Quantitative detection method of pneumococcus polysaccharide
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A technology of pneumococcal polysaccharide and quantitative detection method, which can be applied to measurement devices, preparation of test samples, measurement of color/spectral characteristics, etc., and can solve problems such as large deviation of true values.
Active Publication Date: 2017-08-22
华兰生物疫苗股份有限公司
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[0003] Existing methods for the quantitative detection of polysaccharides include chromogenic methods, such as the anthrone-sulfuric acid method (Wu Kai, Dai Zeyou, the improved anthrone method detects the concentration of polysaccharides in the capsular polysaccharide conjugates of Strep
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Embodiment 1
[0026] Pneumococcal type 12F (PN12F)
[0027] 1. Reagents and standard substances
[0028] (1) Anthrone sulfuric acid solution: slowly add 75ml of concentrated sulfuric acid into 25ml of pure water, fully stir and cool, accurately weigh 0.1g of anthrone, and dissolve it in the above sulfuric acid solution.
[0029] ⑵Pneumococcal polysaccharide standard: PN12F pneumonia polysaccharide standard comes from the Danish National Serum Institute.
[0030] (3) Pneumococcal 12F polysaccharide sample to be tested: 201404002H2, all test indicators are qualified.
[0031] ⑷Pneumococcal polysaccharide combination: according to the structural formula of PN12F pneumococcal polysaccharide, according to the molar ratio of n 葡萄糖 :n 半乳糖 :n 岩藻糖 :n N-乙酰基甘露糖胺 :n N-乙酰基半乳糖胺 = 2:1:1:1:1 ratio to prepare PN12F type combination sugar.
[0032] 2. Instrument
[0033] ⑴UV spectrophotometer (UV-1800)
[0034] ⑵Cuvette: 45*12.5*12.5(mm)
[0035] (3) Test tube: 10ml
[0036] ⑷Analytical balance: S...
Embodiment 2
[0087] Streptococcus pneumoniae type 1 polysaccharide (PN1 type)
[0088] 6. Reagents and standard substances
[0089] (1) Sodium tetraborate sulfuric acid solution: Accurately weigh 9.55g of sodium tetraborate, dissolve in 1000ml of concentrated sulfuric acid, mix the two, and store at room temperature.
[0090] (2) Carbazole solution: Accurately weigh 125mg of carbazole, add 100ml of absolute ethanol, dissolve it, put it in a brown bottle, and store it at 2-8°C.
[0091] (3) Pneumococcal polysaccharide standard (PN1 type): PN1 type pneumonia polysaccharide standard comes from the Danish National Serum Institute.
[0092] ⑷Pneumococcal polysaccharide sample to be tested (PN1 type): JK01B-A201307007, all the test indicators are qualified.
[0093] (5) Pneumococcal composite polysaccharide (PN1 type): According to the structural formula of PN1 type pneumococcal polysaccharide, it is determined that it is composed of two monosaccharides, galacturonic acid and N-acetyl-D-galact...
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Abstract
The invention discloses a quantitative detection method of pneumococcus polysaccharide. According to the method, a combined saccharide prepared from cheap and easily available monosaccharides is used as a standard and replaces a purchased polysaccharide standard, standard curves are built through the combined saccharide and purchased polysaccharide standard, the same batch of pneumococcus polysaccharide samples are detected, the detection results are compared so that a correction coefficient is obtained, in later detection, a standard curve is built through the combined saccharide replacing the purchased standard saccharide, the concentration of the pneumococcus polysaccharide sample is detected, and through correction coefficient correction, the final concentration of the sample to be detected is obtained. The offset between the result and a detection result obtained by purchased standard saccharide as a standard detection sample is less than or equal to 5%. Monosaccharides in the combined saccharide are cheap and easily available, the analysis method is simple, the analysis cost is greatly reduced, the problem of the high cost of the purchased standard saccharide is solved, the experimental result is small and the quantitative detection method is easy to promote. Under appropriate operating conditions, the error is controlled within 0.4%. The quantitative detection method can be used as a quantitative detection method of pneumococcus polysaccharide.
Description
technical field [0001] The invention belongs to the field of drug analysis and relates to a quantitative detection method for pneumococcal polysaccharide. Background technique [0002] Pneumococcus is one of the main pathogenic bacteria that cause invasive meningitis, pneumonia and otitis media in children, and is an important cause of hospitalization and even death of adults and children with pneumonia. Pneumococcus is a Gram-positive bacterium, and its basic virulence factor is Capsular polysaccharides can be divided into different serotypes according to the antigenicity of capsular polysaccharides. So far, 23 serotypes of Streptococcus pneumoniae polysaccharide vaccines have been successfully developed. In the 23-valent pneumococcal polysaccharide vaccine product inspection project, the content of 23-valent pneumococcal polysaccharide is an important detection index of the vaccine. [0003] Existing methods for the quantitative detection of polysaccharides include chromo...
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