Simple and efficient vitamin B12 detection kit and detection method

Abstract

The invention relates to the technical field of chemiluminescence immunoassay, in particular to a simple and efficient vitamin B12 detection kit and a detection method.The simple and efficient vitamin B12 detection kit comprises a pretreatment agent, a magnetic particle system and a marker system, an extracted serum sample is mixed with the protein releasing agent and the complexing agent in the pretreatment agent, and the vitamin B12 in the sample is released from the binding protein; the complexing agent is a complex with cyanide ions, the cyanide ions separated from the complex in an aqueous solution are combined with the released vitamin B12 to obtain stable cyanocobalamin for subsequent detection of the vitamin B12, and the vitamin 12 in a to-be-detected sample is converted into a stable state for detection by the method, so that the detection stability is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescent immunoassay, and in particular to a simple and efficient vitamin B12 detection kit and a detection method. Background Art

[0002] Vitamin B12, also known as cyanocobalamin, is a polycyclic compound containing trivalent cobalt. It is the only vitamin containing metal elements. Its main physiological function is to participate in the production of bone marrow red blood cells, prevent pernicious anemia, and prevent brain nerve damage. Its structural formula is composed of four reducing pyrrole rings connected into a large ring with a cobalt in the center. This large ring is called corrin, which is the core of the vitamin B12 structure. The full chemical name of vitamin B12 is α-5,6-dimethylbenzimidazole-cyanocobalamin, and cyanocobalamin is its abbreviation. The cyano group (CN) in its molecular formula can be replaced by other groups to become different types of cobalamin.

[0003] Existing clinical methods for detecting trace amounts of vitamin B12 in biological samples include radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescent immunoassay. However, since vitamin B12 released from binding proteins has various forms, different R groups can lead to different stabilities. When using the above analytical methods for detection, the different stabilities of vitamin B12 may lead to deviations in the test results. Summary of the invention

[0004] The object of the present invention is to provide a simple and efficient vitamin B12 detection kit and detection method, by which the vitamin 12 in the sample to be detected is converted into a stable state for detection, thereby improving the stability of the detection.

[0005] To achieve the above-mentioned object, in a first aspect, the present invention provides a simple and efficient vitamin B12 detection kit, comprising a pretreatment agent, a magnetic particle system and a marker system, wherein the sample addition ratio is 20:50:50;

[0006] The pretreatment agent is a strong alkaline solution containing a complexing agent, potassium cobalt cyanide; the magnetic particle system contains magnetic microspheres coated with vitamin B12 intrinsic factor or vitamin B12 derivatives; and the marker system is a vitamin B12 derivative or vitamin B12 intrinsic factor labeled with alkaline phosphatase.

[0007] Wherein, the complexing agent is one of potassium cobalt cyanide, potassium ferrocyanide and potassium hexacyanochromate.

[0008] Wherein, the magnetic particle system is superparamagnetic particles coated with streptavidin and suspended in MES buffer.

[0009] Wherein, the particle size of the superparamagnetic particles is 1.0 μm to 5.0 μm.

[0010] The marker system is a MES buffer solution containing a marker of a vitamin B12 derivative with a concentration of 0.2 ug / mL and a pH of 5.4 to 5.6.

[0011] In a second aspect, the present invention also provides a simple and efficient method for detecting vitamin B12, comprising:

[0012] mixing the extracted serum sample with a pretreatment agent to obtain a pretreated serum sample;

[0013] The magnetic microparticle-intrinsic factor conjugate in the magnetic microparticle system is added to the reaction tube, and the vitamin B12 in the pretreated serum sample is combined with the magnetic microparticles;

[0014] Add vitamin B12 derivative labeled with alkaline phosphatase to the reaction tube;

[0015] After incubation, the vitamin B12 derivative of alkaline phosphatase competes with the magnetic particles-intrinsic factor on the complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker;

[0016] Adsorbing magnetic particles through a magnetic field and washing away the part without magnetic particles;

[0017] Add chemiluminescent substrate for incubation;

[0018] The alkaline phosphatase of the magnetic particle-intrinsic factor-vitamin B12-alkaline phosphatase marker catalyzes the substrate to emit light and records the luminescence reading;

[0019] The concentration of vitamin B12 was calculated from the calibration curve.

