium azide and Procline 300;
in one embodiment, the R2 reagent further includes 0.02 to 0.1mol/L of 2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02 to 0.1% of preservative, wherein the preservative is one of sodium azide and Procline 300;
in one embodiment, the R3 reagent further includes 0.02 to 0.1mol/L of a 2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02 to 0.1% of a preservative, and the preservative is one of sodium azide and Procline 300.
In one embodiment, the kit further comprises a calibrator and a quality control product, wherein the calibrator and the quality control product respectively comprise 25-hydroxyvitamin D antigen, serum, preservative and buffer solution, the serum is selected from one of sheep serum, bovine serum, horse serum, donkey serum or serum containing human, the preservative is selected from one of sodium azide and Procline300, the preservative has a mass percentage concentration of 0.02-0.1%, the buffer solution is selected from one of PBS buffer solution, HEPES buffer solution, Tris-Hcl buffer solution, MES buffer solution and MOPS buffer solution, and the pH value of the buffer solution is 7.2-7.4; the concentrations of 25-hydroxy vitamin D antigens in the calibrator liquid series are respectively 0.0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL and 120 ng/mL; the concentration of 25 hydroxy vitamin D antigen in the quality control liquid series is 20ng/mL and 80ng/mL respectively.
The invention also provides a preparation method of the 25-hydroxyvitamin D detection kit, which is characterized in that the preparation method of the alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody in the R2 reagent comprises the following steps:
(1) preparation of ALP-mal:
1) adding DMF into EMCS to prepare an EMCS solution of 60-100 mM;
2) adding the EMCS solution to an ALP-modulating buffer;
3) adding the mixed solution while stirring the ALP solution, heating at 35-38 ℃ for 50-70 minutes;
4) carrying out centrifugal desalination, and recovering the solution after centrifugal desalination;
(2) ALP-mal tag reduction:
1) buffer solution replacement, namely performing the replacement of the 25-hydroxy vitamin D monoclonal antibody buffer solution by using a centrifugal desalting column, and recovering the solution after the buffer solution replacement;
2) dissolving MEA (membrane electrode assembly) by using a buffer solution for gel filtration, and preparing 0.2-0.5M MEA solution;
3) sulfhydrylation of a 25-hydroxy vitamin D monoclonal antibody solution;
4) desalting, namely performing centrifugal desalting after the reaction is finished, and recovering a desalted solution;
5) introducing ALP-mal, adding ALP-mal into the above solution, sealing, and standing at 2-8 deg.C for reaction for about 16-24 hr;
6) stopping, adding 0.1M MEA solution into the solution in the step 5), fully stirring, and reacting for 5-15 minutes at the temperature of 35-38 ℃;
7) and (4) storing the solution after termination at the temperature of 2-8 ℃.
In one embodiment, the 25-hydroxyvitamin D assay kit is characterized in that the preparation method of the biotin-labeled anti-25-hydroxyvitamin D antigen in the R3 reagent comprises the following steps:
(1) desalting the 25-hydroxy vitamin D antigen in a desalting column to obtain a purified solution;
(2) according to DMF: biotin 1: preparing 1-30 mM biotin solution at a ratio of 3-5;
(3) adding the purified solution into a biotin solution, and reacting at normal temperature for 20-40 min;
(4) desalting after reaction, and recovering the centrifugate to obtain the biotin-labeled anti-25-hydroxy vitamin D antigen.
In one embodiment, the preparation method of the 25-hydroxyvitamin D detection kit is characterized in that the preparation method of the magnetic particles coated with streptomycin avidin in the R1 reagent comprises the following steps: and (3) taking the streptavidin magnetic particle solution, and diluting the streptavidin magnetic particle solution by using 0.02-0.1 mol/LTRIS buffer solution to obtain the streptavidin magnetic particles with the concentration of 0.05-1 mg/ml.
In one embodiment, the preparation method of the 25-hydroxyvitamin D detection kit is characterized in that the preparation method of the calibrator liquid series and the quality control liquid series comprises the following steps:
(1) preparing a calibrator diluent by using a buffer solution, serum and a preservative;
(2) dissolving 25-hydroxy vitamin D antigen with DMF;
(3) diluting the dissolved 25 hydroxyvitamin D antigen with the calibrator diluent in the step (1) to obtain calibrator liquid series with the concentrations of the 25 hydroxyvitamin D antigen being 0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL and 120ng/mL respectively; or diluting the dissolved 25 hydroxy vitamin D antigen by using the calibrator diluent in the step (1) to obtain quality control solution series with the concentrations of the 25 hydroxy vitamin D antigen being 20ng/mL and 80ng/mL respectively.
