Preparation method of tetanus standard toxin solution with high stability and uniformity
By dissolving the tetanus standard toxin in normal saline and adding glycerin and human albumin, the problem of poor stability of the tetanus standard toxin solution was solved, and the preparation of a high-stability and uniform tetanus standard toxin solution was achieved, which significantly improved the repetition of the evaluation results.
Patent Information
- Application Number
- CN202411727854.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-03-11
AI Technical Summary
The tetanus standard toxin solution prepared in the prior art has poor stability, which leads to a large fluctuation in the death time of mice, which seriously affects the evaluation results of the tetanus antitoxin titer.
A high-stability and uniform tetanus standard toxin solution was prepared by dissolving the tetanus standard toxin in normal saline, adding glycerin, and then adding human albumin solution.
By adding human albumin, the tetanus toxin molecules are stabilized, aggregation and precipitation are prevented, the solution uniformity and stability are improved, and the coefficient of variation in the time of death in mice is significantly reduced.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing a tetanus standard toxin solution with high stability and uniformity. Background Art
[0002] The tetanus standard toxin solution is a strictly purified and quantified tetanus toxin solution used to calibrate and verify the titer determination of tetanus antitoxin. It plays a key role in the research and development, production and quality control of tetanus vaccines. At present, in the prior art, when preparing a tetanus standard solution, according to the general rule 3508 Tetanus Antitoxin Titer Determination Method (Mouse Test Method) in the Pharmacopoeia of the People's Republic of China (Part III of the 2020 Edition), before the tetanus antibody titer is determined, the tetanus standard toxin prepared by the two methods should be calibrated to determine the optimal dilution multiple of the tetanus standard toxin prepared by the two methods. According to the result judgment criteria, all experimental mice should die within 72 to 120 hours. The time of death of the mice in the experimental control group determines the result of the tetanus antibody titer. However, in actual use, it was found that the tetanus standard toxin solution prepared according to the pharmacopoeia method (prepared using BBS (borate buffer solution)) did not produce the desired results after more than one week, resulting in large fluctuations in the time of death of mice, which seriously affected the evaluation results of the tetanus antitoxin titer (repeatability was only 12%). Therefore, it is particularly important to find a stable and accurate method for preparing a tetanus standard antitoxin solution. Summary of the invention
[0003] The present invention is intended to provide a method for preparing a tetanus standard toxin solution with high stability and uniformity, so as to solve the problem of poor stability of the tetanus standard toxin solution prepared by the prior art method.
[0004] To achieve the above purpose, the present invention adopts the following technical scheme: a method for preparing a tetanus standard toxin solution with high stability and uniformity, wherein the tetanus standard toxin is dissolved in physiological saline and glycerol is added to prepare a toxin solution, and then a human albumin solution is added to the toxin solution.
[0005] Preferably, as an improvement, the concentration of the human albumin solution is 20%, and the volume ratio of the human albumin solution to the toxin solution is 1:3.
[0006] Preferably, as an improvement, the final concentration of human albumin is 5%.
[0007] Preferably, as an improvement, the mass ratio of physiological saline to glycerol is 1:1.
[0008] Preferably, as an improvement, when using physiological saline to dissolve the tetanus standard toxin, first take 1 ml of physiological saline for pre-dissolution, and then add the remaining 4 ml of physiological saline to mix evenly.
[0009] Preferably, as an improvement, the glycerol is sterilized.
[0010] Preferably, as an improvement, the storage temperature of the tetanus standard toxin solution is 2-8°C.
[0011] Preferably, as an improvement, the tetanus standard toxin solution is stored away from light.
[0012] Preferably, as a method for improving a preparation method of a tetanus toxin standard solution with high stability and uniformity, the method comprises the following steps:
[0013] Step 1: Dissolve the cold standard toxin with 5 ml of normal saline. When dissolving, first take 1 ml of normal saline for pre-dissolution, and then add the remaining 4 ml of normal saline and mix well to obtain system I;
[0014] Step 2: Add sterilized glycerol into system I to prepare a toxin solution;
[0015] Step 3, adding a 20% human albumin solution into the toxin solution, with the volume ratio of human albumin to toxin solution being 1:3, to obtain a working toxin containing 5% human albumin;
[0016] Step 4: After mixing, divide the working toxin containing 5% human albumin into smaller packages and store them at 2-8°C in the dark.
