Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product

A meningococcal and conjugated vaccine technology, applied in the biological field, can solve the problems of influence, large differences in the properties of polysaccharide conjugates, influence on immune protection effects, etc., and achieve high accuracy and precision, easy operation, and strong durability. Effect

Active Publication Date: 2012-12-05
云南沃森生物技术股份有限公司
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Problems solved by technology

[0004] Because the properties of different bacterial capsular polysaccharides and different polysaccharide conjugates are quite different, for the content of free polysaccharides in the stock solution of individual conjugates, group A can be obtained by measuring phosphorus content after being treated with cold phenol, and group C, Y or W135 can also be obtained by using cold After phenol treatment, it was obtained by measuring the sialic acid content; the free polysaccharide content of each group of the A+C conjugate vaccine could also be obtained by measuring the phosphorus content of the A group and the sialic acid content of the C group respectively, and in A, C, Y, W135 The conjugates of groups A, C, Y, and W135 in the finished product of group conjugate vaccines are mixed together, and groups C, Y, and W135 all contain sialic acid, so the free polysaccharide content of groups C, Y, and W135 cannot be obtained by measuring the content of sialic acid. Since there are many influencing factors in the process of producing more than 3 groups of polysaccharide conjugated vaccine products, and the stability of free polysaccharides of groups A, C, Y or W135 is different, and the content of free polysaccharides in each group is different from that in the finished conjugate vaccine. The corresponding group antigens are closely related to the immunogenicity of the body, thus affecting the immune protection effect of the final product. Therefore, it is necessary to separately measure the free polysaccharide content of each group of the finished product of the meningococcal polysaccharide conjugate vaccine of groups A, C, Y, and W135. To ensure t

Method used

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  • Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product
  • Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product
  • Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product

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Example Embodiment

[0045] Example 1 Determination of A, C, Y, W135 Group Meningococcal Polysaccharide Conjugate Vaccine Finished Product

[0046] The determination of the free polysaccharide content of group A in the finished product of group A, C, Y, and W135 meningococcal polysaccharide conjugate vaccines was carried out in 3 repetitions, namely repetition 1, repetition 2, and repetition 3. Each repetition was carried out according to the following steps:

[0047] (1) Preparation of test products

[0048] ①Pretreatment: Take 12 bottles of finished meningococcal polysaccharide conjugate vaccine of A, C, Y, W135 group to be tested (labeled volume is 0.5ml per bottle), reconstituted with 6ml of water for injection (that is, use water for injection according to the labeled volume Reconstituted), take 5.0ml of the reconstituted solution in an ultrafiltration cup, centrifuge at 5000×g 4℃ for 40min, collect the concentrate, make up to 1.0ml with water for injection, and obtain the ultrafiltration concentra...

Example Embodiment

[0070] Example 2 Determination of A, C, Y, W135 Group Meningococcal Polysaccharide Conjugate Vaccine Finished Product

[0071] Except for the different operations in the following steps, the rest of the operations are the same as in Embodiment 1, and will not be repeated.

[0072] (1) Preparation of test product

[0073] ②Protease K treatment: Take 70μl of ultrafiltration concentrate, add 4μl of proteinase K, 42μl of proteinase K buffer and supplement with water for injection to 420μl, mix well and incubate at 37°C for 6 hours to obtain the original solution of the test product JY; The amount of proteinase K is 4 times the protein content in the enzymatic hydrolysis reaction system.

[0074] ③Cold phenol treatment: take another 0.6ml of ultrafiltration concentrate, add 1.8ml of sodium acetate saturated cold phenol solution, the volume ratio of ultrafiltration concentrate to sodium acetate saturated cold phenol solution is 1:3, shake and mix for 20 minutes, place on ice Incubate fo...

Example Embodiment

[0081] Example 3 Determination of A, C, Y, W135 Group Meningococcal Polysaccharide Conjugate Vaccine Finished Product

[0082] Except for the different operations in the following steps, the rest of the operations are the same as in Embodiment 1, and will not be repeated.

[0083] (1) Preparation of test product

[0084] ②Protease K treatment: Take 70μl of ultrafiltration concentrate, add 8μl of proteinase K, 42μl of proteinase K buffer and supplement with water for injection to 420μl, mix well and incubate at 37°C for 8 hours to obtain the original solution of the test product JY; The amount of proteinase K is 8 times of the protein content in the enzymolysis reaction system;

[0085] ③Cold phenol treatment: take another 0.6ml of ultrafiltration concentrate, add 1.2ml of sodium acetate saturated cold phenol solution, shake and mix for 30min, place in an ice bath for 20min, 10000rpm, centrifugation at 8℃ for 10min, collect the supernatant, and get the sample The supernatant JS of t...

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Abstract

The invention relates to a method for detecting the content of each group of free polysaccharide in a meningococcus polysaccharide conjugate vaccine finished product and belongs to the technical field of biology. The method comprises the following steps of: during preparation of a detected product sample, carrying out proteolytic enzyme K treatment, wherein the addition amount of the proteolytic enzyme K is 2-8 times the content of the protein in an enzymolysis reaction system; carrying out cold phenol solution treatment, namely separating combined polyose and free polyose in a complex compound by utilizing a sodium acetate saturated cold phenol solution; measuring the content of each group of free polysaccharide in the conjugate vaccine finished product by virtue of the conjunctive use of an immunoelectrophoresis detection technology. The method provided by the invention can be used for eliminating the influence of each group of complex compound carrier protein on relevant factors such as electrophoretic mobility during immunoelectrophoresis, effectively separating the combined polyose from the free polyose in the complex compound and establishing a suitable detection system for each group of free polysaccharide in the conjugate vaccine finished product with more than four groups, and has the characteristics of strong durability, accuracy and high precision, thereby establishing a method for evaluating the quality of the meningococcus polyose conjugate vaccine finished product with more than four groups.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for determining the content of free polysaccharides in each group of finished meningococcal polysaccharide conjugated vaccines. Background technique [0002] Neisseria meningitidis (n. meningitidis ), referred to as meningococcus, meningococcal infection is a common cause of bacterial meningitis in children, with a high mortality rate. Due to the use of meningococcal capsular polysaccharide vaccine, the incidence rate was effectively controlled. However, capsular polysaccharide is a T cell-independent antigen, and re-immunization cannot be boosted, and it cannot induce immune responses in children under 2 years old, which has certain limitations. Covalently binding the protein carrier to the capsular polysaccharide by chemical means can make it a T cell-dependent antigen, thereby having an immune memory response, thus providing immune protection for infants...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/561
Inventor 黄镇吴凯樊会兰张胜祥马波陈磊白梅金栋郭秋岑熊波胡东梅王会梅
Owner 云南沃森生物技术股份有限公司
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