The present invention also provides a chemiluminescent immunoassay method for the detection of 25-hydroxyvitamin D characterized in that the kit according to any one of claims 1 to 9 is used, comprising the steps of:
step A: mixing a sample to be detected, a sample treatment reagent and an R2 reagent, incubating for 20-40 minutes at 36-38 ℃, fully dissociating 25-hydroxyvitamin D in the sample to be detected, fully combining with an alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody, and washing;
and B: adding an R1 reagent and an R3 reagent, incubating for 5-15 minutes at 36-38 ℃, and washing;
and C: adding a chemiluminescent substrate, generating a luminescent signal under the catalysis of enzyme, receiving the luminescent signal by a photon reading system, measuring the number of photons generated by the reaction by a photomultiplier tube, and automatically calculating the content of 25-hydroxyvitamin D in a sample to be detected by software according to the optical signal and a Cutoff value;
the chemiluminescence zymolyte is (3- (2-spiral adamantane) -4-methoxyl-4- (3-phosphorus oxygen acyl) -phenyl-1, 2-dioxetane and AMPPD.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the detection kit for 25-hydroxyvitamin D, the sample treatment reagent comprises a weak acid buffer, a reducing agent and a lower alcohol solvent, and the weak acid buffer, the reducing agent and the lower alcohol solvent are cooperated to play a role, so that 25-hydroxyvitamin D can be effectively dissociated from a sample to be detected, the antigen activity and the solubility of 25-hydroxyvitamin D are enhanced, the activity of an alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody is maintained, the 25-hydroxyvitamin D and the alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody are fully combined, and the detection sensitivity and the detection accuracy are improved.
(2) The chemiluminescence immune method of 25-hydroxyvitamin D adopts the kit, firstly adds the sample to be tested, the sample processing reagent and the R2 reagent, incubates to ensure that the 25-hydroxyvitamin D in the sample to be tested is fully dissociated, fully combining with 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase to form a compound, washing, adding magnetic particles marked by R1 reagent streptavidin and 25-hydroxy vitamin D antigen marked by R3 reagent biotin, the method makes 25-hydroxy vitamin D and 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase fully combined, and then the washing is carried out, thereby avoiding the loss of the 25-hydroxy vitamin D to be detected, and streptavidin magnetic particles and biotin marked 25-hydroxy vitamin D antigen are added, thereby improving the detection sensitivity.
(3) The chemiluminescence detection kit for 25-hydroxyvitamin D realizes chemiluminescence detection by an immunofluorescence competition method, and has high sensitivity, specificity and wide detection range.
(4) The 25-hydroxy vitamin D chemiluminescence detection kit provided by the invention can detect only 40 mu L of serum or plasma, has good patient experience, and is beneficial to the acceptance of patients.
(5) The 25-hydroxyvitamin D test kit provided by the invention can be used with a full-automatic chemiluminescence analyzer, so that automatic monitoring is realized, errors caused by manual operation are avoided, and the detection speed and the detection efficiency are improved.
(6) The 25-hydroxy vitamin D chemiluminescence detection kit provided by the invention has the advantages that the anti-interference capability of a high-quality antibody is strong, the detection range completely covers the existing clinical detection requirements, and the market popularization potential is very strong.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 shows the correlation between the assay value of the 25-hydroxyvitamin D assay kit and the assay value of the imported kit in the example of the present application.
Detailed Description
In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Streptavidin magnetic beads were purchased from roche diagnostics ltd (cat # 11641778001).
Example 1
A25-hydroxy vitamin D detection kit comprises an R1 reagent, an R2 reagent, an R3 reagent, a sample processing reagent, a calibrator and a quality control product, wherein,
the R1 reagent comprises a streptavidin-coated magnetic particle solution, the concentration of the streptavidin-coated magnetic particles is 0.05-1 mg/ml, and the particle size of the magnetic particles is 1-4 μm.
The R2 reagent comprises an alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody solution, the concentration of the alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody is 0.2-1.0 ng/mL, and the labeling ratio of the anti-25-hydroxyvitamin D monoclonal antibody to alkaline phosphatase is 1: 1-10: 1.
the R3 reagent comprises biotin-labeled 25-hydroxyvitamin D antigen, and the concentration of the biotin-labeled 25-hydroxyvitamin D antigen is 0.05-0.2%.
The sample processing reagent comprises a weakly acidic buffer, a reducing agent and a lower alcohol solvent, and is used for dissociating 25-hydroxyvitamin D from conjugated protein.
Wherein the pH value of the weak acid buffer solution is 5.5-6.9; the weak acidic buffer solution is selected from one of a citric acid buffer solution, an acetic acid buffer solution or a phosphoric acid buffer solution; the weak acid buffer is preferably citric acid buffer; the concentration of the citric acid buffer solution is 0.02-2.5%, and the concentration of the citric acid buffer solution is preferably 1.5%.
