CN112485418A - Release agent for detecting content of vitamin B12 in human body and application thereof - Google Patents
Release agent for detecting content of vitamin B12 in human body and application thereof Download PDFInfo
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- CN112485418A CN112485418A CN202011156000.2A CN202011156000A CN112485418A CN 112485418 A CN112485418 A CN 112485418A CN 202011156000 A CN202011156000 A CN 202011156000A CN 112485418 A CN112485418 A CN 112485418A
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- 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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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
Abstract
The invention relates to a releasing agent for detecting the content of vitamin B12 in a human body and application thereof, wherein the releasing agent comprises a denaturant R1 and a neutralizer R3; denaturant R1 is strong base solution of Tris-HCl system, and neutralizer R3 is absolute ethyl alcohol and Tris (2-carboxyethyl) phosphine solution of Tris-HCl system. The vitamin B12 releasing agent provided by the invention dissociates vitamin B12 from conjugated protein through denaturant R1 strong alkali solution, and then realizes magnetic particle chemiluminescence detection under the action of neutralizer R3, and the B12 releasing agent, a corresponding detection reagent and a detection method can effectively and completely release vitamin B12 in a sample, have high sensitivity of a detection result, can detect low-concentration vitamin B12, have good repeatability, and can reach the level of international products in performance. And the pretreatment requirement on the sample is low, and the method can be used for quickly detecting a large batch of samples in high flux and is convenient for clinical application.
Description
Technical Field
The invention relates to the field of medical in-vitro immunodiagnosis reagents, in particular to a preparation method of a release agent for detecting the content of vitamin B12 in serum or heparin plasma, which is applied to a magnetic particle chemiluminescence detection reagent for detecting the content of vitamin B12 in a human body by adopting a magnetic particle chemiluminescence technology and an antigen-antibody combination technology.
Background
Vitamin B12, also known as cyanocobalamin, is a corrinoid compound with four pyrrole rings around one cobalt atom, with a molecular weight of 1355. Cobalamins are mainly derived from animal products such as meat, eggs, milk and other dairy products. The absorption of vitamin B12 in humans requires the formation of a complex by means of a gastrointestinal secretion (intrinsic factor), vitamin B12 and receptors for this protein attached to the ileal mucosa, where a protein called transcobalamin transfers vitamin B12 from mucosal cells into the blood and tissues. Most vitamin B12 is stored in the liver, in addition to bone marrow and other tissues. If desired, they are carried by the B12 binding protein (transcobalamin) and released into the plasma.
The vitamin B12 exists in a coenzyme form in vivo, can increase the utilization rate of folic acid and promote the metabolism of carbohydrate, fat and protein; it is essential for normal DNA (deoxyribonucleic acid) synthesis like folic acid, has the functions of activating amino acid, promoting nucleic acid biosynthesis and promoting protein synthesis, and has important effect on infant growth; promoting the development and maturation of erythrocytes, keeping the hematopoietic function of the body in a normal state, preventing pernicious anemia, and maintaining the health of the nervous system; is involved in metabolism, is an indispensable vitamin for the functional perfection of the nervous system, and is involved in the formation of a lipoprotein in nervous tissues.
Vitamin B12 and folic acid are linked by the reaction pathway of methionine synthesis, so that any lack of either disrupts the metabolic pathway, resulting in the same symptoms and medical problems. Vitamin B12 is often combined with folic acid, homocysteine, ferritin and the like for clinical detection, and is used for clinical analysis of diabetes, anemia and the like. A large number of studies indicate that vitamin B12 deficiency is also a cause of megaloblastic anemia, and is also associated with Alzheimer's disease, depression, coronary heart disease, and the like. Vitamin B12 deficiency is also one of the risk factors for abortion in early pregnancy and recurrent abortion. When a human body lacks vitamin B12, irreversible damage to a nervous system is easily caused, and symptoms such as dysphoria, memory deterioration and the like are caused. Allergic reactions such as asthma, urticaria, eczema, facial edema, chills and tremor can occur when the medicine is taken excessively, and nerve excitation, precordial pain and palpitation can also be caused; it has also been reported that an excess may promote tumorigenesis. Therefore, the vitamin is clinically recommended to be detected and supplemented, and is not lack of supplementation.
At present, methods for quantitatively determining VB12 include microbiology, High Performance Liquid Chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and Radioimmunoassay (RIA). Since vitamin B12 exists in vivo by binding to proteins, it is necessary to detect a biological sample such as serum or plasma by pretreating the sample to release vitamin B12 from the binding proteins. The existing problems are that dissociation is not thorough when a sample is pretreated, so that the detection result is inaccurate, the level of low-concentration vitamin B12 cannot be detected, the repeatability is poor, and the unified standard cannot be formed.
