Method for measuring bovine tuberculin and activity thereof with high performance liquid chromatography
A tuberculin and high-performance liquid phase technology, which can be used in measurement devices, material separation, and analysis of materials, etc., can solve the problems of cumbersome determination, unintuitive data, and low determination specificity.
Inactive Publication Date: 2012-08-29
CHINA INST OF VETERINARY DRUG CONTROL
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Biological methods are used in the "Manufacturing and Testing Procedures for Tuberculin" promulgated by the International Veterinary Health Organization (OIE) and the Ministry of Agriculture of my country, that is, guinea pigs are used to calibrate the activity of purified bovine tuberculin. This method can reflect the However, the disadvantages are that the determination is cumbersome, there are many influencing factors, the parallelism is not good, the specificity of the determination is not strong, the data is not intuitive, etc., and the relative content of tuberculosis protein and nucleic acid cannot be directly determined
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[0023] 1. Liquid chromatography conditions
[0024] High performance liquid chromatography: shimda 2010A high performance liquid chromatography; detector: shimda ultraviolet detector; detection wavelength: 271nm.
[0025] Preparation of mobile phase: Take 250ml of acetonitrile, 750ml of water, add 1ml of trifluoroacetic acid, mix well, filter with a 0.45μm filter membrane, and vacuum degas.
[0026] 2. Preparation of test solution and standard solution
[0027] Preparation of the test solution: Take bovine-type purified tuberculin (provided by the China Veterinary Drug Administration), add sterilized water to dissolve and dilute to make a solution containing about 9,000 IU per 1ml.
[0028] Preparation of standard solution: Take the first-generation bovine puriflorin international standard (58,500 IU, 1.8mg) issued by the British Institute of Biological Products, dissolve and dilute with sterile water to make about 9,000 IU per 1ml The solution.
[0029] 3. Measurement meth...
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Abstract
The invention relates to a method for measuring bovine tuberculin and activity thereof with high performance liquid chromatography. The purified bovine tuberculin is measured with the high performance liquid chromatography, thus the nucleic acid can be completely separated from the tuberculosis protein, the influence of the nucleic acid to the tuberculosis protein can be completely eliminated, and the relative content of the nucleic acid and the tuberculosis protein can be accurately reflected. The valence of the purified bovine tuberculin is currently calculated with the guinea-pig calibration method which is complex to test and operate, can be affected by many factors and has the large measuring result error. The method can easily and directly measure the valence of the purified bovine tuberculin, is easy to operate and can ensure the accurate and reliable data.
Description
technical field [0001] The invention relates to a high-efficiency liquid phase determination method for detecting the relative content of tuberculosis protein and nucleic acid in purified bovine tuberculin (PPD) and the activity of bovine tuberculin. The invention relates to a chromatographic potency determination technique belonging to the field of veterinary biological products. Background technique [0002] Bovine tuberculosis is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis, and it is a zoonotic pathogenic microorganism. Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis and is characterized by the formation of nodular granulomas called tuberculosis. [0003] Purified bovine tuberculin is a liquid or freeze-dried product made from Mycobacterium tuberculosis cultured on the liquid surface, steam sterilized and filtered to remove the bacteria, and purified by precipitation, washing and redissolving. The main compo...
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IPC IPC(8): G01N30/02G01N30/06G01N30/86
Inventor张秀英王在时汪群杰王宛关孚时陆连寿戴志红李翠蒋卉
OwnerCHINA INST OF VETERINARY DRUG CONTROL