Wherein the reducing agent is one of dithiothreitol, tris (2-carboxyethyl) phosphine hydrochloride, reduced glutathione, cysteine or beta-mercaptoethanol; the reducing agent is preferably beta-mercaptoethanol, the concentration of the beta-mercaptoethanol is 0.05 to 5 percent, and the concentration of the beta-mercaptoethanol is preferably 0.1 to 2.5 percent; more preferably, the concentration of β -mercaptoethanol is 0.25% to 1%, and most preferably the concentration of β -mercaptoethanol is 0.5%.
Wherein, the concentration of the lower alcohol solvent is 10-20%, preferably the concentration of the lower alcohol solvent is 10-15%, more preferably the concentration of the lower alcohol solvent is 15%; the lower alcohol solvent is selected from methanol or ethanol.
The sample processing reagent further comprises 0.02-0.1% of a preservative, and the preservative is one of sodium azide or Procline 300.
The R1 reagent further comprises 0.02-0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02-0.1% preservative, wherein the preservative is one of sodium azide or Procline 300;
the R2 reagent further comprises 0.02-0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02-0.1% preservative, wherein the preservative is one of sodium azide or Procline 300;
the R3 reagent further comprises 0.02-0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02-0.1% preservative, and the preservative is one of sodium azide or Procline 300.
The calibrator and the quality control product respectively comprise 25-hydroxy vitamin D antigen, serum, preservative and buffer solution, wherein the serum is selected from one of sheep serum, bovine serum, horse serum, donkey serum or serum containing human serum, the preservative is selected from one of sodium azide and Procline300, the preservative has the mass percentage concentration of 0.02-0.1%, the buffer solution is selected from one of PBS buffer solution, HEPES buffer solution, Tris-Hcl buffer solution, MES buffer solution and MOPS buffer solution, and the pH value of the buffer solution is 7.2-7.4; the concentrations of 25-hydroxy vitamin D antigens in the calibrator liquid series are respectively 0.0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL and 120 ng/mL; the concentration of 25 hydroxy vitamin D antigen in the quality control liquid series is 20ng/mL and 80ng/mL respectively.
The preparation method of the alkaline phosphatase-labeled anti-25-hydroxy vitamin D monoclonal antibody in the R2 reagent comprises the following steps:
(1) preparation of ALP-mal:
1) adding DMF into EMCS to prepare an EMCS solution of 60-100 mM;
2) adding the EMCS solution to an ALP-modulating buffer;
3) adding the mixed solution while stirring the ALP solution, heating at 35-38 ℃ for 50-70 minutes;
4) carrying out centrifugal desalination, and recovering the solution after centrifugal desalination;
(2) ALP-mal tag reduction:
1) buffer solution replacement, namely performing the replacement of the 25-hydroxy vitamin D monoclonal antibody buffer solution by using a centrifugal desalting column, and recovering the solution after the buffer solution replacement;
2) dissolving MEA (membrane electrode assembly) by using a buffer solution for gel filtration, and preparing 0.2-0.5M MEA solution;
3) sulfhydrylation of a 25-hydroxy vitamin D monoclonal antibody solution;
4) desalting, namely performing centrifugal desalting after the reaction is finished, and recovering a desalted solution;
5) introducing ALP-mal, adding ALP-mal into the above solution, sealing, and standing at 2-8 deg.C for reaction for about 16-24 hr;
6) stopping, adding 0.1M MEA solution into the solution in the step 5), fully stirring, and reacting for 5-15 minutes at the temperature of 35-38 ℃;
7) and (4) storing the solution after termination at the temperature of 2-8 ℃.
The preparation method of the biotin-labeled anti-25-hydroxy vitamin D antigen in the R3 reagent comprises the following steps:
(1) desalting the 25-hydroxy vitamin D antigen in a desalting column to obtain a purified solution;
(2) according to DMF: biotin 1: preparing 1-30 mM biotin solution at a ratio of 3-5;
(3) adding the purified solution into a biotin solution, and reacting at normal temperature for 20-40 min;
(4) desalting after reaction, and recovering the centrifugate to obtain the biotin-labeled anti-25-hydroxy vitamin D antigen.
The preparation method of the streptomycin avidin coated magnetic particle in the R1 reagent comprises the following steps: and (3) taking the streptavidin magnetic particle solution, and diluting the streptavidin magnetic particle solution by using 0.02-0.1 mol/LTRIS buffer solution to obtain the streptavidin magnetic particles with the concentration of 0.05-1 mg/ml.
The preparation method of the calibrator liquid series and the quality control liquid series comprises the following steps:
(1) preparing a calibrator diluent by using a buffer solution, serum and a preservative;
(2) dissolving 25 hydroxy vitamin D antigen with DMF;
(3) diluting the dissolved 25 hydroxyvitamin D antigen with the calibrator diluent in the step (1) to obtain calibrator liquid series with the concentrations of the 25 hydroxyvitamin D antigen being 0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL and 120ng/mL respectively; or diluting the dissolved 25 hydroxy vitamin D antigen by using the calibrator diluent in the step (1) to obtain quality control solution series with the concentrations of the 25 hydroxy vitamin D antigen being 20ng/mL and 80ng/mL respectively.