Disclosure of Invention
The invention aims to provide an efficient, simple and quick release agent for detecting the content of vitamin B12 in human serum or heparin plasma, and the release agent is applied to a corresponding immunoassay method so as to solve the technical problems of incomplete release and poor repeatability of the existing product.
In view of the above objects, the present invention provides a releasing agent for detecting the vitamin B12 content in human serum or heparin plasma, comprising a denaturant R1 and a neutralizer R3; denaturant R1 is strong base solution of Tris-HCl system, and neutralizer R3 is absolute ethyl alcohol and Tris (2-carboxyethyl) phosphine solution of Tris-HCl system.
The pH value of the denaturant R1 is 12.0-13.0, and the denaturant R1 comprises: Tris-HCl with the concentration of 7.5-8.2 g/L; sodium hydroxide with concentration of 20.0-22.0 g/L.
The denaturant R1 has a pH value of 12.5 and comprises: Tris-HCl with the concentration of 8.0 g/L; sodium hydroxide with the concentration of 21.0 g/L; the balance of deionized water. Preparation of denaturant R1: weighing 700g of purified water, sequentially adding 8g of Tris-HCl and 21g of sodium hydroxide, uniformly mixing, adjusting the pH value to be within 12.5 +/-0.05, and weighing to 1L by using the purified water.
The pH value of the neutralizer R3 is 6.0-6.6, and the neutralizer comprises: Tris-HCl with the concentration of 90.0-90.8 g/L; anhydrous ethanol with the concentration of 200.0-220.0 g/L; 8-amino-1-sulfonic Acid Naphthylammonium Salt (ANS) with the concentration of 0.8-1.2 g/L; tris (2-carboxyethyl) phosphine, the concentration is 1.0-1.6 g/L; EDTA with concentration of 0.8-1.2 g/L.
The neutralizing agent R3 has a pH of 6.3 and comprises: Tris-HCl with the concentration of 90.4 g/L; absolute ethyl alcohol with the concentration of 210.0 g/L; 8-amino-1-sulfonic acid naphthylammonium salt with the concentration of 1.0 g/L; tris (2-carboxyethyl) phosphine, at a concentration of 1.3 g/L; EDTA with the concentration of 1.0 g/L; the balance of deionized water. Preparation of neutralizing agent R3: weighing 700g of purified water, sequentially adding 90.4g of Tris-HCl, 210g of absolute ethyl alcohol, 1g of 8-amino-1-sulfonic acid naphthalene ammonium salt, 1.3g of Tris (2-carboxyethyl) phosphine and 1g of EDTA, uniformly mixing, adjusting the pH value to be within the range of 6.3 +/-0.05, and weighing to 1L by using the purified water.
The magnetic particle chemiluminescence detection reagent for detecting the content of vitamin B12 in a human body comprises a denaturant R1, a neutralizer R3, a vitamin B12 reagent R2 (a reagent R2 for short), a magnetic separation reagent, a calibrator, a quality control product and chemiluminescence substrate liquid. In the following examples, the vitamin B12 reagent R2, magnetic separation reagents, calibrators, quality controls, and chemiluminescent substrates used were all from reagents manufactured by Ledman.
A detection method of a magnetic particle chemiluminescence detection reagent for determining the content of vitamin B12 in a human body comprises the following steps:
(1) adding the sample to be tested, the calibrator or the quality control product and the denaturant R1 into the reaction tube respectively, and mixing and incubating for 5min at 37 ℃;
(2) sequentially adding components of a neutralizing agent R3 and a reagent R2 into the sample incubated in the step (1), and continuing incubation for 15min at 37 ℃;
(3) adding a magnetic separation reagent into the sample incubated in the step (2), and continuing incubation for 5min at 37 ℃;
(4) washing to remove unbound antibodies and impurities;
(5) adding chemiluminescence substrate solution, and measuring relative luminescence intensity RLU after ALP catalyzes substrate luminescence;
within a certain range, the relative luminous intensity RLU is inversely proportional to the concentration of the vitamin B12 antigen, and the content of the vitamin B12 in the sample to be detected can be read from the standard curve by an interpolation method.
