Vitamin B12 magnetic particle chemiluminescence detection kit and detection method and application thereof

Abstract

The invention relates to the technical field of vitamin detection, in particular to a vitamin B12 magnetic particle chemiluminescence detection kit and a detection method and application thereof. The vitamin B12 magnetic particle chemiluminescence detection kit comprises an M reagent, an R reagent, a first dissociation agent and a second dissociation agent, particularly, the first dissociation agent comprises a protein releasing agent and a complexing agent, and the second dissociation agent comprises a buffer solution and a reducing agent. According to the kit, the safety is improved, the dissociation effect of the vitamin B12 is enhanced, the stability of the free vitamin B12 is improved, and the detection accuracy and the sample coincidence rate are improved. When the kit is used in combination with a full-automatic chemiluminescence immunoassay analyzer, the kit shows excellent comprehensive performance, good low-value segment distinction degree, excellent experimental repeatability and good linear relation. According to the technical scheme, the technical problems that a method and a reagent for detecting the vitamin B12 in the prior art contain highly toxic substances and the detection effect is not ideal can be solved, and the method and the reagent have ideal popularization and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of vitamin detection, and in particular to a vitamin B12 magnetic particle chemiluminescence detection kit and a detection method and application thereof. Background Art

[0002] Vitamin B12 (VB12), also known as cobalamin, belongs to the family of corrinoid compounds containing a corrin ring structure. VB12 plays an important role in human health and is essential for maintaining normal blood production (hematopoiesis) and the functioning of the nervous system. It is deeply involved in the metabolic process of cells, not only affecting the synthesis and regulatory mechanism of DNA, but also directly participating in the production of fatty acids and biochemical reactions of energy conversion. Therefore, in daily life, it is widely used in nutritional supplements and food additives to enhance its nutritional value, and is also used as a pharmaceutical ingredient (Lv Yingjian, Huang Junming. Research Progress of Vitamin B12 [J]. Chinese Journal of Food Hygiene, 2012, 24(04): 394-399.).

[0003] Insufficient intake and deficiency of VB12 are related to a variety of invisible diseases and can also cause a variety of diseases. For example, pernicious anemia, hyperhomocysteinemia, subacute combined degeneration, depression and other neurological diseases, methylmalonic acidemia, acquired malabsorption, birth defects and other diseases are closely related to the lack of VB12. Therefore, VB12 as a diagnostic marker has important clinical significance in the medical field (Feng Xiaoting. Relationship between vitamin B12 deficiency and related diseases [J]. Chinese Journal of Practical Neurological Diseases, 2014, 17 (01): 96-99.).

[0004] However, a considerable portion of the VB12 circulating in the blood is bound to transcobalamin II (TC). Therefore, to detect VB12 in the blood, it is necessary to dissociate VB12 in order to more accurately reflect its content. At the same time, the dissociated VB12 in the body cannot exist stably for a long time and is easily destroyed, which makes detection more difficult. Although mass spectrometry has good accuracy, the pretreatment of VB12 samples is complicated, the detection process is cumbersome, and the time and economic costs are high. Immunochromatography, enzyme-linked detection and other detection methods are restricted by methodology, and various performance indicators are poor, and the sample compliance rate is not good. The magnetic particle chemiluminescence method takes into account time and economic costs, and various performance indicators also show good performance, with a high degree of automation and simple operation (Wang Jue. Research on the Development Status of Vitamin B12 Detection Methods [J]. Food Safety Guide, 2023, (13): 186-189.).

[0005] Due to the particularity of VB12 detection in blood, the current VB12 chemiluminescence detection reagents on the market are all configured with dissociation agents or adapted to commercial dissociation schemes, but the schemes mostly use highly toxic metal cyanides as complexing agents, which are extremely harmful to the human body and the environment. In addition, the reducing agents and other dissociation agent components in these formulas usually use substances that are highly toxic and costly, but the performance of the final dissociated VB12 samples is uneven (Chen Weicai, Li Zhongxin, Qu Hai, et al. Development and current status of folic acid and vitamin B12 detection methods [J]. Journal of Molecular Diagnosis and Therapy, 2015, 7(05): 357-360+356.).

[0006] Based on the above reasons, there is an urgent need to improve the reagents and methods for detecting VB12 in order to improve the detection effect of VB12 without using toxic substances. Summary of the invention

[0007] The present invention is intended to provide a vitamin B12 magnetic particle chemiluminescence detection kit to solve the technical problem that the methods and reagents for detecting vitamin B12 in the prior art contain highly toxic substances and the detection effect is unsatisfactory.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] A vitamin B12 magnetic particle chemiluminescence detection kit, comprising an M reagent, an R reagent, a first dissociation agent, and a second dissociation agent;

[0010] The M reagent includes streptavidin magnetic beads coated with intrinsic factor;

[0011] The R reagent includes VB12 labeled with acridinium ester;

[0012] The first dissociating agent includes a protein releasing agent and a complexing agent;

[0013] The second dissociation agent includes a buffer and a reducing agent.

[0014] In some embodiments, the M reagent is formed by dispersing streptavidin magnetic beads coated with intrinsic factor in MES buffer; the concentration of the streptavidin magnetic beads coated with intrinsic factor is 0.2-0.8 mg / mL;

[0015] MES buffer is a solution containing 0.03-0.8 M MES and a pH of 5.5-6.7;

[0016] Preferably, the concentration of MES is 0.5 M; the pH value is 5.8-6.4;

[0017] More preferably, the pH value is 6.0-6.2.