Example 2
Detection method of 25-hydroxy vitamin D detection kit
Step A: mixing a sample to be detected, a sample treatment reagent and an R2 reagent, incubating for 20-40 minutes at 36-38 ℃, fully dissociating 25-hydroxyvitamin D in the sample to be detected, fully combining with an alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody, and washing;
and B: adding an R1 reagent and an R3 reagent, incubating for 5-15 minutes at 36-38 ℃, and washing;
and C: adding a chemiluminescent substrate, generating a luminescent signal under the catalysis of enzyme, receiving the luminescent signal by a photon reading system, measuring the number of photons generated by the reaction by a photomultiplier tube, and automatically calculating the content of 25-hydroxyvitamin D in a sample to be detected by software according to the optical signal and a Cutoff value;
the chemiluminescence zymolyte is (3- (2-spiral adamantane) -4-methoxyl-4- (3-phosphorus oxygen acyl) -phenyl-1, 2-dioxetane and AMPPD.
Test example 1
Selection of citrate buffer concentration:
after the mother solutions of the components of the 25-hydroxyvitamin D detection reagent are prepared into corresponding working concentrations, citric acid buffer solutions with different concentrations are respectively added into a sample processing reagent, and the detection method of the embodiment 2 is adopted to detect the 25-hydroxyvitamin D in a sample to be detected under the condition that other conditions are not changed.
A sample to be detected: 40 μ L of test serum.
Working concentration of each component: the concentration of the streptavidin-labeled magnetic particles is 0.5 mg/mL; the concentration of the 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase is 0.2 ng/mL; concentration of biotinylated 25-hydroxyvitamin D antigen 0.2%; the concentration of the citric acid buffer solution is 0.02-2.5%, the concentration of the reducing agent is 0.1%, the concentration of the lower alcohol solvent is 10%, and the incubation temperature is 36-38 ℃.
And (3) detecting data: see table 1 below.
| Selection of |
0 | 0.02％ | 0.05％ | 0.1％ | 1.5％ | 2％ | 2.50％ |
| Detection limit (ng/mL) | 5 | 4.6 | 4.2 | 3.8 | 3 | 3.2 | 3.5 |
TABLE 1
And (3) analyzing a detection result: as can be seen from table 1 above, when the concentration of the citrate buffer is 0.05 to 2.5%, the sensitivity of the kit is high, and preferably, the concentration of the citrate buffer is 1.5%.
Test example 2
Selection of the reducing agent:
after mother solutions of components of a 25-hydroxyvitamin D detection reagent are prepared into corresponding working concentrations, 1% of reducing agents (including Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine hydrochloride (TECP-HCL), beta-mercaptoethanol, reductive glutathione and cysteine) are respectively added into a sample treatment reagent, and the 25-hydroxyvitamin D in a sample to be detected is detected by adopting the detection method of the embodiment 2 under the condition that other conditions are not changed.
A sample to be detected: 40 mu L of serum to be detected;
working concentration of each component: the concentration of the streptavidin-labeled magnetic particles is 0.5 mg/mL; the concentration of the 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase is 0.2 ng/mL; concentration of biotinylated 25-hydroxyvitamin D antigen 0.2%; the concentration of the citric acid buffer solution is 1.5 percent, the concentration of the reducing agent is 0.1 percent, the concentration of the lower alcohol solvent is 10 percent, and the incubation temperature is 36-38 ℃.
And (3) detecting data: see table 2 below.
TABLE 2
And (3) analyzing a detection result: as can be seen from table 2 above, by using no reducing agent as a control, the addition of reducing agents (including Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine hydrochloride (TECP-HCL), β -mercaptoethanol, reduced glutathione, cysteine) increases the binding effect of the 25-hydroxyvitamin D antigen and the alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody, and increases the sensitivity of the kit; wherein the reduction capability of the beta-mercaptoethanol is optimal.
Test example 3
Selection of the concentration of reducing agent (. beta. -mercaptoethanol):
after mother solutions of components of the 25-hydroxyvitamin D detection reagent are prepared into corresponding working concentrations, reducing agents (beta-mercaptoethanol) with different concentrations are added into antigen diluent, the detection method of the embodiment 2 is adopted, and under the condition that other conditions are not changed, the 25-hydroxyvitamin D in a sample to be detected is detected.
A sample to be detected: 40 mu L of serum to be detected;
working concentration of each component: the concentration of the streptavidin-labeled magnetic particles is 0.5 mg/mL; the concentration of the 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase is 0.2 ng/mL; concentration of biotinylated 25-hydroxyvitamin D antigen 0.2%; the concentration of the citric acid buffer solution is 1.5%, the concentration of beta-mercaptoethanol is 0.05-5%, the concentration of the lower alcohol solvent is 15%, and the incubation temperature is 36-38 ℃.