The data of the methodological identification when the vitamin B12 detection reagent is used for determining the content of the vitamin B12 in the human body can reach the following indexes:
sensitivity: the minimum detection amount is 20.00 pg/ml;
repeatability: the precision of the quality control QC1 is 5.24% (n is 10, which is the number of repetitions), the precision of the quality control QC2 is 4.62% (n is 10, which is the number of repetitions), the precision is far below the industry requirements, and the detection reagent using the diluent of the invention has good repeatability in the experimental process;
correlation: 77 samples are simultaneously detected by using the reagent in the embodiment and an imported chemiluminescence magnetic particle immunoassay kit, the concentration of vitamin B12 measured by using the reagent is used as an ordinate, the result measured by the imported chemiluminescence immunoassay kit is used as an abscissa for regression analysis, the correlation equation is that y is 1.0473x +5.18, and the correlation coefficient R is2The results of statistical treatment showed good correlation when the sample was 0.9799.
From the above, it can be seen that the advantages of the present invention are:
1. the denaturant R1 is a strong alkali solution of a Tris-HCl system, can fully dissociate the conjugated protein after denaturation, and the neutralizer R3 is absolute ethyl alcohol of the Tris-HCl system, a Tris (2-carboxyethyl) phosphonic acid solution, and 8-amino-1-sulfonic acid naphthalene ammonium salt, so that a buffer system with proper pH is ensured before the specific antibody is added, wherein the absolute ethyl alcohol is a low-toxicity organic solvent, and the polarity of hydroxyl can dissolve a plurality of ionic compounds; the 8-amino-1-sulfonic acid naphthylammonium salt and the tri (2-carboxyethyl) phosphine and the absolute ethyl alcohol have synergistic effect to play the role of further dissociation and stabilization, and meanwhile, the tri (2-carboxyethyl) phosphine is used as an effective disulfide bond quantitative reducing agent, can quantitatively reduce disulfide compounds in a wide pH value range, and keeps other functional groups intact.
2. The vitamin B12 releasing agent provided by the invention dissociates vitamin B12 from conjugated protein through denaturant R1 strong alkali solution, and then realizes magnetic particle chemiluminescence detection under the action of neutralizer R3, and the B12 releasing agent, a corresponding detection reagent and a detection method can effectively and completely release vitamin B12 in a sample, have high sensitivity of a detection result, can detect low-concentration vitamin B12, have good repeatability, and can reach the level of international products in performance. And the pretreatment requirement on the sample is low, and the mass samples (the batch is 160 tests/hour) can be rapidly detected in a high-throughput manner, so that the clinical application is facilitated.
3. The release agent provided by the invention is applied to a magnetic particle chemiluminescence platform, can be used together with a full-automatic chemiluminescence instrument, greatly simplifies the operation steps, increases the detection speed and the detection flux, improves the detection efficiency, and can also avoid errors caused by manual operation; the magnetic microspheres are used as solid phase carriers, so that the cleaning is convenient, and the nonspecific binding can be effectively removed.
Drawings
FIG. 1 is a graph of concentration-luminescence values of a standard substance when the releasing agent of the comparative example is used for detecting vitamin B12;
FIG. 2 is a graph of concentration versus luminescence for a standard substance when the release agent of example 2 of the present invention is used to detect vitamin B12.
FIG. 3 is a scatter plot of the correlation of vitamin B12 concentration with contrast agent when vitamin B12 was tested using the release agent of the comparative example.
FIG. 4 is a scattergram showing the correlation between the vitamin B12 concentration detected by the detection reagent obtained in example 4 of the present invention and a contrast agent.
Detailed Description
The present invention is further explained with reference to the following examples and drawings, but the scope of the present invention is not limited thereto.
Example 1
The releasing agent for detecting the content of the vitamin B12 in human serum or heparin plasma comprises a denaturant R1 and a neutralizer R3.
1) The denaturant R1 comprises: Tris-HCl with the concentration of 7.5-8.2 g/L; sodium hydroxide with the concentration of 20.0-22.0 g/L; the balance of deionized water. The pH value of the denaturant R1 is 12.0-13.0.
2) The neutralizer R3 includes: Tris-HCl with the concentration of 90.0-90.8 g/L; anhydrous ethanol with the concentration of 200.0-220.0 g/L; 8-amino-1-sulfonic Acid Naphthylammonium Salt (ANS) with the concentration of 0.8-1.2g/L, and tri (2-carboxyethyl) phosphine with the concentration of 1.0-1.6 g/L; EDTA with the concentration of 0.8-1.2 g/L; the balance of deionized water. The pH value of the neutralizer R3 is 6.0-6.6.
Example 2
The release agent for detecting the content of vitamin B12 in human serum or heparin plasma comprises a denaturant R1 (also called reagent R1) and a neutralizer R3 (also called reagent R3).