[0018] In some embodiments, the streptavidin magnetic beads coated with intrinsic factor are prepared by the following method: washing the streptavidin magnetic beads 2-5 times with a washing buffer, then adding the intrinsic factor in a ratio of 1:3000-1:10000 to the streptavidin magnetic beads, mixing, and incubating at 20-37° C. for 25-35 minutes; washing with a washing buffer 2-5 times, and then resuspending with a MES buffer to obtain;

[0019] The washing buffer is a solution containing 0.01-0.0.06 M PBS and 0.02%-3% Tween 20, pH 6.8-7.6;

[0020] Preferably, the concentration of PBS is 0.02 M, the concentration of Tween is 0.05%, and the pH value is 7.4.

[0021] In some embodiments, the R reagent is formed by diluting acridinium ester-labeled VB12 with a HEPES buffer at a dilution ratio of 1:5000-1:20000; preferably, the dilution ratio is 1:12500;

[0022] HEPES buffer is a solution containing 0.01-0.2 M HEPES and a pH of 6.8-8.2;

[0023] Preferably, the concentration of HEPES is 0.1 M and the pH value is 6.8-8.2;

[0024] Further preferably, the pH value is 7.2-7.6;

[0025] More preferably, the pH value is 7.4.

[0026] In some embodiments, the protein releasing agent is sodium hydroxide and the complexing agent is a cyanide-containing organic compound;

[0027] The first dissociating agent is prepared with water as solvent, the concentration of sodium hydroxide is 0.1-0.8M, and the concentration of the organic compound containing cyanide is 1-8mg / L;

[0028] Preferably, the concentration of sodium hydroxide is 0.3-0.6M, and the concentration of the cyanide-containing organic compound is 3-6mg / L;

[0029] More preferably, the concentration of sodium hydroxide is 0.4-0.5 M, and the concentration of the cyanide-containing organic compound is 4-5 mg / L.

[0030] In some embodiments, the cyanide-containing organic compound is methyl cyanate and / or ethyl cyanate.

[0031] In some embodiments, the buffer is a citric acid buffer and the reducing agent is mercaptoethanol;

[0032] The concentration of mercaptoethanol in the citrate buffer is 10-40 g / L;

[0033] Preferably, the concentration of mercaptoethanol is 20-30 g / L;

[0034] More preferably, the concentration of mercaptoethanol is 25 g / L.

[0035] In some embodiments, the citrate buffer solution is prepared by mixing 0.1-0.5 mol / L disodium hydrogen phosphate solution and 0.1-0.5 mol / L citric acid solution in a volume ratio of 59-83:17-41; the pH value of the citrate buffer solution is 5.0-6.0;

[0036] Preferably, the pH value of the citrate buffer solution is 5.5.

[0037] In some embodiments, the vitamin B12 magnetic microparticle chemiluminescent detection kit further includes a calibrator and a quality control product;

[0038] The calibrator is a vitamin B12 solution with a concentration of 0, 100, 200, 400, 800 or 1600 pg / mL of vitamin B12 prepared using a calibrator buffer as a solvent;

[0039] The calibrator buffer is a solution containing 0.01-0.06 M PBS and 0.02%-3% Tween 20, pH 6.8-7.6;

[0040] Preferably, the concentration of PBS is 0.02M, the concentration of Tween is 0.05%, and the pH value is 7.4;

[0041] The quality control product is a vitamin B12 solution with a concentration of 200 or 800 pg / mL of vitamin B12 prepared using a quality control product buffer as a solvent;

[0042] The quality control buffer is a solution containing 0.01-0.06M PBS and 0.02%-3% Tween 20, with a pH of 6.8-7.6;

[0043] Preferably, the concentration of PBS is 0.02 M, the concentration of Tween is 0.05%, and the pH value is 7.4.

[0044] This protocol also provides a method for non-disease quality or diagnosis of vitamin B12 detection using a vitamin B12 magnetic particle chemiluminescence detection kit, comprising the following steps in sequence:

[0045] S1: Add 10-50 μl of sample or calibrator or quality control into the reaction cup.

[0046] S2: Add the first dissociation agent into the reaction cup, mix well, and incubate at 37°C for 0.5-2 minutes;

[0047] S3: Add the second dissociation agent into the reaction cup, mix well, and incubate at 37°C for 2-5 minutes;

[0048] S4: Add reagent M to the reaction cup and mix well, then incubate at 37°C for 5-15 minutes;

[0049] S5: Add R reagent into the reaction cup, mix well, and incubate at 37°C for 5-15 minutes;

[0050] S6: Under the action of an external magnetic field, the reactants in the reaction cup are cleaned 3-5 times to obtain a final reaction product;

[0051] S7: Add chemiluminescent excitation into the reaction cup and detect the luminescence intensity after 1-2 minutes;

[0052] S8: Draw a standard curve based on the test results of the calibration substance, and calculate the vitamin B12 concentration value in the sample based on the standard curve and the sample light intensity;

[0053] The volume ratio of the sample or calibrator or quality control product to the first dissociation agent and the second dissociation agent is 1-3:1-3:1-3, and the volume ratio of the sample or calibrator or quality control product to the M reagent and the R reagent is 1-3:1-3:1-3;

[0054] Preferably, the volume ratio of the sample or calibrator or quality control product to the first dissociation agent and the second dissociation agent is 1:1:1; the volume ratio of the sample or calibrator or quality control product to the M reagent and the R reagent is 1:2:2.

[0055] The principle and beneficial effects of this technical solution are:

[0056] The present invention uses intrinsic factor to directly coat streptavidin magnetic beads, uses acridinium ester to label vitamin B12, uses cyanate as a chelating agent, and uses mercaptoethanol as a stabilizer. While reducing the biological hazards and costs of reagents, the dissociation rate of vitamin B12 in the sample is increased, so that VB12 is retained more stably in vitro, is more suitable for in vitro diagnosis, has a better sample compliance rate, good repeatability, is applicable to many sample types, and has a wide range of application scenarios.