[0020] The specific steps of mixing the extracted serum sample with the pretreatment agent to obtain the pretreated serum sample include:

[0021] Take the serum sample to be tested and add it into the reaction tube;

[0022] Adding sodium hydroxide aqueous solution to react at a suitable temperature to release vitamin B12 in the serum sample from the binding protein;

[0023] The complexing agent combines with the released vitamin B12 to dissociate the trace cyanide ion to form stable cyanocobalamin.

[0024] The present invention provides a simple and efficient vitamin B12 detection kit and detection method. The extracted serum sample is mixed with the protein releaser and the complexing agent in the pretreatment agent, and the vitamin B12 in the sample is released from the binding protein. The complexing agent is a complex with cyanide ions. The complex separates cyanide ions in an aqueous solution and combines with the released vitamin B12 to obtain stable cyanocobalamin. Subsequently, the magnetic particle-intrinsic factor conjugate in the magnetic particle system is added to a reaction tube, and the vitamin B12 in the pretreated serum sample is combined with the magnetic particle-intrinsic factor. The vitamin B12 derivative labeled with alkaline phosphatase in the marker system is added to the reaction tube. In the tube, after incubation, the vitamin B12 derivative of alkaline phosphatase competes with the magnetic particles-intrinsic factor on the binding complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker, the magnetic particles are adsorbed by a magnetic field, the part without magnetic particles is washed away, and a chemiluminescent substrate is added for incubation. The alkaline phosphatase of the magnetic particles-intrinsic factor-vitamin B12-alkaline phosphatase marker catalyzes the luminescence of the substrate and records the luminescence reading. The amount of analysis in the sample is calculated from the calibration curve to obtain the concentration of vitamin B12. The stability of the detection is improved by converting the vitamin 12 in the sample to be detected into a stable state for detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0026] Figure 1 The present invention is a flowchart of a simple and efficient vitamin B12 detection method.

[0027] Figure 2 The present invention is a flow chart of mixing the extracted serum sample with the pretreatment agent to obtain the pretreated serum sample.

[0028] Figure 3 It is a diagram of the serum sample pretreatment process of the present invention.

[0029] Figure 4 It is a comparison chart of VB12 sample concentrations of the present invention.

[0030] Figure 5 It is a test value diagram of the VB12 sample of the present invention.

[0031] Figure 6 It is a graph showing the detection results of samples of different concentrations according to the present invention.

[0032] Figure 7 It is a concentration value diagram of the present invention.

[0033] Figure 8 It is the extreme weather preservation test chart of the present invention. DETAILED DESCRIPTION

[0034] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0035] In the first aspect, a simple and efficient vitamin B12 detection kit of the present invention comprises a pretreatment agent, a magnetic particle system and a marker system; the pretreatment agent is a strong alkaline solution containing a complexing agent which is potassium cobalt cyanide; the magnetic particle system contains magnetic microspheres coated with vitamin B12 intrinsic factor or vitamin B12 derivative; the marker system is a vitamin B12 derivative or vitamin B12 intrinsic factor labeled with alkaline phosphatase; the extracted serum sample is mixed with the protein releaser and complexing agent in the pretreatment agent, and the vitamin B12 in the sample is released from the binding protein; the complexing agent is a complex with cyanide ion, and the complexing agent is a complex with cyanide ion. The compound separates cyanide ions in an aqueous solution and combines with the released vitamin B12 to obtain stable cyanocobalamin. The magnetic particle system combined with the vitamin B12 binding protein (vitamin B12 intrinsic factor) can be a magnetic particle that directly coats the vitamin B12 binding protein (vitamin B12 intrinsic factor) on the magnetic particle, or can be connected in an indirect manner, such as a bridge that can bind biotin and streptavidin to each other in an indirect binding manner. The magnetic particle system includes magnetic particles connected with streptavidin and combined with biotinylated vitamin B12 binding protein (vitamin B12 intrinsic factor). The magnetic particle-intrinsic factor conjugate in the magnetic particle system is added to a reaction tube, and vitamin B12 in the pretreated serum sample is combined with the magnetic particle-intrinsic factor. The marker system is a marker that can directly emit light used in chemiluminescent immunoassay, such as luminol and its derivatives, isoluminol or its derivatives, and acridinium ester; or a marker that can emit light in combination with a corresponding substrate used in chemiluminescent enzyme immunoassay, such as alkaline phosphatase or peroxidase, preferably alkaline phosphatase. The vitamin B12 derivative labeled with alkaline phosphatase in the marker system is added to the reaction tube, and after incubation, the alkaline phosphatase The vitamin B12 derivative competes with the magnetic particles-intrinsic factor on the binding complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker. The magnetic particles are adsorbed by a magnetic field, the part without magnetic particles is washed away, and a chemiluminescent substrate is added for incubation. The alkaline phosphatase of the magnetic particles-intrinsic factor-vitamin B12-alkaline phosphatase marker catalyzes the luminescence of the substrate and records the luminescence reading. The amount of analysis in the sample is calculated from the calibration curve to obtain the concentration of vitamin B12. The stability of the detection is improved by converting the vitamin 12 in the sample to be detected into a stable state for detection.