1) The denaturant R1 has a pH value of 12.5 and comprises: Tris-HCl with the concentration of 8.0 g/L; sodium hydroxide with the concentration of 21.0 g/L; the balance of deionized water. Preparation of denaturant R1: weighing 700g of purified water, sequentially adding 8g of Tris-HCl and 21g of sodium hydroxide, uniformly mixing, adjusting the pH value to be within 12.5 +/-0.05, and weighing to 1L by using the purified water.
2) The neutralizing agent R3 has a pH of 6.3 and comprises: Tris-HCl with the concentration of 90.4 g/L; absolute ethyl alcohol with the concentration of 210.0 g/L; 8-amino-1-sulfonic acid naphthylammonium salt with the concentration of 1.0 g/L; tris (2-carboxyethyl) phosphine, at a concentration of 1.3 g/L; EDTA with the concentration of 1.0 g/L; the balance of deionized water. Preparation of neutralizing agent R3: weighing 700g of purified water, sequentially adding 90.4g of Tris-HCl, 210g of absolute ethyl alcohol, 1g of 8-amino-1-sulfonic acid naphthalene ammonium salt, 1.3g of Tris (2-carboxyethyl) phosphine and 1g of EDTA, uniformly mixing, adjusting the pH value to be within the range of 6.3 +/-0.05, and weighing to 1L by using the purified water.
Comparative example
The releasing agent of the comparative example comprises a denaturant R1 and a neutralizer R3, wherein the denaturant R1 has the same composition as that of example 2, the neutralizer has the different composition from that of example 2, the neutralizer R3 in the comparative example is Tris-HCl only, the concentration is 90.4g/L, and the pH is adjusted to 6.0 by using HCl.
Example 3
The present embodiment is a method for preparing a magnetic particle chemiluminescence detection reagent for determining vitamin B12 content in a human body, wherein the formulation of the release agent in the present embodiment is the formulation in embodiment 2, the detection reagent comprises a denaturant R1, a neutralizer R3, a reagent R2, a magnetic separation reagent, a calibrator, a quality control material, and a chemiluminescence substrate solution, and the preparation of the detection reagent comprises the following steps:
preparing a reagent R2: 1) the buffer solution of the reagent R2 adopts a commercial AP Conjugate Stabilizer, and the pH value is 7.0; 2) preparing an alkaline phosphatase-labeled anti-vitamin B12 antibody; 3) the alkaline phosphatase-labeled anti-vitamin B12 antibody was diluted with a buffer of reagent R2.
Preparing a magnetic separation reagent: 1) the magnetic particle buffer is a commercial AP Conjugate Stabilizer, and the pH value is 7.0; 2) preparing a magnetic particle concentrated solution coated by vitamin B12-BSA (bovine serum albumin); 3) and diluting the magnetic particle concentrated solution with a magnetic particle buffer solution of the magnetic separation reagent to obtain the magnetic separation reagent.
Preparing a calibrator/quality control product: vitamin B12 antigen was dissolved in DMSO (dimethyl sulfoxide) and configured to different concentrations in calibrator/quality control buffer to obtain different concentrations of calibrator and quality control QC1, QC 2.
Preparing a chemiluminescent substrate solution: 1) preparing a Tris-HCl buffer solution with the value of 0.2M, pH being 9.3, and adding dioxane with the value of 0.3 mg/mL; 2) a chemiluminescent substrate that catalyzes luminescence by alkaline phosphatase is dissolved in a buffer.
Wherein the coupling of the anti-vitamin B12 antibody and alkaline phosphatase is a common coupling process of a chemiluminescence platform.