[0057] More specifically, the technical solution first uses a first dissociator and a second dissociator to process and dissociate the sample. The first dissociator includes a protein releaser and a cyanate chelating agent. The protein releaser is intended to destroy any complexes that vitamin B12 may be bound to in the sample, ensuring that free vitamin B12 can be captured in subsequent steps. Cyanate chelating agents are used to complex the dissociated vitamin B12 and stabilize it, thereby protecting the free vitamin B12. The second dissociator contains a buffer and a reducing agent, mercaptoethanol, to maintain suitable reaction conditions, and may further promote the release of vitamin B12, as well as maintain the stability of the released vitamin B12. Mercaptoethanol has the function of reducing disulfide bonds, greatly reducing the situation where the dissociated vitamin B12 is re-coated by large proteins, and has low cost and is suitable for mass production. The first dissociator and the second dissociator are combined with other components of the vitamin B12 magnetic particle chemiluminescence detection kit to improve the ability to dissociate and stabilize vitamin B12 samples. While reducing the risk and cost of the reagent formulation, it greatly increases the reliability and convenience of vitamin B12 clinical testing, showing a high sample compliance rate, good repeatability, and is applicable to a wide range of sample types and application scenarios, providing a new method for the clinical diagnosis of related diseases.

[0058] The M reagent contains streptavidin (SA) magnetic beads coated with intrinsic factor (IF). The intrinsic factor on these magnetic beads can specifically bind to the dissociated vitamin B12 to form a vitamin B12-intrinsic factor-magnetic bead complex. The R2 reagent contains acridinium ester-labeled vitamin B12. This acridinium ester-labeled vitamin B12 recognizes and binds to the intrinsic factor that has been fixed on the magnetic beads to form a complex. Since magnetic beads are used, they are directly precipitated in an external magnetic field to separate the complex formed by the immune reaction from other unbound substances. Next, a cleaning solution is used to remove unbound components and other interfering substances to ensure that only the specifically bound complex remains on the magnetic beads (the complex formed by "acridinium ester-vitamin B12-intrinsic factor-magnetic bead complex" + "vitamin B12-intrinsic factor-magnetic bead complex"). After adding the chemiluminescent excitation, the acridinium ester is oxidized and emits a light signal. The light intensity is inversely proportional to the amount of vitamin B12 in the sample (this protocol uses a competitive design, where acridinium ester-labeled vitamin B12 competes with vitamin B12 in the sample and binds to magnetic beads coated with intrinsic factor). By measuring the intensity of the light signal, the actual concentration of vitamin B12 in the sample can be calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The standard curves are determined for Experimental Example 1 and Comparative Examples 1, 2, 3 and 4.

[0060] Figure 2This is a correlation analysis of the measurement results of 20 samples using the Roche reagent of Experimental Example 3 and the kits of Example 1 and Comparative Examples 5-8.

[0061] Figure 3 This is a comparison of the test results of serum and plasma homologous samples using the kit of Example 1 in Experimental Example 6.

[0062] Figure 4 This is a comparison of the test results of serum and plasma homologous samples using the kit of Comparative Example 1 in Experimental Example 6.

[0063] Figure 5 This is a comparison of the test results of serum and plasma homologous samples using the kit of Comparative Example 2 in Experimental Example 6.

[0064] Figure 6 This is a comparison of the test results of serum and plasma homologous samples using the kit of Comparative Example 3 in Experimental Example 6.

[0065] Figure 7 This is a comparison of the test results of serum and plasma homologous samples using the kit of Comparative Example 4 in Experimental Example 6.

[0066] Figure 8 This is a correlation analysis of the measurement results of 300 samples using the Roche reagent in Experimental Example 7 and the kit in Example 1. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0068] Overall technical solution:

[0069] (I) Composition and preparation method of vitamin B12 magnetic microparticle chemiluminescence detection kit

[0070] A vitamin B12 magnetic particle chemiluminescence detection kit comprises an M reagent, an R reagent, a first dissociation agent and a second dissociation agent. The kit also comprises a calibration product and a quality control product.

[0071] (1) The M reagent includes streptavidin magnetic beads (SA magnetic beads) coated with intrinsic factor, and the concentration of the SA magnetic beads coated with intrinsic factor in the M reagent is 0.2-0.8 mg / mL. Among them, intrinsic factor (IF) is a glycoprotein secreted by the parietal cells of the gastric mucosa, which plays a vital role in the absorption of vitamin B12. Intrinsic factor is a conventional component in the vitamin B12 quantitative detection kit and can be obtained by methods familiar to those skilled in the art, which will not be repeated here. SA magnetic beads are specifically: magnetic microbeads coated with streptavidin (SA), which are a tool commonly used in biochemistry, molecular biology and immunology experiments. Streptavidin is a protein isolated from Streptomyces, which has a very high ability to specifically bind to biotin.

[0072] In the SA magnetic beads coated with intrinsic factor, the mass ratio of intrinsic factor to SA magnetic beads is 1:3000-1:10000, and is prepared by the following method:

[0073] Wash the SA magnetic beads 2-5 times with a washing buffer, which is a solution containing 0.01-0.06M PBS (preferably 0.02M) and 0.02-3% Tween 20 (preferably 0.05%), with a pH of 6.8-7.6 (preferably 7.4).