[0036] Secondly, the complexing agent is one of potassium cobalt cyanide, potassium ferrocyanide and potassium hexacyanochromate.

[0037] Again, the magnetic particle system is superparamagnetic particles coated with streptavidin and suspended in MES buffer.

[0038] In addition, the superparamagnetic particles have a particle size of 1.0 μm to 5.0 μm.

[0039] Furthermore, the marker system is a MES buffer solution with a concentration of 0.2 ug / mL of a vitamin B12 derivative marker and a pH of 5.4-5.6.

[0040] In the use of a simple and efficient vitamin B12 detection kit described in the present invention, the extracted serum sample is mixed with the pretreatment agent to obtain a pretreated serum sample, and then the magnetic particle-intrinsic factor conjugate in the magnetic particle system is added to a reaction tube, and the vitamin B12 in the pretreated serum sample is combined with the magnetic particle-intrinsic factor, and the vitamin B12 derivative labeled with alkaline phosphatase in the marker system is added to the reaction tube. After incubation, the vitamin B12 derivative of alkaline phosphatase competes with the magnetic particle-intrinsic factor on the complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker, the magnetic particles are adsorbed by a magnetic field, the part without magnetic particles is washed away, and a chemiluminescent substrate is added for incubation. The alkaline phosphatase of the magnetic particle-intrinsic factor-vitamin B12-alkaline phosphatase marker catalyzes the substrate to emit light and records the luminescence reading, the amount of analysis in the sample is calculated by the calibration curve to obtain the concentration of vitamin B12, and the vitamin 12 in the sample to be detected is converted into a stable state for detection, thereby improving the stability of the detection. Compared with vitamin B12 (VB12) detection kits on the market, the present invention has simpler reagent components, higher safety, higher detection efficiency, stronger specificity, and better clinical relevance.

[0041] In a second aspect, the present invention also provides a simple and efficient method for detecting vitamin B12. Figure 1-Figure 8 ,in, Figure 1 It is a flow chart of a simple and efficient vitamin B12 detection method of the present invention. Figure 2 is a flow chart of the present invention of mixing the extracted serum sample with the pretreatment agent to obtain the pretreated serum sample, Figure 3 is a diagram of the serum sample pretreatment process of the present invention, Figure 4 This is a comparison chart of the concentration of VB12 samples of the present invention. Figure 5 is a test value diagram of the VB12 sample of the present invention, Figure 6 is a graph showing the test results of samples of different concentrations according to the present invention, Figure 7 is a concentration value diagram of the present invention, Figure 8 It is the extreme weather preservation test chart of the present invention.