The preparation method of the magnetic particle reagent concentrated solution comprises the following steps:
(1) preparing a coupling buffer solution 1: 0.1M sodium borate buffer solution, with pH of 9.5, and storing at 2-8 deg.C;
(2) preparing a coupling buffer solution 2: 3M ammonium sulfate buffer solution, pH9.5, 2-8 deg.C storage;
(3) preparing a coupling sealing liquid: 0.2M phosphate buffer, pH7.5, containing 1% bovine serum albumin, 0.1% Tween20, 10% sucrose;
(4) preparing a washing buffer solution and a storage buffer solution: 0.2M phosphate buffer, pH7.5, containing 1% bovine serum albumin, 10% sucrose (mass%);
(5) resuspending tosyl modified magnetic bead microspheres by vortexing;
(6) transferring 100uL of magnetic bead microspheres into a 2mL centrifuge tube, and adding 1mL of coupling buffer solution 1;
(7) placing the centrifugal tube on a magnetic separator until the magnetic bead microspheres migrate to the side close to the magnet, and clarifying the solution in about 5 minutes;
(8) carefully removing the supernatant to avoid suspending the magnetic bead microspheres;
(9) removing the centrifugal tube from the magnet, and suspending the magnetic bead microspheres in 1mL of coupling buffer solution 1 by a vortex oscillation method for 5 minutes;
(10) repeating the steps (7) to (9) for 3 times;
(11) the magnetic beads are weighed to 100uL by using the coupling buffer solution 1, 322uL of the coupling buffer solution 2 and 400ug of vitamin B12-BSA are added, and then the weight is weighed to 1mL by using the coupling buffer solution 1;
(12) fully shaking and uniformly mixing, and then incubating overnight in an incubator at 37 ℃;
(13) adding 5uL of ethanolamine into the coupling reaction system incubated overnight in the step (12) in a ventilation kitchen, and after vortex oscillation, uniformly mixing the ethanolamine on a vortex mixer for 30 minutes to terminate the reaction;
(14) placing the centrifuge tube on a magnetic separator until the microspheres migrate to the side near the magnet, the solution becomes clear in about 5 minutes;
(15) carefully removing the supernatant to avoid suspending the magnetic bead microspheres;
(16) removing the centrifugal tube from the magnet, and suspending the magnetic bead microspheres in 1mL of coupling buffer solution 1 by a vortex oscillation method for 5 minutes;
(17) repeating steps (14) - (16) 3 times;
(18) adding 1mL of coupling sealing solution, shaking on a vortex mixer for 5 minutes to resuspend the magnetic beads, and then incubating in an incubator at 37 ℃ overnight;
(19) placing the centrifuge tube on a magnetic separator until the microspheres migrate to the side near the magnet, the solution becomes clear in about 5 minutes;
(20) carefully removing the supernatant to avoid suspending the magnetic bead microspheres;
(21) removing the centrifugal tube from the magnet, and suspending the magnetic bead microspheres in 1mL of washing and storage buffer solution by a vortex oscillation method for 5 minutes;
(22) repeating the steps (19) to (21) for 3 times, weighing the coupled magnetic beads to 1mL by using washing and storage buffer solution, and storing for later use;
the prepared magnetic particle concentrated solution is 10mg/mL, the technical data of the reaction system is 1mL, and the large sample is coupled according to the technical data (the corresponding parameters and the sample adding amount of the coupling buffer solution are increased proportionally along with the reaction system).
Example 4
The magnetic particle chemiluminescence detection reagent with the vitamin B12 content prepared in example 3 is used for detecting the vitamin B12 content of a human body, and the detection method is as follows:
(1) first, 30. mu.l of the calibrator series of example 3 (concentrations of 0, 50, 200, 500, 1000, 2000pg/ml, respectively) and 30. mu.L of reagent R1 of example 2 were added to a reaction tube, and mixed and incubated at 37 ℃ for 5 min;
(2) adding the above series of reagents respectively with 50 μ l of reagent R3 of example 2 and 50 μ l of reagent R2 of example 3 into a reaction tube, mixing and incubating at 37 deg.C for 15 min;
(3) respectively combining the reagent series obtained in the step (2) with 25 μ l of the magnetic separation reagent of the example 3, and then continuing the incubation at 37 ℃ for 5 min;
(4) washing with a washing solution 3 times to remove unbound antibodies and impurities;
(5) after adding 150. mu.l of the luminescent substrate solution of example 3 and allowing the ALP-catalyzed substrate to emit light, the relative luminescence intensity (RLU) was measured using a Lindman chemiluminescence detector to obtain a series of values of vitamin B12 concentration-luminescence.
(6) Fitting is performed according to the series of values of vitamin B12 concentration-luminescence value to obtain a concentration-luminescence value standard curve of vitamin B12 based on the standard, as shown in FIG. 2.
(7) And calculating the concentration value of the sample to be detected according to the fitted concentration-luminous value standard curve of the vitamin B12.
Within a certain range, the RLU is in inverse proportion to the concentration of the vitamin B12 antigen, and the content of the vitamin B12 in the sample to be detected can be read from the standard curve by an interpolation method.