[0074] After washing is completed, add gastric factor to the SA magnetic beads in a specified proportion and mix thoroughly. Next, mix and incubate at 20-37°C for 25-35 minutes. After incubation, wash 2-5 times with washing buffer, then resuspend the incubated magnetic beads with MES buffer, and store at 2-8°C for use. Among them, MES buffer is a solution containing 0.03-0.8M MES (preferably 0.5M) and a pH value of 5.5-6.7 (preferably 5.8-6.4, more preferably 6.0-6.2).

[0075] (2) The R reagent includes vitamin B12 labeled with acridinium ester. Vitamin B12 (VB12) labeled with acridinium ester is a marker commonly used in chemiluminescent immunoassay. Acridinium ester is used as a luminescent substrate. When acridinium ester is oxidized, a light signal is generated. By detecting these light signals, the concentration of the target molecule in the sample can be quantitatively determined.

[0076] Use HEPES buffer as a solvent to dilute the acridinium ester-labeled vitamin B12, and then prepare R reagent. Wherein, the HEPES buffer is a solution containing 0.01-0.2M HEPES (preferably 0.1M) and a pH value of 6.8-8.2 (preferably 7.2-7.6, and more preferably 7.4). The dilution ratio of the acridinium ester-labeled vitamin B12 is 1:5000-1:20000, preferably 1:12500.

[0077] (3) The first dissociator includes a protein releaser and a complexing agent. The protein releaser is sodium hydroxide, and the complexing agent is an organic compound containing cyanide (preferably methyl cyanate, ethyl cyanate). The first dissociator is prepared with water as solvent, the concentration of sodium hydroxide in the first dissociator is 0.1-0.8M (preferably 0.3-0.6M, more preferably 0.4-0.5M), and the concentration of the organic compound containing cyanide in the first dissociator is 1-8mg / L (preferably 3-6mg / L, more preferably 4-5mg / L).

[0078] During preparation, sodium hydroxide is added to water and mixed thoroughly to form a sodium hydroxide solution. Then, a cyanide-containing organic compound is added to the sodium hydroxide solution, mixed thoroughly and placed at 2-8°C for use, and then packaged as needed.

[0079] (4) The second dissociation agent includes a dissociation buffer and a reducing agent. The dissociation buffer is preferably a citrate buffer solution, and the reducing agent is preferably mercaptoethanol. The pH value of the citrate buffer solution is 5.0-6.0 (preferably 5.5), and it is prepared by mixing 0.1-0.5 mol / L disodium hydrogen phosphate solution and 0.1-0.5 mol / L citric acid solution in a volume ratio of 59-83:17-41. In the second dissociation agent, the concentration of the reducing agent mercaptoethanol is 10-40 g / L (preferably 20-30 g / L, and more preferably 25 g / L).

[0080] The second dissociation agent is prepared by the following method: disodium hydrogen phosphate solution and citric acid solution are fully mixed to prepare a citrate buffer solution with a specified pH value. Then, mercaptoethanol is added to the citrate buffer solution at a specified concentration to obtain the second dissociation agent.

[0081] (5) Calibrator

[0082] The vitamin B12 solutions have concentrations of 0, 100, 200, 400, 800 and 1600 pg / mL, respectively, and the solvent used for preparation is a calibration buffer. The calibration buffer can be selected to be completely consistent with the washing buffer, that is, the calibration buffer is a solution containing 0.01-0.06M PBS (preferably 0.02M) and 0.02-3% Tween 20 (preferably 0.05%) and a pH of 6.8-7.6 (preferably 7.4).

[0083] (6) Quality control products

[0084] The vitamin B12 solutions have concentrations of 200 and 800 pg / mL, respectively, and the solvent used for preparation is a quality control buffer. The quality control buffer can be completely consistent with the washing buffer, that is, the quality control buffer is a solution containing 0.01-0.06M PBS (preferably 0.02M) and 0.02-3% Tween 20 (preferably 0.05%), and a pH of 6.8-7.6 (preferably 7.4).

[0085] (II) Detection process using vitamin B12 magnetic microparticle chemiluminescence detection kit

[0086] The volume ratio of the test sample stock solution to the first dissociation agent and the second dissociation agent is 1-3:1-3:1-3 (preferably 1:1:1), and the volume ratio of the test sample stock solution to the M reagent and the R reagent is 1-3:1-3:1-3 (preferably 1:2:2).

[0087] The method for detecting according to the aforementioned vitamin B12 magnetic microparticle chemiluminescence detection kit comprises the following steps:

[0088] (1) Add 10-50 μl of sample / calibrator / quality control material into the reaction cup of the fully automatic chemiluminescence immunoassay analyzer.

[0089] (2) Continue to add the first dissociation agent into the reaction cup of the fully automatic chemiluminescence immunoassay analyzer, mix well, and incubate at 37° C. for 0.5-2 min (preferably 1 min).

[0090] (3) Add the second dissociation agent to the reaction cup of the fully automatic chemiluminescence immunoassay analyzer, mix well, and incubate at 37° C. for 2-5 min (preferably 3.5 min).

[0091] (4) Continue to add M reagent into the reaction cup of the fully automatic chemiluminescence immunoassay analyzer, mix well, and incubate at 37° C. for 5-15 min (preferably 8-12 min).

[0092] (5) Continue to add R reagent into the reaction cup of the fully automatic chemiluminescence immunoassay analyzer, mix well, and incubate at 37° C. for 5-15 min (preferably 8-12 min).

[0093] (6) After step (5) is completed, the fully automatic chemiluminescence immunoassay analyzer automatically perfuses the cleaning solution (PBS as the cleaning solution) 3-5 times for cleaning to obtain the final reaction product. This step is a conventional operation step in the prior art and is a conventional cleaning step after the above reaction is completed.

