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Method for measuring content of free polysaccharides in meningococcal polysaccharide combo

A technology for the content of meningococcus and polysaccharides, applied in biological testing, testing water, measuring devices, etc., can solve problems such as cumbersome operation, large error in measurement results, damage to liver and kidney functions, etc., and achieve the effect of simple and fast operation method

Inactive Publication Date: 2011-09-07
BEIJING MINHAI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In foreign literature reports, hydrophobic chromatography and HPLC are used for the determination of free polysaccharides in polysaccharide conjugates, but because the chromatography process is easily affected by impurities with similar molecular sizes, the measurement results have large errors
[0005] Domestic Zhu Wei and others introduced a method for the determination of group A meningococcal polysaccharide-tetanus toxoid binding by ethanol precipitation However, the operation of this method is cumbersome, and it takes several days for a sample to obtain the result, and there may be some errors for samples with low free polysaccharide content;
[0006] Domestic Huang Zhen et al disclosed in Chinese patent 200610048876.9 "a method for the determination of free polysaccharides in meningococcal group A and C polysaccharide conjugates". This method needs to add cold phenol solution to the sample for shaking, but cold phenol is toxic It is strong and has a strong corrosive effect on the skin and mucous membranes. Long-term exposure can inhibit the central nervous system or damage the liver and kidney functions. Inhalation of phenol can also cause headaches, dizziness, fatigue, blurred vision, pulmonary edema, etc.
[0007] In addition, due to the large differences in the properties of different polysaccharide conjugates, the determination of free polysaccharides in polysaccharide conjugate vaccines has always been a technical difficulty. At present, there is no report on the determination of free polysaccharide content in polysaccharide conjugates by using sodium deoxycholate extraction method

Method used

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  • Method for measuring content of free polysaccharides in meningococcal polysaccharide combo
  • Method for measuring content of free polysaccharides in meningococcal polysaccharide combo
  • Method for measuring content of free polysaccharides in meningococcal polysaccharide combo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Sampling: Take 1ml of the polysaccharide conjugate of group A meningococcus to be tested as sample 1, and take the derivative solution corresponding to the polysaccharide conjugate of group A meningococcus as sample 3;

[0053] 2. Centrifugation: take another 1ml of the group A meningococcal polysaccharide conjugate and place it in a 1.5ml centrifuge tube, and take 1ml of the derivative solution corresponding to the group A meningococcal polysaccharide conjugate to be tested and place it in another 1.5ml centrifuge tube Add 100 μl of 1% DOC solution to each centrifuge tube, keep in ice bath for 30 minutes, then add 25 μl of 0.5M HCL solution, shake fully, centrifuge at 5000 rpm for 15-20 minutes, collect 1ml of supernatant in each centrifuge tube separately, and combine The product supernatant is sample 2, and the derivative supernatant is sample 4.

[0054] 3. Calculate the polysaccharide concentration: measure the phosphorus content of sample 1, sample 2, sample 3,...

Embodiment 2

[0062] 1. Sampling: Take 1ml of the polysaccharide conjugate of group C meningococcus to be tested as sample 1, and take the derivative solution corresponding to the polysaccharide conjugate of group C meningococcus as sample 3;

[0063] 2. Centrifugation: take another 1ml of the group C meningococcal polysaccharide conjugate and place it in a 1.5ml centrifuge tube, and take 1ml of the derivative solution corresponding to the group C meningococcal polysaccharide conjugate to be tested and place it in another 1.5ml centrifuge tube Add 100 μl of 1% DOC solution to each centrifuge tube, keep in ice bath for 30 minutes, then add 25 μl of 0.5M HCL solution, shake fully, centrifuge at 5000 rpm for 15-20 minutes, collect 1ml of supernatant in each centrifuge tube separately, and combine The product supernatant is sample 2, and the derivative supernatant is sample 4;

[0064] 3. Calculate the polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sample 3...

Embodiment 3

[0072] 1. Sampling: Take 1ml of the W135 group meningococcal polysaccharide conjugate to be tested as sample 1, and take the derivative solution corresponding to the W135 group meningococcal polysaccharide conjugate as sample 3;

[0073] 2. Centrifugation: Take another 1ml of the W135 group meningococcal polysaccharide conjugate and place it in a 1.5ml centrifuge tube, and take 1ml of the derivative solution corresponding to the W135 group meningococcal polysaccharide conjugate to be tested and place it in another 1.5ml centrifuge tube Add 100 μl of 1% DOC solution to each centrifuge tube, keep in ice bath for 30 minutes, then add 25 μl of 0.5M HCL solution, shake fully, centrifuge at 5000 rpm for 15-20 minutes, collect 1ml of supernatant in each centrifuge tube separately, and combine The product supernatant is sample 2, and the derivative supernatant is sample 4;

[0074] 3. Calculate the polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sa...

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Abstract

The invention provides a method for measuring the content of free polysaccharides in A, C, W135 and Y cluster meningococcal polysaccharide combo, wherein the method comprises the following steps: 1) sampling: respectively taking 1ml of a meningococcal polysaccharide combo to be measured and a corresponding derivative solution of the meningococcal polysaccharide combo to be measured as a sample 1 and a sample 3; 2) centrifuging: additionally placing the meningococcal polysaccharide combo and the corresponding derivative solution of the meningococcal polysaccharide combo to be measured in centrifuge tubes; adding a sodium deoxycholate solution into each centrifuge tube, performing ice bath for 30 minutes and adding a hydrochloric acid solution; fully oscillating, centrifuging and collecting the supernatant of each centrifuge tube as a sample 2 and a sample 4; 3) calculating the concentration of the polysaccharides; and 4) calculating the content of the free polysaccharides. The method provided by the invention can measure the content of free polysaccharides in A, C, W135 and Y cluster meningococcal polysaccharide combo accurately and simply, so that the quality of products is evaluated.

Description

technical field [0001] The invention relates to a method for determining free polysaccharides, in particular to a method for determining the content of free polysaccharides in polysaccharide conjugates of meningococcal groups A, C, W135 and Y. Background technique [0002] Meningococcal infection is the leading cause of bacterial meningitis and fulminant sepsis in children. According to statistics, the annual incidence of meningitis in the world is about 500,000, especially in developing countries. Worldwide, almost all meningitis diseases are caused by group A, group B, group C, group Y and group W135 meningococci, and vaccination with safe and effective vaccines is an ideal measure to prevent and control meningitis. [0003] The meningitis vaccines currently on the market at home and abroad mainly include polysaccharide vaccines and polysaccharide conjugate vaccines. The antigens of polysaccharide vaccines are haptens, which are T cell-independent antigens. The immune res...

Claims

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Application Information

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IPC IPC(8): G01N33/48
CPCG01N33/50G01N2333/22G01N33/18G01N2400/10G01N33/1826
Inventor 王婷婷魏文进何佳琦
Owner BEIJING MINHAI BIOTECH
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