In the above manner, the corresponding concentration-luminescence value profile was obtained using the releasing agent in the comparative example, as shown in FIG. 1. The results of comparison of the standard curves obtained with and without addition of absolute ethanol, 8-amino-1-sulfonic acid naphthylammonium salt and tris (2-carboxyethyl) phosphine to the specific neutralizing agents are as follows:
TABLE 1 Standard Curve (before addition)
| Calibrator (pg/ml) | 0 | 50 | 200 | 500 | 1000 | 2000 |
| RLU(100000) | 56.123 | 36.147 | 18.374 | 8.731 | 4.532 | 2.295 |
TABLE 2 Standard Curve (after addition)
| Calibrator (pg/ml) | 0 | 50 | 200 | 500 | 1000 | 2000 |
| RLU(100000) | 62.215 | 31.254 | 15.287 | 7.318 | 3.914 | 1.504 |
Adding 30 ul of sample/quality control to be tested and 30 ul of reagent R1 of example 2 into a reaction tube, and mixing and incubating for 5min at 37 ℃; repeating the steps (2) to (5) to determine the relative luminous intensity RLU of the sample to be detected;
and calculating the vitamin B12 concentration value corresponding to the sample RLU to be detected according to the concentration-luminous value standard curve of the vitamin B12.
By adopting the detection method, the data identified by methodology can reach the following indexes:
sensitivity: the minimum detected amount is 20.00 pg/ml;
repeatability: the precision of the quality control QC1 is 5.24% (n is 10), the precision of QC2 is 4.62% (n is 10), the precision is far lower than the national requirement, and the kit has good repeatability in the experimental process;
correlation: 77 samples were simultaneously detected by using the detection reagent obtained from the detection reagent of the present example and the release agent of the comparative example, and the same test kit as the imported chemiluminescence magnetic particle immunoassay, and correlation analysis was performed: the vitamin B12 concentration measured by the reagent is used as an ordinate, the result measured by the immune test kit of the imported chemiluminescence method is used as an abscissa for regression analysis, as shown in figure 4, the correlation equation is that y is 1.0473x +5.18, the correlation coefficient R2 is 0.9799, and the statistical processing result shows that the correlation is good. Whereas, the correlation analysis result obtained using the releasing agent of the comparative example, as shown in fig. 3, the correlation was poor.
TABLE 3 sensitivity (before addition)
As can be seen from table 3, point a has an average value of emission values X-6870925, SD-775464, and X-2 SD-5319997; point B has an average of 3138698 light values. The AB point continuous point fitting equation is that y is-74644 x +6870925, R2＝1。
Substituting the luminous value of M-2SD into the equation according to an A-B point concentration-luminous value linear fitting equation to obtain a corresponding concentration value, namely a minimum detection limit, namely 20.778 pg/mL:
TABLE 4 sensitivity (after addition)
As can be seen from table 4, point a has an average value of emission values X-6159629, SD-248462, and X-2 SD-5662705; point B has an average of 3180931 light values. The AB point continuous point fitting equation is that y is-59574 x +6159629, R2＝1。
And substituting the luminous value of M-2SD into the equation according to the linear fitting equation of the concentration-luminous value of the A-B point to obtain a corresponding concentration value, namely the lowest detection limit of the kit is 8.341 pg/mL.
And (4) conclusion: the sensitivity is obviously improved after the neutralizing agent is added with absolute ethyl alcohol, 8-amino-1-sulfonic acid naphthalene ammonium salt and tri (2-carboxyethyl) phosphine.
Table 5 repeatability (before addition):
as can be seen from Table 5, the CV was 13.41% in 10 replicates of the quality control QC1 test at a target concentration range of 200. + -. 20% (pg/mL) and 11.63% in 10 replicates of the quality control QC2 test at a target concentration range of 1000. + -. 20% (pg/mL).
Table 6 repeatability (after addition):
as can be seen from Table 6, the CV was 5.24% in 10 replicates of the quality control QC1 test at a target concentration range of 200. + -. 20% (pg/mL) and 4.62% in 10 replicates of the quality control QC2 test at a target concentration range of 1000. + -. 20% (pg/mL).
And (4) conclusion: the repeatability is obviously improved after the absolute ethyl alcohol, the 8-amino-1-sulfonic acid naphthalene ammonium salt and the tri (2-carboxyethyl) phosphine are added into the neutralizer.
Table 7 clinical sample alignment correlation (absolute ethanol, 8-amino-1-sulfonic acid naphthylammonium salt, and tris (2-carboxyethyl) phosphine before addition):
as can be seen from table 7, 77 samples were tested simultaneously by using the reagents of the present invention to determine the concentration of vitamin B12 as ordinate and the results of the test using the chemiluminescence immunoassay kit as abscissa, and the correlation equation is y 1.0877x +51.358, and the correlation coefficient R2 is 0.8539, which are shown by statistical processing results.