[0094] (7) Add chemiluminescent excitant to the reaction cup of the fully automatic chemiluminescent immunoassay analyzer and detect the luminescence intensity after 1-2 minutes.

[0095] (8) Using the working curve calibrated by the kit program (the luminescence value is the vertical axis and the concentration of the calibration substance is the horizontal axis, the curve drawn is the working curve), the vitamin B12 concentration value can be automatically calculated based on the sample light intensity.

[0096] According to the overall situation of the above technical solution, the following examples and comparative examples were prepared and tested:

[0097] Example 1

[0098] First, the composition and preparation method of the vitamin B12 magnetic microparticle chemiluminescence detection kit of this embodiment are described as follows:

[0099] (1) Reagent M

[0100] Wash the SA magnetic beads 3 times with washing buffer, then add intrinsic factor and mix thoroughly. The mass ratio of intrinsic factor to SA magnetic beads is 1:6000. Mix and incubate at 30°C for 30 minutes. After incubation, wash 4 times with washing buffer. Next, resuspend the incubated magnetic beads with MES buffer to a concentration of 0.4 mg / mL and place at 2-8°C for use. The washing buffer is a solution containing 0.02M PBS and 0.05% Tween 20 with a pH of 7.4. The MES buffer is a solution containing 0.5M MES with a pH of 6.1.

[0101] (2) R reagent

[0102] Acridinium ester-labeled vitamin B12 was diluted in a HEPES buffer at a ratio of 1:12500, mixed thoroughly, and placed at 2-8°C for use. The HEPES buffer was a solution containing 0.1 M HEPES and a pH of 7.4.

[0103] (3) First dissociator

[0104] Add 36g of sodium hydroxide into 800mL of deionized water, mix thoroughly and adjust the volume to 1L to prepare a 0.45M sodium hydroxide solution. Add methyl cyanate into the prepared 1L 0.45M NaOH solution to obtain the first dissociator. The concentration of methyl cyanate in the first dissociator is 4.5mg / L. After mixing thoroughly, place at 2-8℃ for use and dispense when needed.

[0105] (4) Second dissociation agent

[0106] 2.29 parts by volume of 0.2 mol / L sodium hydrogen phosphate solution and 1.71 parts by volume of 0.2 mol / L citric acid solution are fully mixed to obtain a citric acid buffer solution with a pH of 5.5. 1 part by volume of mercaptoethanol is added to 40 parts by volume of a citric acid buffer solution with a pH of 5.5 to obtain a second dissociation agent with a mercaptoethanol volume percentage of about 2.5%, which is converted into a mercaptoethanol concentration of about 25 g / L (based on the density of β-mercaptoethanol being 1 g / mL). After fully mixing, place at 2-8°C for use and dispense when needed.

[0107] (5) Calibrator

[0108] Vitamin B12 was serially diluted into 0, 100, 200, 400, 800, and 1600 pg / mL calibrators using a calibrator buffer (pH 7.4, containing 0.02 M PBS and 0.05% Tween 20).

[0109] (6) Quality control products

[0110] Vitamin B12 was serially diluted into 200 and 800 pg / mL quality control products using quality control buffer (pH 7.4, containing 0.02 M PBS and 0.05% Tween 20).

[0111] Next, the specific process of the detection using the vitamin B12 magnetic microparticle chemiluminescence detection kit of this embodiment is described as follows:

[0112] In the present technical solution, the volume ratio of the original solution of the sample to be tested to the first dissociation agent and the second dissociation agent is 1:1:1, and the volume ratio of the original solution of the sample to be tested to the M reagent and the R reagent is 1:2:2.

[0113] The fully automatic chemiluminescence analyzer automatically draws 30μl of sample / calibrator / quality control into the reaction cup. Then, add the first dissociation agent equal to the volume of the sample / calibrator / quality control, mix well, and incubate at 37°C for 1min. Then, add the second dissociation agent equal to the volume of the first dissociation agent, mix well, and incubate at 37°C for 3.5min. Then, add M reagent equal to 2 times the volume of the second dissociation agent, mix well, and incubate at 37°C for 10min. Next, add R reagent equal to the volume of M reagent, mix well, and incubate at 37°C for 10min. After completing the above reaction, the fully automatic chemiluminescence immunoassay analyzer automatically perfuses the cleaning solution into the reaction cup 4 times for cleaning to obtain the final reaction product. The fully automatic chemiluminescence immunoassay analyzer automatically adds chemiluminescent excitation into the reaction cup and detects the relative light intensity emitted after 1.5min. The working curve (also known as the standard curve) calibrated by the kit program can automatically calculate the VB12 concentration value according to the sample light intensity.

[0114] Comparative Example 1:

[0115] The only difference from Example 1 is that the mass of methyl cyanate in the first dissociator is replaced by sodium cyanide.

[0116] Comparative Example 2:

[0117] The only difference from Example 1 is that the mass of methyl cyanate in the first dissociator is replaced by potassium hexacyanocobaltate.

[0118] Comparative Example 3:

[0119] The only difference from Example 1 is that the mass of mercaptoethanol in the second dissociation agent is replaced by dithiothreitol.

[0120] Comparative Example 4:

[0121] The only difference from Example 1 is that the mass of mercaptoethanol in the second dissociating agent is replaced by thioglycerol.

[0122] Comparative Example 5

[0123] The only difference from Example 1 is that the citric acid buffer in the second dissociation agent is replaced with a PBS buffer with a pH of 6.8 and a concentration of 0.02M.

[0124] Comparative Example 6

[0125] The only difference from Example 1 is that the citric acid buffer in the second dissociation agent is replaced with a MES buffer with a pH of 6.0 and a concentration of 0.5 M.