[0017] The principle and advantages of this solution are: in actual application, in this technical solution, the problems of poor stability and high coefficient of variation of mouse death time in the traditional pharmacopoeia-guided preparation method in the prior art are analyzed: tetanus toxin may aggregate or precipitate in the solution, which will affect the uniformity and stability of the solution. This technical solution breaks away from the barriers of the existing pharmacopoeia-guided technology and creatively proposes to add human albumin: human albumin is a macromolecular protein with high hydrophilicity and stability. It can bind to tetanus toxin molecules to form a complex, thereby protecting the toxin molecules from environmental factors; in addition, human albumin can prevent the interaction between toxin molecules through steric hindrance effect, reducing the occurrence of aggregation and precipitation; not only that, the interaction between human albumin and tetanus toxin molecules (such as hydrogen bonds, van der Waals forces, etc.) can stabilize the conformation of toxin molecules and prevent them from denaturing; and the macromolecular structure of human albumin can provide steric hindrance to prevent direct contact between toxin molecules and reduce the possibility of aggregation. The buffering capacity of human albumin can resist the interference of external acid-base substances and maintain the stability of the pH value of the solution, thereby protecting the toxin molecules from the influence of pH changes. It can also provide an appropriate amount of ions to adjust the ionic strength of the solution, which is beneficial to maintaining the solubility and stability of the toxin molecules.
[0018] During the technical research and development stage, the confirmation of the ratio of physiological saline to glycerol was one of the difficulties and key points in the research and development of this technical solution. The inventors found that increasing the amount of glycerol added would increase the viscosity of the mixed system, which is not conducive to dissolution and monitoring; while reducing the amount of glycerol added would affect the storage stability of the tetanus standard toxin solution, resulting in an impact on the detection effect. Through continuous screening and comparison, the pre-dissolution, mixed dissolution and sterilized glycerol addition strategies of this technical solution were confirmed. In addition, the inventors also tried to dilute the toxin directly with borates, but the effect was not obvious. DETAILED DESCRIPTION
[0019] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.
[0020] Program Overview:
[0021] A method for preparing a highly stable and uniform tetanus toxin standard solution comprises the following steps:
[0022] Step 1: Dissolve 1 vial of tetanus standard toxin in 5 ml of normal saline. During this process, first take 1 ml of normal saline for pre-dissolution, and then add the remaining 4 ml of normal saline and mix well to obtain system I.
[0023] Step 2: Add 5 ml of sterilized glycerol into system I to prepare a toxin solution;
[0024] Step 3, adding a 20% human albumin solution into the toxin solution, with the volume ratio of human albumin to toxin solution being 1:3, to obtain a working toxin containing 5% human albumin;
[0025] Step 4: After mixing, pack the working toxin containing 5% human albumin into light-proof tubes and store them in a dark place at 2-8°C.
[0026] Example 1
[0027] Dilute 1 tetanus human immunoglobulin national standard with 10 ml borate buffer to make a working standard, mix well and store at 2-8°C.
[0028] Dissolve 1 vial of tetanus standard toxin in 5 ml of normal saline (first take 1 ml of normal saline for pre-dissolution, and then add the remaining 4 ml of normal saline to mix evenly), add 5 ml of sterilized glycerol to make a toxin solution after mixing, take 9 ml of the toxin solution and add 3 ml of 20% human albumin solution to make a working toxin containing 5% human albumin, mix well and store at 2-8°C away from light.
[0029] Comparative Example 1
[0030] Dilute 1 tetanus human immunoglobulin national standard with 10 ml borate buffer to make a working standard, mix well and store at 2-8°C.