[0042] The simple and efficient vitamin B12 detection method comprises:

[0043] S1 mixes the extracted serum sample with the pretreatment agent to obtain a pretreated serum sample;

[0044] The specific steps include:

[0045] S11 Take the serum sample to be tested and add it into the reaction tube;

[0046] S12 adds sodium hydroxide aqueous solution to react at a suitable temperature to release vitamin B12 in the serum sample from the binding protein;

[0047] The S13 complexing agent combines with the released vitamin B12 to dissociate the trace cyanide ion and form stable cyanocobalamin;

[0048] In the embodiment of the present invention, 20 μl of the serum sample to be tested is taken, and 20 μl of the vitamin B12 pretreatment agent is added into the reaction tube. An aqueous solution with a sodium hydroxide concentration of 1.0 mol / L and a K3Co(CN)6 concentration of 3.0 g / L (i.e., 3.0 mg of the complex is added per milliliter of the sample solution to be tested) is added and reacted at 37°C for 5 minutes to release the vitamin B12 in the serum sample from the binding protein. The released protein combines with the trace cyanide ions dissociated from K3Co(CN)6 in the solution to form stable cyanocobalamin.

[0049] S2 adds the magnetic microparticle-intrinsic factor conjugate in the magnetic microparticle system to the reaction tube, and at the same time, vitamin B12 in the pretreated serum sample is combined with the magnetic microparticle;

[0050] S3: adding the vitamin B12 derivative labeled with alkaline phosphatase in the labeling system to the reaction tube for incubation;

[0051] After incubation with S4, the vitamin B12 derivative of alkaline phosphatase competes with the magnetic particles-intrinsic factor on the complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker;

[0052] S5 absorbs magnetic particles through a magnetic field and washes away the part without magnetic particles;

[0053] S6 added chemiluminescent substrate for incubation;

[0054] S7 magnetic microparticles-intrinsic factor-vitamin B12-alkaline phosphatase labeled alkaline phosphatase catalyzes the substrate to emit light and records the luminescence readings;

[0055] S8 calculates the amount of vitamin B12 analyzed in the sample using the calibration curve to obtain the concentration.

[0056] In an embodiment of the present invention, superparamagnetic particles coated with streptavidin are suspended in MES buffer, the concentration of the superparamagnetic particles coated with streptavidin and bound to biotinylated intrinsic factor is 0.2 g / L, the pH is 5.4-5.6, the magnetic particle-intrinsic factor conjugate is added to the reaction tube, and the vitamin B12 in the sample is bound to the magnetic particle-intrinsic factor. A vitamin B12 derivative labeled with alkaline phosphatase is added to the reaction tube for incubation. After incubation, the vitamin B12 derivative of alkaline phosphatase competes for the magnetic particle-intrinsic factor on the binding complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker. The magnetic particles are adsorbed by the magnetic field, and the part without magnetic particles is washed away. A chemiluminescent substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane, AMPPD) is added and incubated at 37°C for 5 minutes. The alkaline phosphatase of the magnetic particle-intrinsic factor-vitamin B12-alkaline phosphatase marker can catalyze the substrate to emit light, and the luminescence value is directly proportional to the alkaline phosphatase content. The luminescence reading is recorded. The number of photons generated is inversely proportional to the concentration of vitamin B12 in the sample. The amount of analyte in the sample is calculated by the calibration curve to obtain the concentration of vitamin B12.

[0057] In the present invention, in order to better ensure that vitamin B12 and intrinsic factor react within a more appropriate pH range, when designing the magnetic particle reagent buffer and alkaline phosphatase marker buffer system, when adding the sample pretreatment solution, the strong alkaline solution of the sample pretreatment solution can be neutralized so that the pH of the mixed reaction system is within an appropriate range. This method saves reagents and ensures the accuracy of the test, while improving the economic benefits and ease of use.

[0058] A comparative test was conducted with a two-component sample pretreatment solution and a single-component sample pretreatment solution. A comparative determination experiment of vitamin B12 in serum was conducted using treatment solution 1 containing KCN and treatment solution 2 containing DTT, as well as a treatment solution containing only K3Co(CN)6 as sample pretreatment agents. The specific method of using K3Co(CN)6 as a sample pretreatment agent was in accordance with the sample pretreatment method described above in this embodiment; the specific method of using treatment solution 1 containing KCN and treatment solution 2 containing DTT as sample pretreatment agents was in accordance with the sample pretreatment method described above, except that a sample treatment solution 2 was added; and the content of cyanocobalamin in the sample to be tested was determined according to the above-mentioned chemiluminescent immunoassay. The curves of treatment solution 1 containing KCN and treatment solution 2 containing DTT as sample pretreatment agents, as well as a treatment solution containing only K3Co(CN)6, had high similarity at each concentration gradient, indicating that a treatment solution containing only K3Co(CN)6 can be used to achieve a better pretreatment effect.