[0126] Comparative Example 7

[0127] The only difference from Example 1 is that the citric acid buffer in the second dissociator is replaced with an acetic acid / sodium acetate buffer solution, the acetic acid / sodium acetate buffer solution is prepared by mixing 0.1 mol / L acetic acid solution and 0.1 mol / L sodium acetate solution in a volume ratio of 3:17, and the pH value of the acetic acid / sodium acetate buffer solution is 5.5.

[0128] Comparative Example 8

[0129] The only difference from Example 1 is that the citric acid buffer in the second dissociation agent is replaced with a HEPES buffer with a pH of 7.4 and a concentration of 0.1 M.

[0130] Experimental Example 1: Standard Curve

[0131] The kits of Example 1 and Comparative Examples 1-8 were used to test the samples / calibrators / quality control products according to the method of Example 1, and the working curve (standard curve) was drawn using the test results of the calibrators. Figure 1 and Table 1.

[0132] Table 1: Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, Comparative Example 8 Determination of luminescence values ​​of calibration products

[0133]

[0134] The results show that the standard curves of Example 1 and Comparative Example 1 are close, and both show excellent detection results, but the chelating agent used in the example is less biohazardous than that used in Comparative Example 1 and has lower cost; the chelating and stabilization effects of Comparative Examples 2-4 are not good, and the changes in the components of the buffer solutions of Comparative Examples 5-8 affect the detection results, so the luminescence value ratio is not good, resulting in poor discrimination between the concentrations.

[0135] Experimental Example 2: Blank Limit

[0136] The reagents prepared in Example 1 and Comparative Examples 1-8 were used to detect the zero-concentration calibrator, and the determination was repeated 20 times to obtain the RLU values ​​of the 20 determination results, and the mean (M) and standard deviation (SD) were calculated to obtain the luminescence value of (M-2SD); the luminescence value of the second-point calibrator was then measured, and the luminescence value of the zero concentration and the luminescence value of the second-point calibrator were fitted to obtain a linear equation; the luminescence value of (M-2SD) was then substituted into the linear equation to calculate the corresponding concentration value, which is the blank limit. The experimental results are shown in Table 2. Blank limit detection was performed on Example 1 and Comparative Examples 1-8, and it was found that the blank limit of Example 1 was the lowest, which was 9.63pg / mL. Among them, the blank limits of Comparative Examples 5, Comparative Examples 6, and Comparative Examples 8 were all as high as 20pg / mL or even more than 30pg / mL, which was much higher than the level of Example 1, which shows that the choice of buffer type has a very important influence on the detection effect, and it can be seen that the dissociation agent scheme in Example 1 can make the blank limit lower.

[0137] Table 2: Blank limit results for Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, Comparative Example 8

[0138]

[0139]

[0140]

[0141] Experimental Example 3: Relationship between buffer type selection and sample dissociation effect

[0142] The type of dissociation agent buffer can affect the properties of the dissociation agent components, which is obviously related to the dissociation effect. The measurement of the calibrator cannot reflect the actual dissociation situation, so the actual serum sample is measured and the correlation with the Roche measurement is observed to determine the effect of different dissociation agent buffers on the dissociation effect. The reagents prepared in Example 1 and Comparative Examples 5-8 were used to detect the zero concentration calibrator (no additional free vitamin B12 was added to the sample). According to Table 3 and Figure 2 It can be seen that the measured value of the sample in Example 1 has a good correlation with the measured value of Roche (R 2 The above results show that the kit of Example 1 has a good effect on the dissociation of vitamin B12 in the sample. Comparative Example 5 has an incomplete dissociation (R 2 The value is too low; for the same sample, the sample value of Comparative Example 5 is significantly different from the Roche value. The incomplete dissociation of Comparative Example 6 is more obvious (R 2 The value is too low; for the same sample, the sample value of Comparative Example 6 is significantly different from the Roche value. The sample value of Comparative Example 7 is reasonably correlated with the Roche value (R2 Value>0.97), but the deviation is large (for the same sample, the difference between the test value and the Roche test value is relatively large). Comparative Example 8 has no dissociation effect at all (the luminescence value is almost equivalent to the luminescence value at 0 o'clock, which can be understood as the test results of samples 1-20 are almost 0). It shows that the buffer system used in Comparative Examples 5, 6, 7 and 8 affects the dissociation effect and the final detection effect of the components therein, and further illustrates that the kit of this scheme has good detection performance, and it is very important to use a specific buffer in combination with a reducing agent. In addition to Comparative Examples 5-8, the inventor also tried to use ethanol as a solvent to prepare a second dissociation solution (ethanol replaced the citric acid solution of Example 1, and the others were completely the same as Example 1), and used this kit for sample detection. It was found that the luminescence value of the test result was almost close to the local luminescence value of the instrument. It is speculated that under the composition and use of the kit of this scheme, ethanol destroys the structure of the intrinsic factor, resulting in the inability of specific substances to bind to the magnetic beads, and it is impossible to use it to detect vitamin B12 in the sample.

[0143] Table 3: Samples with value determined in Example 1, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8

[0144]

[0145]

[0146] Experimental Example 4: Precision

[0147] The reagents prepared in Example 1 and Comparative Examples 1-4 were used to measure samples with concentrations of 200 pg / mL and 800 pg / mL, respectively. The test was repeated 10 times, and the mean (M) of the 10 test results was calculated, and the standard deviation (SD) and the coefficient of variation (CV) were calculated, where CV is the precision of the measurement. The experimental results are shown in Table 4. The precision of Example 1 and Comparative Examples 1-4 was tested, and it was found that the precision of Example 1 was the best, with the precision of both points being less than 3%, and the precision of Comparative Example 4 was the worst, which was due to the poor effect of the stabilizer in Comparative Example 4, which shows that the stabilizer used in Example 1 brought good results.