Table 8 clinical sample alignment correlation (after addition):
as can be seen from table 8, 77 samples were simultaneously tested by adding the neutralizing agent components (anhydrous ethanol, 8-amino-1-sulfonic acid naphthylammonium salt and tris (2-carboxyethyl) phosphine) in the examples and the imported chemiluminescence magnetic particle immunoassay kit, the concentration of vitamin B12 measured by the reagent of the present invention was taken as the ordinate, the results measured by the imported chemiluminescence immunoassay kit were taken as the abscissa for regression analysis, the correlation equation was y 1.0473x +5.18, and the correlation coefficient R2 was 0.9799, and the statistical results showed that the correlation was good.
And (4) conclusion: the correlation is obviously improved after the neutralizer is added with absolute ethyl alcohol, 8-amino-1-sulfonic acid naphthalene ammonium salt and tri (2-carboxyethyl) phosphine.
The steps show that the denaturant R1 and the neutralizer R3 are used, the reaction mode of a competition method is adopted, the principle that a chemiluminescence detection technology is combined with a magnetic particle immune separation technology is utilized, the content of vitamin B12 in a human serum or heparin plasma sample is quantitatively detected, the detection sensitivity is ensured, the requirement on pretreatment of the sample is low, a large number of samples can be rapidly detected in a high-throughput manner, and the clinical application is facilitated. The invention provides a more accurate, precise, convenient, rapid and simple method for dissociating the bound vitamin B12 in human serum or heparin plasma into free vitamin B12.
The magnetic particle chemiluminescence detection reagent for preparing the human vitamin B12 release agent and measuring the content can be used together with a full-automatic chemiluminescence analyzer, the operation steps are greatly simplified, the detection speed and the detection flux are increased, the detection efficiency is improved, and errors caused by manual operation are avoided.
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 those 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.
Nothing in this specification is said to apply to the prior art.
Claims (7)
1. A release agent for detecting the content of vitamin B12 in human body and application thereof are characterized by comprising a denaturant R1 and a neutralizer R3; denaturant R1 is strong base solution of Tris-HCl system, and neutralizer R3 is absolute ethyl alcohol and Tris (2-carboxyethyl) phosphine solution of Tris-HCl system.
2. The releasing agent according to claim 1, wherein the pH value of the denaturant R1 is 12.0-13.0, and the releasing agent comprises: Tris-HCl with the concentration of 7.5-8.2 g/L; sodium hydroxide with concentration of 20.0-22.0 g/L.
3. The release agent according to claim 2, characterized in that the denaturant R1 has a pH value of 12.5, comprising: Tris-HCl with the concentration of 8.0 g/L; sodium hydroxide with the concentration of 21.0 g/L; the balance of deionized water; preparation of denaturant R1: weighing 700g of purified water, sequentially adding 8g of Tris-HCl and 21g of sodium hydroxide, uniformly mixing, adjusting the pH value to be within 12.5 +/-0.05, and weighing to 1L by using the purified water.
4. The releasing agent according to claim 1, wherein the neutralizing agent R3 has a pH value of 6.0-6.6, and comprises: Tris-HCl with the concentration of 90.0-90.8 g/L; anhydrous ethanol with the concentration of 200.0-220.0 g/L; 8-amino-1-sulfonic Acid Naphthylammonium Salt (ANS) with the concentration of 0.8-1.2 g/L; tris (2-carboxyethyl) phosphine, the concentration is 1.0-1.6 g/L; EDTA with concentration of 0.8-1.2 g/L.
5. The release agent according to claim 4, wherein the neutralizing agent R3 has a pH value of 6.3 and comprises: Tris-HCl with the concentration of 90.4 g/L; absolute ethyl alcohol with the concentration of 210.0 g/L; 8-amino-1-sulfonic acid naphthylammonium salt with the concentration of 1.0 g/L; tris (2-carboxyethyl) phosphine, at a concentration of 1.3 g/L; EDTA with the concentration of 1.0 g/L; the balance of deionized water; preparation of neutralizing agent R3: weighing 700g of purified water, sequentially adding 90.4g of Tris-HCl, 210g of absolute ethyl alcohol, 1g of 8-amino-1-sulfonic acid naphthalene ammonium salt, 1.3g of Tris (2-carboxyethyl) phosphine and 1g of EDTA, uniformly mixing, adjusting the pH value to be within the range of 6.3 +/-0.05, and weighing to 1L by using the purified water.
6. A magnetic particle chemiluminescence detection reagent for determining vitamin B12 content in human body, which is characterized in that the release agent of any claim 1-5 is used, and comprises denaturant R1, neutralizer R3 and vitamin B12 reagent R2, magnetic separation reagent, calibrator, quality control material, chemiluminescence substrate solution;
the sensitivity of the detection reagent is as follows: the minimum detection amount is 20.00 pg/mL; repeatability: the precision of the quality control QC1 with the target value concentration range of 200 +/-20% (pg/mL) is 5.24%, and the precision of the quality control QC2 with the target value concentration range of 1000 +/-20% (pg/mL) is 4.62%.
