Method for measuring content of free protein in bacterial capsule protein-polysaccharide ligature

A technology for bacterial capsular polysaccharide and protein content, applied in the biological field, can solve problems such as large differences in properties, and achieve good repeatability

Inactive Publication Date: 2007-07-18
云南沃森生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the properties of different bacterial capsular polysaccharides, different carrier proteins, and different bacterial capsular polysaccharide-protein conjugates are quite different, the determination of free protein content in polysaccharide-conjugated vaccines is a technical difficulty. At present, there is no literature on bacterial capsular polysaccharides at home and abroad. Method for determination of free protein content in protein conjugates

Method used

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  • Method for measuring content of free protein in bacterial capsule protein-polysaccharide ligature
  • Method for measuring content of free protein in bacterial capsule protein-polysaccharide ligature
  • Method for measuring content of free protein in bacterial capsule protein-polysaccharide ligature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Utilize the Lowry method to measure the total protein concentration in the meningococcal group A polysaccharide tetanus toxoid conjugate to be tested, it is 275.5 μ g / ml (C 0 ); get the tetanus toxoid solution corresponding to the conjugate, and dilute to 50 μg / ml with normal saline according to its protein concentration (C 1 ), as sample 1; take the meningococcal group A polysaccharide tetanus toxoid conjugate to be tested, as sample 2;

[0021] Load sample 1 and sample 2 respectively on a high-efficiency gel chromatography column TSKgel G5000PW (30cm×7.5mm) fully balanced with physiological saline, with a sample volume of 20μl, an elution flow rate of 0.3ml / min, and record the chromatogram at a wavelength of 214nm; The time corresponding to the top of the carrier protein elution peak was 27.89 minutes, and the corresponding time to the end of the peak was 34.76 minutes;

[0022] Integrate the sample 1 chromatogram, adjust the integration window to be the second half ...

Embodiment 2

[0028] Utilize the Lowry method to measure the total protein concentration in the meningococcal group C polysaccharide tetanus toxoid conjugate to be tested, it is 320.4 μ g / ml (C 0 ); get the tetanus toxoid solution corresponding to the conjugate, and dilute to 1000 μg / ml with normal saline according to its protein concentration (C 1 ), as sample 1; take the meningococcal group C polysaccharide tetanus toxoid conjugate to be tested, as sample 2;

[0029] Load sample 1 and sample 2 respectively on a high-efficiency gel chromatographic column TSKgelG5000PWxl (30cm×7.8mm) fully balanced with physiological saline, with a sample volume of 200μl, an elution flow rate of 1.0ml / min, and record the chromatogram at a wavelength of 214nm; carrier The corresponding time of protein elution peak peak is 8.37min, and the corresponding time of peak end is 10.22min;

[0030] Integrate the chromatogram of sample 1, and adjust the integration window to be the second half peak from the correspo...

Embodiment 3

[0036] Utilize the Lowry method to measure the total protein concentration in the Haemophilus influenzae type b polysaccharide tetanus toxoid conjugate to be tested, it is 183.8 μ g / ml (C 0 ); get the tetanus toxoid solution corresponding to the conjugate, and dilute to 500 μg / ml with normal saline according to its protein concentration (C 1 ), as sample 1; take the polysaccharide tetanus toxoid conjugate of Haemophilus influenzae type b to be tested, as sample 2;

[0037]Load sample 1 and sample 2 respectively on a high-efficiency gel chromatographic column TSKgelG4000PWxl (30cm×7.8mm) fully balanced with physiological saline, with a sample volume of 100μl, an elution flow rate of 0.5ml / min, and record the chromatogram at a wavelength of 214nm; carrier The corresponding time of protein elution peak peak is 16.75min, and the corresponding time of peak end is 20.46min;

[0038] Integrate the chromatogram of sample 1, and adjust the integration window to be the second half peak...

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Abstract

A method for measuring content of free protein in solder of bacterial capsule protein-polysaccharide includes determining out carrier protein concentration in bacterial capsule protein-polysaccharide solder to be detected by utilizing Lowry manner, using carrier protein corresponding to solder to be detected as sample one and using solder to be detected as sample two, using high efficiency gel chromatography to obtain chromatogram of sample one and two separately then applying external-labeling means to calculate out content of free protein.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for measuring free protein content in bacterial capsular polysaccharide protein conjugates. Background technique: [0002] Meningitis, pneumonia, and sepsis caused by bacterial infection are common diseases in children, with a high mortality rate. Bacterial capsular polysaccharides are the main protective antigens of bacteria, but bacterial capsular polysaccharides are T cell-independent antigens, which cannot induce immune responses in children under 2 years old, and have certain limitations. The carrier protein is covalently bound by chemical methods On the capsular polysaccharide, it can be made into a T cell-dependent antigen, so that it can be effectively used to prevent diseases caused by corresponding pathogen infection in infants aged 3 months to 5 years old. [0003] The content of free protein in bacterial capsular polysaccharide protein conjugate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N30/02G01N1/38G06F19/00G06F19/10
Inventor 黄镇向左云吴凯
Owner 云南沃森生物技术股份有限公司
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