[0031] Dissolve 1 vial of tetanus standard toxin in 5 ml of normal saline, mix well and add 5 ml of sterilized glycerol to make working toxin. Mix well and store at 2-8°C away from light.
[0032] Experimental Example 1
[0033] Experimental methods:
[0034] 1. Dilute the working toxin prepared in Example 1 and Comparative Example 1 into 5 dilutions with borate buffer solution, so that each 1 ml contains about 5 experimental amounts (1 / 10L+), add them into small test tubes respectively, add an equal amount of diluted working standard control solution to each tube, mix evenly, seal, combine at 37°C for 1 hour, and immediately inject into mice. Inject 0.4 ml subcutaneously into the abdomen of 17-19g mice, be careful not to let the injection solution flow out, and inject 3 mice for each dilution. Observe the mice at least once in the morning and afternoon every day for 5 consecutive days. Select the dilution that causes all deaths within 72-120 hours as the optimal dilution multiple of the liquid toxin, and accurately record the death time of the mice at the optimal dilution multiple.
[0035] 2. Take the preparation time of the standard reagent as Day 0, and use the best dilution multiple obtained from the experiment on Day 0 as a reference. Perform toxin calibration experiments every 7 days thereafter, for a total of 6 experiments. Conduct one group of experiments for each method, and determine the time of death of mice at the same dilution multiple and record it accurately.
[0036] 3. Calculate the coefficient of variation based on the six experimental data of each method (the time of death of mice when the toxin dilution multiples are the same), and compare the coefficients of variation obtained by the two methods. The method with a smaller coefficient of variation is better.
[0037] The formula for calculating the coefficient of variation is:
[0038] Coefficient of variation = (standard deviation SD of the same group of data / arithmetic mean) × 100%
[0039] The experimental results are shown in Table 1:
[0040] Table 1
[0041]
[0042]
[0043] 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. A method for preparing a tetanus standard toxin solution with high stability and uniformity, characterized in that: The tetanus standard toxin was dissolved in physiological saline and glycerol was added to prepare a toxin solution, and then human albumin solution was added to the toxin solution.
2. The method for preparing a tetanus standard toxin solution with high stability and uniformity according to claim 1, characterized in that: The concentration of the human albumin solution is 20%, and the volume ratio of the human albumin solution to the toxin solution is 1:
3.
3. The method for preparing a tetanus standard toxin solution with high stability and uniformity according to claim 2, characterized in that: The final concentration of the human albumin is 5%.
4. The method for preparing a highly stable and uniform tetanus toxin standard solution according to claim 3, characterized in that: The mass ratio of the physiological saline to glycerol is 1:
1.
5. The method for preparing a highly stable and uniform tetanus toxin standard solution according to claim 4, characterized in that: When using normal saline to dissolve the tetanus toxin, first take 1 ml of normal saline for pre-dissolution, and then add the remaining 4 ml of normal saline and mix well.
6. The method for preparing a highly stable and uniform tetanus toxin standard solution according to claim 5, characterized in that: The glycerol is sterilized.
7. The method for preparing a highly stable and uniform tetanus toxin standard solution according to claim 6, characterized in that: The storage temperature of tetanus standard toxin solution is 2-8℃.
8. The method for preparing a highly stable and uniform tetanus toxin standard solution according to claim 7, characterized in that: Tetanus toxin standard solution should be stored away from light.
9. The method for preparing a highly stable and uniform tetanus standard toxin solution according to claim 8, characterized in that: The steps include: Step 1: Dissolve the cold standard toxin with 5 ml of normal saline. When dissolving, first take 1 ml of normal saline for pre-dissolution, and then add the remaining 4 ml of normal saline and mix well to obtain system I; Step 2: Add sterilized glycerol into system I to prepare a toxin solution; Step 3, adding a 20% human albumin solution into the toxin solution, with the volume ratio of human albumin to toxin solution being 1:3, to obtain a working toxin containing 5% human albumin; Step 4: After mixing, divide the working toxin containing 5% human albumin into smaller packages and store them at 2-8°C in the dark.