7. The method for detecting the magnetic particle chemiluminescence detection reagent for detecting the content of vitamin B12 in a human body according to claim 6, comprising the steps of:
(1) adding the sample to be tested, the calibrator or the quality control product and the denaturant R1 into the reaction tube respectively, and mixing and incubating for 5min at 37 ℃;
(2) sequentially adding components of a neutralizing agent R3 and a reagent R2 into the sample incubated in the step (1), and continuing incubation for 15min at 37 ℃;
(3) adding a magnetic separation reagent into the sample incubated in the step (2), and continuing incubation for 5min at 37 ℃;
(4) washing to remove unbound antibodies and impurities;
(5) adding chemiluminescence substrate solution, and measuring relative luminescence intensity RLU after ALP catalyzes substrate luminescence;
within a certain range, the relative luminous intensity RLU is inversely proportional to the concentration of the vitamin B12 antigen, and the content of the vitamin B12 in the sample to be detected is read from the standard curve by an interpolation method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113848330A (en) * | 2021-11-30 | 2021-12-28 | 山东子峰生物技术有限公司 | Detection method of homocysteine and vitamin B12, detection test strip and application thereof |
| CN114011111A (en) * | 2022-01-05 | 2022-02-08 | 广州科方生物技术股份有限公司 | Extraction liquid for extracting vitamins from binding protein and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100068725A1 (en) * | 2007-02-01 | 2010-03-18 | Franz Paul Armbruster | Direct determination of vitamin d in serum or plasma |
| CN105636595A (en) * | 2013-08-07 | 2016-06-01 | 欧根泰克诊断有限公司 | 25-oh vitamin d derivatives for determination of vitamin d metabolites |
| US20170184615A1 (en) * | 2014-07-10 | 2017-06-29 | Shenzhen New Industries Biomedical Engineering Co., Ltd | Detection agent for detecting 25-hydroxy vitamin d, preparation method and use |
| CN108362688A (en) * | 2018-01-02 | 2018-08-03 | 北京利德曼生化股份有限公司 | A kind of 25(OH)VD magnetic microparticle chemiluminescence detection kit |
| CN111307789A (en) * | 2019-12-18 | 2020-06-19 | 郑州安图生物工程股份有限公司 | Folic acid detection kit and detection method |
| CN111458525A (en) * | 2020-01-16 | 2020-07-28 | 卢氏实验室公司 | Test strip and kit for detecting human body physiological substance concentration and preparation method |
| CN111721944A (en) * | 2020-06-18 | 2020-09-29 | 深圳市宇诺生物技术有限公司 | 25-hydroxy vitamin D detection kit, preparation method and detection method |
-
2020
- 2020-10-26 CN CN202011156000.2A patent/CN112485418B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100068725A1 (en) * | 2007-02-01 | 2010-03-18 | Franz Paul Armbruster | Direct determination of vitamin d in serum or plasma |
| CN105636595A (en) * | 2013-08-07 | 2016-06-01 | 欧根泰克诊断有限公司 | 25-oh vitamin d derivatives for determination of vitamin d metabolites |
| US20170184615A1 (en) * | 2014-07-10 | 2017-06-29 | Shenzhen New Industries Biomedical Engineering Co., Ltd | Detection agent for detecting 25-hydroxy vitamin d, preparation method and use |
| CN108362688A (en) * | 2018-01-02 | 2018-08-03 | 北京利德曼生化股份有限公司 | A kind of 25(OH)VD magnetic microparticle chemiluminescence detection kit |
| CN111307789A (en) * | 2019-12-18 | 2020-06-19 | 郑州安图生物工程股份有限公司 | Folic acid detection kit and detection method |
| CN111458525A (en) * | 2020-01-16 | 2020-07-28 | 卢氏实验室公司 | Test strip and kit for detecting human body physiological substance concentration and preparation method |
| CN111721944A (en) * | 2020-06-18 | 2020-09-29 | 深圳市宇诺生物技术有限公司 | 25-hydroxy vitamin D detection kit, preparation method and detection method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113848330A (en) * | 2021-11-30 | 2021-12-28 | 山东子峰生物技术有限公司 | Detection method of homocysteine and vitamin B12, detection test strip and application thereof |
| CN114011111A (en) * | 2022-01-05 | 2022-02-08 | 广州科方生物技术股份有限公司 | Extraction liquid for extracting vitamins from binding protein and preparation method and application thereof |
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