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Method for determining content of free polysaccharides in conjugate vaccines

A technology that combines the content of vaccines and polysaccharides, which is applied in the field of biomedicine and can solve problems such as affecting the accuracy of measurement results.

Inactive Publication Date: 2020-10-30
艾美探索者生命科学研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing polysaccharide content determination method is usually the anthrone method, and the determination results obtained by the anthrone method will include the polysaccharide content in the bound state, thus affecting the accuracy of the determination results

Method used

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  • Method for determining content of free polysaccharides in conjugate vaccines
  • Method for determining content of free polysaccharides in conjugate vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] From the prepared samples, take 0.75ml sample 1 and 0.75ml sample 2 and place them in two centrifuge tubes with a capacity of 50ml.

[0028] Add 0.5 ml of a sodium chloride solution with a concentration of 5 mol / L to the aforementioned sample 1 and sample 2, respectively, and after mixing, let it stand at room temperature for 2 hours.

[0029] Add 9.0ml of absolute ethanol to sample 1 and sample 2 after salting out treatment to obtain mixed solution A 1 And mixed solution A 2 .

[0030] The mixture A 1 And mixed solution A 2 Placed in a centrifuge, centrifuged for 1 hour under the conditions of a centrifugal force of 5500×g and a temperature of 5°C to remove the supernatant to obtain mixed precipitate 1 and mixed precipitate 2.

[0031] Add 3ml of water to the mixed sediment 1 and the mixed sediment 2 respectively to obtain a mixed liquid B 1 And mixed solution B 2 .

[0032] The mixture B 1 And mixed solution B 2 , Placed in a centrifuge, centrifuged for 15 minutes at a speed of...

Embodiment 2

[0036] From the prepared samples, take 0.75ml sample 1 and 0.75ml sample 2 and place them in two centrifuge tubes with a capacity of 50ml.

[0037] Add 0.5 ml of a sodium chloride solution with a concentration of 5 mol / L to the aforementioned sample 1 and sample 2, respectively, and after mixing, let it stand at room temperature for 2 hours.

[0038] Add 9.0ml of absolute ethanol to sample 1 and sample 2 after salting out treatment to obtain mixed solution A 1 And mixed solution A 2 .

[0039] The mixture A 1 And mixed solution A 2 Placed in a centrifuge and centrifuged for 1 hour under the conditions of a centrifugal force of 6000×g and a temperature of 5°C to remove the supernatant to obtain mixed precipitate 1 and mixed precipitate 2.

[0040] Add 3ml of water to the mixed sediment 1 and the mixed sediment 2 respectively to obtain a mixed liquid B 1 And mixed solution B 2 .

[0041] The mixture B 1 And mixed solution B 2 , Placed in a centrifuge, centrifuged for 15 minutes at a speed...

Embodiment 3

[0045] From the prepared samples, take 0.75ml sample 1 and 0.75ml sample 2 and place them in two centrifuge tubes with a capacity of 50ml.

[0046] 0.5ml of sodium chloride solution with a concentration of 5mol / L was added to the aforementioned sample 1 and sample 2, respectively, and after mixing, let it stand at room temperature for 2 hours.

[0047] Add 9.0ml of absolute ethanol to sample 1 and sample 2 after salting out treatment to obtain mixed solution A 1 And mixed solution A 2 .

[0048] The mixture A 1 And mixed solution A 2 Placed in a centrifuge, centrifuged for 1 hour under the conditions of a centrifugal force of 6500×g and a temperature of 5°C to remove the supernatant to obtain mixed precipitate 1 and mixed precipitate 2.

[0049] Add 3ml of water to the mixed sediment 1 and the mixed sediment 2 respectively to obtain a mixed liquid B 1 And mixed solution B 2 .

[0050] The mixture B 1 And mixed solution B 2 , Placed in a centrifuge, centrifuged for 15 minutes at a speed ...

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Abstract

The invention discloses a method for determining the content of free polysaccharides in a conjugate vaccine. The method comprises the following steps: carrying out salting-out treatment on a conjugatevaccine stock solution; adding absolute ethyl alcohol into the stock solution subjected to salting-out treatment, and obtaining a mixed solution A; carrying out centrifugal treatment and separation on the mixed solution A to obtain a mixed precipitate of a polysaccharide protein conjugate and free sugar; S4, adding the mixed precipitate into water to obtain a mixed solution B; carrying out centrifugal treatment on the mixed solution B, and collecting supernatant as a test solution; and determining the polysaccharide content of the test solution, and recording the polysaccharide content as thefree polysaccharide content in the conjugate vaccine. According to the method for determining the content of free polysaccharide in the conjugate vaccine, polysaccharide binding protein in the conjugate vaccine is irreversibly denatured by adding ethanol to precipitate and separate the polysaccharide binding protein in the conjugate vaccine, thereby reducing influence of the polysaccharide binding protein in the conjugate vaccine on the determination of the free polysaccharide content and obtaining more accurate free polysaccharide content.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for determining the content of free polysaccharides in a combined vaccine. Background technique [0002] The active ingredient of the conjugate vaccine is the polysaccharide protein conjugate, which can cause the body's thymus-dependent immune response and play a long-term protective effect on young children; while the free polysaccharide has no immunogenicity and immune memory, and it will be caused when the free polysaccharide is too much. The immunogenicity of the conjugate vaccine has a certain influence, so the free polysaccharide content is an important quality control parameter in the development and production of the conjugate vaccine. The existing polysaccharide content determination method is usually the anthrone method, and the determination result obtained by the anthrone method will include the bound polysaccharide content, thereby affecting the ac...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/78G01N1/34G01N1/40
CPCG01N33/6827G01N21/78G01N1/34G01N1/4044G01N1/4077G01N2001/4083
Inventor 张凡曹方蒋苏顾芸菲
Owner 艾美探索者生命科学研发有限公司
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