A dissociation agent for vitamin B12 detection and its preparation and application

Abstract

The application provides a dissociation agent for vitamin B12 detection, which is a single-component freeze-dried reagent; a stock solution of the single-component freeze-dried reagent comprises a buffer, a binding agent, a reducing agent, a protective agent and a pH regulator; wherein the molar concentration of the buffer is 20-100 mM, the weight-volume percentage of the binding agent is 0.05-0.2 %, the weight-volume percentage of the reducing agent is 0.05-0.2 %, the weight-volume percentage of the protective agent is 3-8 %, and the weight-volume percentage of the pH regulator is 1-2 %. The application also provides a preparation method and application of the dissociation agent. The dissociation agent has a simple formula, adopts a single-component reagent and uses a freeze-drying process, and can be used in the reaction after being redissolved with ultrapure water and placed in a chemiluminescence reagent rack R1, so that vitamin B12 in a sample can be dissociated in one step.

Claims

1. A dissociation agent for the detection of vitamin B12, characterized in that, The dissociation agent is a single-component lyophilized reagent; the stock solution of the single-component lyophilized reagent consists of a buffer, a binding agent, a reducing agent, a protecting agent, and a pH adjuster; wherein, the buffer is TRIS with a molar concentration of 20~100mM; the binding agent is potassium cyanide with a weight / volume percentage of 0.05%~0.2%; the reducing agent is tris(2-carboxyethyl)phosphine with a weight / volume percentage of 0.05%~0.2%; the protecting agent is trehalose with a weight / volume percentage of 3%~8%; and the pH adjuster is sodium hydroxide with a weight / volume percentage of 1%~2%, used to adjust the pH of the dissociation agent mixture to 12.0~14.

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2. The dissociation agent for vitamin B12 detection according to claim 1, characterized in that, In the stock solution of the single-component lyophilized reagent, the weight-volume percentage of the binder is 0.1% to 0.15%, the weight-volume percentage of the reducing agent is 0.1% to 0.15%, and the weight-volume percentage of the protective agent is 4% to 6%.

3. A method for preparing a dissociation agent for vitamin B12 detection as described in any one of claims 1 to 2, characterized in that, The buffer, binder, reducing agent, protective agent, and pH adjuster are mixed and then prepared into corresponding freeze-dried products using the following specific method: Step (1) Weigh out the buffer, pH adjuster, reducing agent, binding agent and protective agent, add them to the container and stir evenly. Add purified water to make up to the required total volume, and then adjust the pH of the solution to 12.0~14.

0. Then place the resulting mixed sample evenly in the tray, put it in the drying room, put the temperature probe in place, and close the drying room door and the air inlet valve. Step (2) Turn on the freeze dryer for pre-freezing. When the sample temperature and the partition temperature reach the set temperature, turn on the vacuum pump; the vacuum level is displayed as ≤50Pa. Step (3) is then vacuum freeze-drying. When the sample temperature and the partition temperature reach the set temperature, observe the freeze-drying curve. When the moisture content is stable, turn off the vacuum pump. Step (4) Activate the hydraulic system to seal the sample, open the air inlet valve and wait for the internal and external air pressure to balance, then open the drying chamber door and take out the sample.

4. The method for preparing the dissociation agent for vitamin B12 detection according to claim 3, characterized in that, In step (1), the mixed sample is dispensed into vials and then freeze-dried. In step (2), the pre-freezing time is 2~6h, and the set temperature is -50~-65℃; In step (3), the vacuum freeze-drying time is 18~24h.

5. A magnetic microparticle chemiluminescence detection kit for determining vitamin B12 content, characterized in that, Includes reagent R1, reagent R2, magnetic separation reagent M, calibrators, quality control materials, and chemiluminescent substrate solution; The reagent R1 is the dissociation agent for vitamin B12 detection as described in any one of claims 1 to 2; The preparation method of reagent R2 is as follows: take acridinium ester NSP-DMAE-NHS, dissolve it in DMF to prepare a solution, add vitamin B12 intrinsic factor, and react with shaking at room temperature in the dark. Add a blocking agent and react with shaking at room temperature in the dark. Purify using molecular sieve, filter through a 0.22μm membrane, and dilute to the working concentration with diluent to obtain reagent R2. The magnetic separation reagent M is prepared as follows: Vitamin B12 is mixed with magnetic microparticles in 2-(N-Marin)ethanesulfonic acid (MES) buffer at a ratio of 1:20-50; EDC is then added and the mixture is reacted with shaking at room temperature; TRIS buffer containing bovine serum albumin is then added and the mixture is reacted with shaking at room temperature; the mixture is then diluted to the working concentration using a diluent to obtain the magnetic separation reagent M.

6. A method of using the magnetic particle chemiluminescence detection kit for determining vitamin B12 content as described in claim 5 for non-diagnostic purposes, characterized in that, Includes the following steps: First, the dissociation agent for vitamin B12 detection is mixed with ultrapure water and dissolved. Then, the sample to be tested, calibrator and quality control are mixed with the dissolved reagent R1 and incubated. Then, the vitamin B12 reagent R2 and the magnetic separation reagent M were added to the incubated solution, and the incubation was continued. Washing removes unbound substances, then the chemiluminescent substrate solution is added for light excitation, and the light intensity is detected. Establish a calibration curve and calculate the concentration of vitamin B12 in the sample based on the luminescence value of the sample.

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