[0059] A simple and efficient vitamin B12 detection method of the present invention uses a complex containing cyanide ions but being difficult to dissociate as a donor of cyanide ions, and utilizes the dissociated trace cyanide ions to react with the vitamin B12 in the sample to be tested to generate stable cyanocobalamin for subsequent determination. And by selecting a complex with a specific cyanide ion dissociation constant, the purpose of satisfying the sample processing requirements, fully processing the sample, and not producing greater toxicity is achieved. In the present invention, the sample pretreatment liquid of vitamin B12 has only one component reagent, which can achieve a good dissociation effect. Compared with the sample pretreatment reagents of two components and three components, it has low cost, simple preparation, and efficient treatment effect. In the pretreatment, it is not necessary to use highly toxic drugs or a large amount of cyanide ion donors, and vitamin B12 can be well combined to form stable cyanocobalamin for determination, which has the advantage of high safety.

[0060] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A simple and efficient vitamin B12 detection kit, characterized in that: It includes a pretreatment agent, a magnetic particle system and a marker system, and the sample addition ratio is 20:50:50; The pretreatment agent is a strong alkaline solution containing a complexing agent, potassium cobalt cyanide; the magnetic particle system contains magnetic microspheres coated with vitamin B12 intrinsic factor or vitamin B12 derivatives; and the marker system is a vitamin B12 derivative or vitamin B12 intrinsic factor labeled with alkaline phosphatase.

2. A simple and efficient vitamin B12 detection kit as claimed in claim 1, characterized in that: The complexing agent is one of potassium cobalt cyanide, potassium ferrocyanide and potassium hexacyanochromate.

3. A simple and efficient vitamin B12 detection kit as claimed in claim 2, characterized in that: The magnetic particle system is superparamagnetic particles coated with streptavidin and suspended in MES buffer.

4. A simple and efficient vitamin B12 detection kit as claimed in claim 3, characterized in that: The particle size of the superparamagnetic particles is 1.0 μm to 5.0 μm.

5. A simple and efficient vitamin B12 detection kit as claimed in claim 4, characterized in that: The marker system is a MES buffer solution of a vitamin B12 derivative marker with a concentration of 0.2 ug / mL and a pH of 5.4-5.

6.

6. A simple and efficient vitamin B12 detection method, applied to a simple and efficient vitamin B12 detection kit as claimed in any one of claims 1 to 5, characterized in that: include: mixing the extracted serum sample with a pretreatment agent to obtain a pretreated serum sample; The magnetic particle-intrinsic factor conjugate in the magnetic particle system is added to the reaction tube, and vitamin B12 in the pretreated serum sample is combined with the magnetic particle-intrinsic factor; Adding a vitamin B12 derivative labeled with alkaline phosphatase in the labeling system to a reaction tube for incubation; After incubation, the vitamin B12 derivative of alkaline phosphatase competes with the magnetic particles-intrinsic factor on the complex to form a mixture of intrinsic factor-vitamin B12-alkaline phosphatase marker and vitamin B12-alkaline phosphatase marker; Adsorbing magnetic particles through a magnetic field and washing away the part without magnetic particles; Add chemiluminescent substrate for incubation; The alkaline phosphatase of the magnetic particle-intrinsic factor-vitamin B12-alkaline phosphatase marker catalyzes the substrate to emit light and records the luminescence reading; The concentration of vitamin B12 was calculated from the calibration curve.

7. A simple and efficient vitamin B12 detection method as claimed in claim 6, characterized in that: The specific steps of mixing the extracted serum sample with the pretreatment agent to obtain the pretreated serum sample include: Take the serum sample to be tested and add it into the reaction tube; Adding sodium hydroxide aqueous solution to react at a suitable temperature to release vitamin B12 in the serum sample from the binding protein; The complexing agent combines with the released vitamin B12 to dissociate the trace cyanide ion to form stable cyanocobalamin.

Images