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Method for measuring free polysaccharide content in polysaccharide protein conjugate

A polysaccharide protein and determination method technology, applied in the field of vaccine detection, can solve the problems of deviation of free sugar content determination results, numerous steps, large sample volume, etc., and achieve the effects of saving sample amount, wide application range and simple determination.

Active Publication Date: 2014-06-04
TASLY BIOPHARMACEUTICALS CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

We use this method to measure the content of free polysaccharides in the stock solution of pneumonia polysaccharide protein conjugates, but the effect is not good, the main reason is that the protein cannot be completely precipitated after adding HCl, which leads to certain deviations in the results of the determination of free sugar content
There are also literature reports that utilize ethanol precipitation method to measure the method of free polysaccharide of group A meningococcus polysaccharide-tetanus toxoid conjugate, and this method is time-consuming, and steps are numerous, and the sample amount that needs is bigger
According to the literature, there is no report on the determination of the content of free polysaccharides in the stock solution of polysaccharide-protein conjugates by using the trichloroacetic acid precipitation method

Method used

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  • Method for measuring free polysaccharide content in polysaccharide protein conjugate
  • Method for measuring free polysaccharide content in polysaccharide protein conjugate
  • Method for measuring free polysaccharide content in polysaccharide protein conjugate

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experiment example

[0035] Determination of free polysaccharide content in experimental example 9V-CRM197 conjugate

[0036] Take 1ml of 9V-CRM197, in which the concentration of 9V is 110ug / ml. Under the condition of ice-water bath, add 100ul of 1% sodium deoxycholate solution, mix thoroughly, and let stand for 30min. Then add 5ul of 100% trichloroacetic acid solution, mix well and centrifuge at 12000rpm at 4°C for 15min. Take the supernatant as the test sample A, take the conjugate stock solution to be tested as the test sample B, use the sulfate anthrone method to measure the polysaccharide content a and b in sample A and sample B respectively, and use the BCA method to measure the polysaccharide content a and b in A and B respectively. The protein content is c and d, according to the formula:

[0037] Free polysaccharide content=a / b *100% Calculate the free polysaccharide content in the stock solution of 9V-CRM197 conjugate as 4.5%.

[0038] The prerequisite for determining the content of f...

Embodiment 1

[0066] Take 1ml of 9V-CRM197, in which the concentration of 9V is 110ug / ml, add 100ul of 0.3% sodium deoxycholate solution in an ice-water bath, the addition amount is 5% of the volume of the original solution of the conjugate, mix well, and stand still for 20min; then add 5ul of 100% trichloroacetic acid solution, the amount added is 0.1% of the volume of the original solution of the conjugate, after mixing, centrifuge at 4°C, 8000rpm for 15;

[0067] Take the centrifuged supernatant as the test sample A, take the conjugate stock solution to be tested as the test sample B, use the sulfate anthrone method to measure the polysaccharide content in sample A and sample B respectively as a and b; use the BCA method to measure The protein content in A and B is c and d, according to the formula:

[0068] The free polysaccharide content in the polysaccharide-protein conjugate = a / b × 100% Calculate the free polysaccharide content in the stock solution of the 9V-CRM197 conjugate to be ...

Embodiment 2

[0071] Take 1ml of 9V-CRM197, in which the concentration of 9V is 110ug / ml, add 100ul of 3% sodium deoxycholate solution in an ice-water bath, the addition amount is 15% of the volume of the original solution of the conjugate, mix well, let stand for 40min; then add 100% trichloroacetic acid solution 5ul, the amount added is 1% of the volume of the conjugate stock solution, after mixing, centrifuge at 8°C, 12000rpm for 20min;

[0072] Take the centrifuged supernatant as the test sample A, take the conjugate stock solution to be tested as the test sample B, use the sulfate anthrone method to measure the polysaccharide content in sample A and sample B respectively as a and b; use the BCA method to measure The protein content in A and B is c and d, according to the formula:

[0073] Free polysaccharide content in the polysaccharide-protein conjugate = a / b × 100% Calculate the free polysaccharide content in the stock solution of the 9V-CRM197 conjugate to be 6.5%.

[0074] Protei...

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Abstract

The invention relates to a method for measuring free polysaccharide content in polysaccharide protein conjugate, which comprises the following steps: under ice bath conditions, adding a sodium deoxycholate solution into a conjugate stock solution with a certain concentration, mixing, standing, adding a trichloroacetic acid solution, evenly mixing, centrifugating, and taking the supernatant, wherein the supernatant is a sample A to be measured, and the conjugate stock solution to be measured is a sample B to be detected; respectively measuring polysaccharide contents a and b in the sample A and sample B by a chemical method or immunologic method; and calculating the free polysaccharide content in the conjugate according to the formula free polysaccharide content=a / b*100%.

Description

Technical field: [0001] The invention relates to the detection of vaccines, in particular to the determination of the free polysaccharide content in a polysaccharide-protein conjugate. Background technique: [0002] The use of various polysaccharide vaccines has effectively reduced the incidence of corresponding infectious diseases. However, because capsular polysaccharide is a T-cell-independent antigen, it does not have immune memory response, and cannot produce protective antibodies to children under 2 years old, so its use has certain limitations. Studies have shown that covalently binding protein carriers to capsular polysaccharides by chemical methods to form polysaccharide-protein conjugates can convert them into T-cell-dependent antigens, thereby providing protection for infants under 5 years old. [0003] Experiments have shown that too high content of free polysaccharide in the polysaccharide-protein conjugate will lead to a decrease in the effective antigen conte...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N1/38
CPCG01N33/50G01N33/6803G01N2333/4722
Inventor 赵秋敏孙倩李军强金永杰李亚冰刘贺军李剑
Owner TASLY BIOPHARMACEUTICALS CO LTD
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