And (3) detecting data: see table 3 below.
| β selection of the concentration of |
0 | 0.05％ | 0.10％ | 0.25％ | 0.50％ | 1％ | 2.50％ | 5％ |
| Detection limit (ng/mL) | 4.2 | 3.7 | 3.6 | 3.1 | 3.0 | 3.1 | 3.3 | 3.2 |
TABLE 3
And (3) analyzing a detection result: as can be seen from table 4 above, the sensitivity of the kit was improved by adding β -mercaptoethanol at concentrations of 0.05%, 0.10%, 0.25%, 0.50%, 1%, 2.50%, and 5% to β -mercaptoethanol at a concentration of 0% as a control. The concentration of beta-mercaptoethanol is determined to be 0.05% to 5%, preferably 0.1% to 2.5%, more preferably 0.25% to 1%, most preferably 0.5%.
Test example 4
Selection of the concentration of the lower alcohol solvent:
preparing mother liquor of each component of the 25-hydroxyvitamin D detection reagent into corresponding working concentration, respectively adding lower alcohol solvents with different concentrations into the antigen diluent, and detecting the 25-hydroxyvitamin D in a sample to be detected by adopting the detection method of the embodiment 2 under the condition that other conditions are not changed.
A sample to be detected: 40 mul of blood plasma to be detected;
working concentration of each component: the concentration of the streptavidin-labeled magnetic particles is 1 mg/mL; the concentration of the 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase is 1 ng/mL; concentration of biotinylated 25-hydroxyvitamin D antigen 0.2%; the concentration of the citric acid buffer solution is 1.5 percent, the concentration of the beta-mercaptoethanol is 0.5 percent, the concentration of the lower alcohol solvent is 0-25 percent, the lower alcohol solvent is selected from one of methanol or ethanol, and the incubation temperature is 36-38 ℃.
And (3) detecting data: see table 4 below.
| Selection of |
0 | 5％ | 10％ | 15％ | 20％ | 25％ |
| Detection limit (ng/mL) | 5 | 3.5 | 3.2 | 3.0 | 3.3 | 3.5 |
TABLE 4
And (3) analyzing a detection result: as can be seen from table 4 above, the sensitivity of the kit was improved by using a lower pure solvent with a concentration of 0% as a control and adding lower alcohol solvents with concentrations of 5%, 10%, 15%, 20%, and 25%, respectively. Determining the concentration of the lower alcohol to be 10-20%, preferably the concentration of the lower alcohol solvent to be 10-15%, more preferably the concentration of the lower alcohol solvent to be 15%, wherein the lower alcohol solvent is selected from one of methanol or ethanol.
Test example 5 precision test
In-batch precision test
The reagent prepared by the reagent composition and preparation method of example 1 is used for measuring sample series with high and low concentrations in the same batch by the detection method of example 2, and 10-hole parallel measurement is carried out.
Working concentration of each component: the concentration of the streptavidin-labeled magnetic particles is 0.2 mg/mL; the concentration of the 25-hydroxy vitamin D monoclonal antibody marked by alkaline phosphatase is 0.2 percent; concentration of biotinylated 25-hydroxyvitamin D antigen 0.1%; the concentration of the weak acid buffer solution is 1.5 percent, the concentration of the reducing agent is 0.5 percent, the concentration of the lower alcohol solvent is 15 percent, and the incubation temperature is 36-38 ℃.
And (3) detecting data: as in table 5 below.
| Same batch kit | Low concentration sample | High concentration sample |
| 1 | 15.76 | 38.31 |
| 2 | 16.46 | 37.75 |
| 3 | 15.89 | 38.80 |
| 4 | 17.07 | 38.71 |
| 5 | 16.33 | 40.03 |
| 6 | 16.51 | 36.95 |
| 7 | 15.71 | 37.90 |
| 8 | 15.94 | 36.88 |
| 9 | 15.84 | 35.89 |
| 10 | 15.45 | 37.79 |
| AVE. | 16.10 | 37.90 |
| SD. | 0.48 | 1.16 |
| CV. | 3.01％ | 3.07％ |
TABLE 5
As shown in table 5 above, the detection kits of the same batch respectively measure the low-concentration sample and the high-concentration sample, and the obtained intra-batch variation coefficients are 3.01% and 3.07%, respectively, and the intra-batch precision is good.
Inter-batch precision test
Three batches of the kit in example 1 were taken, samples with high and low concentrations were measured in each batch of the kit, and 10 wells were measured in parallel, and 30 measurement values were obtained for each sample.
The measurement data of the high concentration samples are shown in Table 6 below; the data for the low concentration samples are shown in table 7 below:
TABLE 6
TABLE 7
From the above table 6 and table 7, the detection kits of different batches respectively measure the high concentration sample and the low concentration sample, and the obtained intra-batch variation coefficients are 5.14% and 2.69%, respectively, and the inter-batch precision is good.