[0148] Table 4: Determination precision results of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4

[0149]

[0150]

[0151] Experimental Example 5: Linearity

[0152] The reagents prepared by Example 1 and Comparative Examples 1-4 were mixed into samples of at least 5 diluted concentrations for determination of high concentration samples close to the upper limit of the linear range and low concentration samples or 0 concentration samples close to the lower limit of the linear range. Each concentration sample was tested 3 times, and the average value was calculated. The average value (yi) of the measured concentration was taken and the corresponding theoretical concentration value or dilution multiple (xi) was subjected to regression analysis to obtain a linear regression equation and calculate the linear correlation coefficient r. The experimental results are shown in Table 5. Linear detection was performed on Example 1 and Comparative Examples 1-4. It was found that the linearity of Example 1 was optimal, and Comparative Example 2 seemed to have linearity, but according to the results, it was found that the concentration value CV calculated from the luminescence value was unacceptable, and the difference between the calculated value and the theoretical concentration value was huge, presenting a chaotic situation. This was caused by the incomplete complexation of vitamin B12 by the complexing agent in Comparative Example 2, and there was a situation in which the calibration curve and the main curve were too different in Comparative Example 4, and the calculated concentration value was very different from the theoretical concentration value. This was caused by the poor effect of the stabilizer in Comparative Example 4. It can be seen that the linearity of the reagent can be optimized by using the first dissociating agent and the second dissociating agent scheme.

[0153] Table 5: Linearity results of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4

[0154]

[0155]

[0156]

[0157] Experimental Example 6: Accuracy

[0158] The reagents prepared in Example 1 and Comparative Examples 1-4 were used to determine the accuracy. The low-value sample and the high-value sample were measured three times each and the average value was taken. The high-value sample was added to the low-value sample, wherein the volume of the added high-value sample did not exceed 10% of the total volume of the high-value and low-value samples. The mixed sample was measured three times and the average value was taken. The recovery rate R was calculated according to the formula (Formula (1)).

[0159]

[0160] Among them, R is the recovery rate, C is the detection concentration after the high-value sample is added to the low-value sample, V0 is the volume of the low-value sample, V is the volume of the high-value sample, C0 is the concentration of the low-value sample, and C S is the concentration of the high value sample.

[0161] The experimental results are shown in Table 6. The accuracy of Example 1 and Comparative Examples 1-4 was tested. The results showed that Example 1 had the best accuracy, with a recovery rate of 98.69%, reflecting that the reagent formula described in Example 1 showed excellent accuracy.

[0162] Table 6: Determination accuracy results of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4

[0163]

[0164]

[0165] Experimental Example 7: Applicable Sample Types

[0166] The reagents prepared in Example 1 and Comparative Examples 1-4 were used to measure the homologous serum and plasma samples. 20 homologous serum and plasma samples were measured once for each sample, and the concentration was fitted with the theoretical concentration of the sample to observe the correlation and determine the sample type suitable for the scheme. The experimental results are shown in Table 7 and Figure 3-Figure 7 Homologous serum and plasma samples of Example 1 and Comparative Examples 1-4 were measured, and the results showed that the homologous sample comparison results of Example 1 were the best, reflecting that the reagent formula solution described in Example 1 is applicable to many types of samples and has a wide range of application scenarios.

[0167] Table 7: Results of homologous samples measured in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4

[0168]

[0169] Experimental Example 8: Sample Matching Rate

[0170] The reagent prepared in Example 1 was used together with the vitamin B12 detection kit (electrochemiluminescence method) of Roche Diagnostics to detect 300 samples with values. The experimental results are detailed in Tables 8 and Figure 8 The results of the test results of Roche reagent and Example 1 on 300 samples showed that the correlation R between Example 1 of the present invention and the vitamin B12 detection kit (electrochemiluminescence method) of Heshi Diagnostics Company was 2 The coefficient A is 0.9902 and 0.9989, respectively. The sample comparison results are excellent, indicating that the detection kit of Example 1 fully meets the clinical testing requirements for VB12.

[0171] Table 8: Roche reagent and Example 1 for measuring 300 samples with value

[0172]

[0173]

[0174]

[0175] In summary (Experimental Examples 1-8), by performing blank limit detection on the reagents of Example 1 and Comparative Examples 1-8, analyzing the relationship between buffer type selection and sample dissociation effect of the reagents of Example 1 and Comparative Examples 5-8, and performing precision, linearity, accuracy, and applicable sample type detection and sample comparison tests on the reagents of Example 1 and Comparative Examples 1-4, it was found that the first dissociator and the second dissociator schemes proposed in Example 1 help improve the various performance indicators of the reagents, and the results are better than those of other comparative examples, and the sample compliance rate is excellent. It can be seen that the kit of the present invention has great application value in clinical testing scenarios for vitamin B12.

[0176] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Vitamin B12 magnetic microparticle chemiluminescence detection kit, characterized in that, It includes an M reagent, an R reagent, a first dissociating agent, and a second dissociating agent; The M reagent includes streptavidin magnetic beads coated with intrinsic factor; The R reagent includes VB12 labeled with acridinium ester; The first dissociating agent includes a protein releasing agent and a complexing agent; The second dissociation agent includes a buffer and a reducing agent.

2. The vitamin B12 magnetic particle chemiluminescence detection kit according to claim 1, characterized in that: The M reagent is formed by dispersing streptavidin magnetic beads coated with intrinsic factor in MES buffer; the concentration of streptavidin magnetic beads coated with intrinsic factor is 0.2-0.8 mg / mL; MES buffer is a solution containing 0.03-0.8 M MES and a pH of 5.5-6.7; Preferably, the concentration of MES is 0.5 M; the pH value is 5.8-6.4; More preferably, the pH value is 6.0-6.