Test example 6 accuracy measurement test
In this test, the concentration of the international reference substance SRM 972a was measured by the detection method of example 2 using the reagent composition and the reagent prepared by the preparation method of example 1, and the relative deviation between the detected value and the standard value of the reference substance should not exceed ± 10%, and the results are shown in table 8:
TABLE 8
As can be seen from Table 8 above, the relative deviation of the measured value from the reference value of the reference substance was not more than. + -. 5.5%, and was much less than. + -. 10%, and the accuracy of the kit was good.
Test example 7 specificity analysis test
Interference analysis of common abnormal samples
Respectively adding interference substance storage solutions into fresh clinical serum samples of healthy individuals with the concentrations of 12ng/mL and 45ng/mL to prepare interference substances serving as interference samples; a control sample was prepared by adding an equal volume of a matrix-like diluent to each of fresh clinical serum samples of healthy individuals at concentrations of 12ng/mL and 45ng/mL, respectively, as an interfering substance-containing stock solution.
The test kit of example 1 was used, and the control sample and the interference sample were measured three times for each sample, and the average value and the relative deviation were calculated. When the relative deviation is within. + -. 10%, it can be determined that there is no cross or interference in the detection result of the reagent by the added substance, and the results are shown in tables 9 and 10.
| Additive material | Mean value ng/ml of detection concentration | Deviation of |
| Control sample 1 | 12.26 | / |
| 200mg/dL hemoglobin | 12.84 | 4.78％ |
| 450mg/dL triglyceride | 12.61 | 7.77％ |
| 150mg/dL Total bilirubin | 12.92 | 5.41％ |
TABLE 9
| Additive material | Mean value ng/ml of detection concentration | Deviation of |
| Control sample 2 | 44.47 | / |
| 200mg/dL hemoglobin | 48.08 | 8.10 |
| 450mg/dL triglyceride | 47.72 | 7.29 |
| 150mg/dL Total bilirubin | 42.95 | -3.43 |
Watch 10
As can be seen from the above results, the deviation between the measured value of the interference sample and the measured value of the control sample is less than + -10%. The kit has good anti-interference effect.
Test example 8 interference test
A25-hydroxy vitamin D calibrator (not more than 10% of the volume) is added into certain concentration of rheumatoid factor plasma and HAMA plasma to prepare experimental samples with the concentrations of 15ng/mL and 45 ng/mL. To an equivalent volume of zero concentration calibrator, 25-hydroxyvitamin D calibrator (no more than 10% by volume) was added to make control samples at 15ng/mL and 45ng/mL (made up to the same experimental group).
The test sample and the control sample were measured using the kit of example 1 in triplicate for each sample, and the mean and relative deviation were calculated. When the relative deviation is within ± 10%, it can be determined that there is no cross or interference in the detection result of the reagent by the added substance, and the results are shown in tables 11 and 12:
| additive material | Mean value ng/ml of detection concentration | Deviation of |
| Control sample 1 | 15.28 | / |
| 790IU/mL rheumatoid factor plasma | 16.06 | 3.40％ |
| 800ng/ml HAMA plasma | 16.22 | 4.44％ |
TABLE 11
| Additive material | Mean value ng/ml of detection concentration | Deviation of |
| Control sample 2 | 45.96 | / |
| 790IU/mL rheumatoid factor plasma | 48.36 | 4.11％ |
| 800ng/ml HAMA plasma | 48.76 | 6.25％ |
TABLE 12
As can be seen from the above results, the deviation between the measured value of the interference sample and the measured value of the control sample is less than + -10%. The kit has good anti-interference effect.
Test example 9 stability test
The stability test of placing the kit in example 1 at 2-8 ℃ is carried out, the calibration of the standard substance is normal when the kit is placed at 2-8 ℃ for 12 months, the measured value of the quality control substance is within the specified range, the detection limit is not higher than 3ng/mL, the precision of the intra-analysis and inter-analysis is not more than +/-8.0% and +/-10.0%, and the deviation of the measured value of the accuracy is not more than +/-10%. Therefore, the test result of the kit is in accordance with the requirement when the kit is placed at 2-8 ℃ for 12 months, and the effective period of the kit can reach 12 months.
The margin is the dose that can be distinguished from the zero dose at a given level of significance. The zero concentration calibration sample is used as a sample for detection, the measurement is repeated for 20 times to obtain the relative luminous intensity (RLU) value of 20 measurement results, the average value (M) and the Standard Deviation (SD) are calculated to obtain M-2SD, the RLU value of the M-2SD is substituted into a curve equation to obtain the corresponding concentration value, namely the blank limit, and the result is shown in Table 13.
The detection limit is verified by detecting 5 samples close to the detection limit according to the proportion of the number of results smaller than the blank limit at a certain probability (0.05), and the results are shown in a table 14.