2.

3. The vitamin B12 magnetic particle chemiluminescence detection kit according to claim 2, characterized in that: The streptavidin magnetic beads coated with intrinsic factor are prepared by the following method: washing the streptavidin magnetic beads 2-5 times with a washing buffer, then adding the intrinsic factor in a ratio of 1:3000-1:10000 to the streptavidin magnetic beads, mixing, and incubating at 20-37°C for 25-35 minutes; washing with a washing buffer 2-5 times, and resuspending with a MES buffer to obtain the product; The washing buffer is a solution containing 0.01-0.06 M PBS and 0.02%-3% Tween 20, pH 6.8-7.6; Preferably, the concentration of PBS is 0.02 M, the concentration of Tween is 0.05%, and the pH value is 7.

4.

4. The vitamin B12 magnetic particle chemiluminescence detection kit according to claim 1, characterized in that: The R reagent is formed by diluting acridinium ester-labeled VB12 with HEPES buffer at a dilution ratio of 1:5000-1:20000; preferably, the dilution ratio is 1:12500; HEPES buffer is a solution containing 0.01-0.2 M HEPES and a pH of 6.8-8.2; Preferably, the concentration of HEPES is 0.1 M and the pH value is 6.8-8.2; Further preferably, the pH value is 7.2-7.6; More preferably, the pH value is 7.

4.

5. The vitamin B12 magnetic particle chemiluminescence detection kit according to claim 1, characterized in that: The protein release agent is sodium hydroxide, and the complexing agent is an organic compound containing cyanide; The first dissociating agent is prepared with water as solvent, the concentration of sodium hydroxide is 0.1-0.8M, and the concentration of the organic compound containing cyanide is 1-8mg / L; Preferably, the concentration of sodium hydroxide is 0.3-0.6M, and the concentration of the cyanide-containing organic compound is 3-6mg / L; More preferably, the concentration of sodium hydroxide is 0.4-0.5 M, and the concentration of the cyanide-containing organic compound is 4-5 mg / L.

6. The vitamin B12 magnetic particle chemiluminescence detection kit according to claim 5, characterized in that: The organic compound containing cyanide is methyl cyanate and / or ethyl cyanate.

7. The vitamin B12 magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that: The buffer was citric acid buffer and the reducing agent was mercaptoethanol; The concentration of mercaptoethanol in the citrate buffer is 10-40 g / L; Preferably, the concentration of mercaptoethanol is 20-30 g / L; More preferably, the concentration of mercaptoethanol is 25 g / L.

8. The vitamin B12 magnetic microparticle chemiluminescence detection kit according to claim 7, characterized in that: The citrate buffer solution is prepared by mixing 0.1-0.5 mol / L disodium hydrogen phosphate solution and 0.1-0.5 mol / L citric acid solution in a volume ratio of 59-83:17-41; the pH value of the citrate buffer solution is 5.0-6.0; Preferably, the pH value of the citrate buffer solution is 5.

5.

9. The vitamin B12 magnetic particle chemiluminescence detection kit according to claim 8, characterized in that: Also includes calibrators and quality controls; The calibrator is a vitamin B12 solution with a concentration of 0, 100, 200, 400, 800 or 1600 pg / mL of vitamin B12 prepared using a calibrator buffer as a solvent; The calibrator buffer is a solution containing 0.01-0.06 M PBS and 0.02%-3% Tween 20, pH 6.8-7.6; Preferably, the concentration of PBS is 0.02M, the concentration of Tween is 0.05%, and the pH value is 7.4; The quality control product is a vitamin B12 solution with a concentration of 200 or 800 pg / mL of vitamin B12 prepared using a quality control product buffer as a solvent; The quality control buffer is a solution containing 0.01-0.06M PBS and 0.02%-3% Tween 20, with a pH of 6.8-7.6; Preferably, the concentration of PBS is 0.02 M, the concentration of Tween is 0.05%, and the pH value is 7.

4.

10. A method for non-disease quality or diagnosis of vitamin B12 using the vitamin B12 magnetic particle chemiluminescence detection kit according to any one of claims 1 to 9, characterized in that: The method includes the following steps: S1: Add 10-50 μl of sample or calibrator or quality control into the reaction cup. S2: Add the first dissociation agent into the reaction cup, mix well, and incubate at 37°C for 0.5-2 minutes; S3: Add the second dissociation agent into the reaction cup, mix well, and incubate at 37°C for 2-5 minutes; S4: Add reagent M to the reaction cup and mix well, then incubate at 37°C for 5-15 minutes; S5: Add R reagent into the reaction cup, mix well, and incubate at 37°C for 5-15 minutes; S6: Under the action of an external magnetic field, the reactants in the reaction cup are cleaned 3-5 times to obtain a final reaction product; S7: Add chemiluminescent excitation into the reaction cup and detect the luminescence intensity after 1-2 minutes; S8: Draw a standard curve based on the test results of the calibration substance, and calculate the vitamin B12 concentration value in the sample based on the standard curve and the sample light intensity; The volume ratio of the sample or calibrator or quality control product to the first dissociation agent and the second dissociation agent is 1-3:1-3:1-3, and the volume ratio of the sample or calibrator or quality control product to the M reagent and the R reagent is 1-3:1-3:1-3; Preferably, the volume ratio of the sample or calibrator or quality control product to the first dissociation agent and the second dissociation agent is 1:1:1; the volume ratio of the sample or calibrator or quality control product to the M reagent and the R reagent is 1:2:2.

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