Watch 13
TABLE 14
As can be seen from the results in the table above, the detection limit of the kit of the invention is not higher than 3 ng/mL; the blank limit is not more than 2 ng/mL.
Test example 10 Linear Range test
The high-value samples were diluted 2 times, 4 times, 8 times, 16 times and 32 times, respectively, and each diluted sample was repeatedly tested 3 times by the test method in example 2 using the kit in example 1, the average value was calculated, the average value of the measured concentration and the dilution multiple were linearly fitted by the least square method, and the linear correlation coefficient r was calculated, and the results are shown in table 15 below.
Watch 15
The results in the table show that the kit line has a wide linear range, and the linear range reaches 3-120 ng/mL.
Test example 11 comparative test
The kit of example 1 and the imported electrochemiluminescence kit (Roche Diagnostics GmbH, full-automatic electrochemiluminescence immunoassay Cobas e411) were used to simultaneously detect and compare 110 human serum samples. The results are shown in Table 16:
TABLE 16
The serum 25-hydroxy vitamin D concentration measured by the kit is the ordinate, the result measured by the kit of the imported electrochemical luminescence method is the abscissa, the regression analysis is carried out, and the correlation equation is as follows as shown in figure 1: y is 1.0054x-0.0206, and the correlation coefficient R is 0.9970. The statistical processing result shows that the correlation between the measured value of the clinical sample of the kit and the measured value of the imported kit is good.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A25-hydroxyvitamin D detection kit, comprising: r1 reagent, R2 reagent, R3 reagent and sample processing reagent,
the R1 reagent comprises a streptavidin-coated magnetic particle solution, the concentration of the streptavidin-coated magnetic particles is 0.05-1 mg/ml, and the particle size of the magnetic particles is 1-4 μm;
the R2 reagent comprises an alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody solution, the concentration of the alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody is 0.2-1.0 ng/mL, and the labeling ratio of the anti-25-hydroxyvitamin D monoclonal antibody to alkaline phosphatase is 1: 1-10: 1.
the R3 reagent comprises a biotin-labeled 25-hydroxyvitamin D antigen, and the concentration of the biotin-labeled 25-hydroxyvitamin D antigen is 0.05-0.2%;
the sample processing reagent comprises a weakly acidic buffer, a reducing agent and a lower alcohol solvent, and is used for dissociating 25-hydroxyvitamin D from conjugated protein.
2. The kit for detecting 25-hydroxyvitamin D according to claim 1, wherein the pH value of the weakly acidic buffer is 5.5 to 6.9; the weak acidic buffer solution is selected from one of a citric acid buffer solution, an acetic acid buffer solution or a phosphoric acid buffer solution; the weak acid buffer is preferably a citric acid buffer; the concentration of the citric acid buffer solution is 0.02-2.5%, and the concentration of the preferable citric acid buffer solution is 1.5%;
the reducing agent is selected from one of dithiothreitol, tris (2-carboxyethyl) phosphine hydrochloride, reduced glutathione, cysteine or beta-mercaptoethanol; the reducing agent is preferably beta-mercaptoethanol, the concentration of the beta-mercaptoethanol is 0.05% -5%, and the concentration of the beta-mercaptoethanol is preferably 0.1% -2.5%; more preferably, the concentration of beta-mercaptoethanol is 0.25 to 1 percent, and most preferably, the concentration of beta-mercaptoethanol is 0.5 percent;
the concentration of the lower alcohol solvent is 10-20%, preferably the concentration of the lower alcohol solvent is 10-15%, and more preferably the concentration of the lower alcohol solvent is 15%; the lower alcohol solvent is selected from one of methanol or ethanol.
3. The 25-hydroxyvitamin D detection kit according to claim 1, wherein the sample processing reagent further comprises 0.02-0.1% of a preservative, and the preservative is one of sodium azide or Procline 300.
4. The 25-hydroxyvitamin D detection kit according to claim 1, wherein the R1 reagent further comprises 0.02-0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02-0.1% preservative, and the preservative is one of sodium azide and Procline 300;
the R2 reagent further comprises 0.02-0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02-0.1% preservative, wherein the preservative is one of sodium azide or Procline 300;
the R3 reagent further comprises 0.02-0.1 mol/L2- (N-morpholine) ethanesulfonic acid buffer solution and 0.02-0.1% preservative, and the preservative is one of sodium azide or Procline 300.
5. The 25-hydroxyvitamin D detection kit according to claim 1, wherein the kit further comprises a calibrator and a quality control,
the calibrator and the quality control product respectively comprise 25-hydroxy vitamin D antigen, serum, preservative and buffer solution, wherein the serum is selected from one of sheep serum, bovine serum, horse serum, donkey serum or serum containing human serum, the preservative is selected from one of sodium azide and Procline300, the preservative has the mass percentage concentration of 0.02-0.1%, the buffer solution is selected from one of PBS buffer solution, HEPES buffer solution, Tris-HCl buffer solution, MES buffer solution and MOPS buffer solution, and the pH value of the buffer solution is 7.2-7.4; the concentrations of 25-hydroxy vitamin D antigens in the calibrator liquid series are respectively 0.0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL and 120 ng/mL; the concentration of 25 hydroxy vitamin D antigen in the quality control liquid series is 20ng/mL and 80ng/mL respectively.
6. The 25-hydroxyvitamin D detection kit according to claim 1, wherein the preparation method of the alkaline phosphatase-labeled anti-25-hydroxyvitamin D monoclonal antibody in the R2 reagent comprises the following steps:
(1) preparation of ALP-mal:
1) adding DMF into EMCS to prepare an EMCS solution of 60-100 mM;
2) adding the EMCS solution to an ALP-modulating buffer;
3) adding the mixed solution while stirring the ALP solution, heating at 35-38 ℃ for 50-70 minutes;
4) carrying out centrifugal desalination, and recovering the solution after centrifugal desalination;
(2) ALP-mal tag reduction:
1) buffer solution replacement, namely performing the replacement of the 25-hydroxy vitamin D monoclonal antibody buffer solution by using a centrifugal desalting column, and recovering the solution after the buffer solution replacement;
2) dissolving MEA (membrane electrode assembly) by using a buffer solution for gel filtration, and preparing 0.2-0.5M MEA solution;
3) sulfhydrylation of a 25-hydroxy vitamin D monoclonal antibody solution;
4) desalting, namely performing centrifugal desalting after the reaction is finished, and recovering a desalted solution;
5) introducing ALP-mal, adding ALP-mal into the above solution, sealing, and standing at 2-8 deg.C for reaction for about 16-24 hr;
6) stopping, adding 0.1M MEA solution into the solution in the step 5), fully stirring, and reacting for 5-15 minutes at the temperature of 35-38 ℃;
7) and (4) storing the solution after termination at the temperature of 2-8 ℃.
7. The 25-hydroxyvitamin D assay kit of claim 1, wherein the preparation of the anti-25 hydroxyvitamin D antigen labeled with biotin in the R3 reagent comprises the steps of:
(1) desalting the 25-hydroxy vitamin D antigen in a desalting column to obtain a purified solution;
(2) according to DMF: biotin 1: preparing 1-30 mM biotin solution at a ratio of 3-5;
(3) adding the purified solution into a biotin solution, and reacting at normal temperature for 20-40 min;
(4) desalting after reaction, and recovering the centrifugate to obtain the biotin-labeled anti-25-hydroxy vitamin D antigen.
8. The 25-hydroxyvitamin D detection kit according to claim 1, wherein the preparation method of the streptomycin avidin-coated magnetic particles in the R1 reagent comprises the following steps: and (3) taking the streptavidin magnetic particle solution, and diluting the streptavidin magnetic particle solution by using 0.02-0.1 mol/LTRIS buffer solution to obtain the streptavidin magnetic particles with the concentration of 0.05-1 mg/ml.
9. The 25-hydroxyvitamin D detection kit according to claim 1, wherein the preparation method of the calibrator liquid series and the quality control liquid series comprises:
(1) preparing a calibrator diluent by using a buffer solution, serum and a preservative;
(2) dissolving 25 hydroxy vitamin D antigen with DMF;
(3) diluting the dissolved 25 hydroxyvitamin D antigen with the calibrator diluent in the step (1) to obtain calibrator liquid series with the concentrations of the 25 hydroxyvitamin D antigen being 0ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 100ng/mL and 120ng/mL respectively; or diluting the dissolved 25 hydroxy vitamin D antigen by using the calibrator diluent in the step (1) to obtain quality control solution series with the concentrations of the 25 hydroxy vitamin D antigen being 20ng/mL and 80ng/mL respectively.
10. A chemiluminescent immunoassay for the detection of 25-hydroxyvitamin D using the kit of any one of claims 1-9 comprising the steps of:
step A: mixing a sample to be detected, a sample treatment reagent and an R2 reagent, incubating for 20-40 minutes at 36-38 ℃, fully dissociating 25-hydroxyvitamin D in the sample to be detected, fully combining with an alkaline phosphatase-labeled 25-hydroxyvitamin D monoclonal antibody, and washing;
and B: adding an R1 reagent and an R3 reagent, incubating for 5-15 minutes at 36-38 ℃, and washing;
and C: adding a chemiluminescent substrate, generating a luminescent signal under the catalysis of enzyme, receiving the luminescent signal by a photon reading system, measuring the number of photons generated by the reaction by a photomultiplier tube, and automatically calculating the content of 25-hydroxyvitamin D in a sample to be detected by software according to the optical signal and a Cutoff value;
the chemiluminescence zymolyte is (3- (2-spiral adamantane) -4-methoxyl-4- (3-phosphorus oxygen acyl) -phenyl-1, 2-dioxetane and AMPPD.
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