Screening method and content measurement method for bordetella pertussis tracheal cytotoxin

A technology of tracheal cytotoxin and determination method, which is applied in the field of vaccine quality evaluation, and can solve problems such as difficulty in implementing the detection method of tracheal cytotoxin of B. pertussis

Active Publication Date: 2020-02-18
SHIMADZU (CHINA) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the current situation that the Bacillus pertussis tracheal cytotoxin detection method is difficult to implement due to the lack of reference substances for Bacillus pertussis tracheal cytotoxins, the present invention provides an HPLC-MS / MS method for detecting Bacillus pertussis tracheal cytotoxins in pertussis or DTP vaccines method, this method only needs to use the B. pertussis tracheal cytotoxin reference substance when the method is established, and the method does not need the B. pertussis tracheal cytotoxin reference substance during the implementation of the method to screen whether there is B.

Method used

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  • Screening method and content measurement method for bordetella pertussis tracheal cytotoxin
  • Screening method and content measurement method for bordetella pertussis tracheal cytotoxin
  • Screening method and content measurement method for bordetella pertussis tracheal cytotoxin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] 1. Experimental instruments and equipment:

[0093] High pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.

[0094] 2. Experimental reagents:

[0095] The reference substance Bacillus pertussis tracheal cytotoxin was purified in the laboratory, and its structure was confirmed by multi-stage characterization of high-resolution mass spectrometry.

[0096] 3. Detection conditions:

[0097] Chromatographic column: stationary phase Ⅰ, R1 is silica gel;

[0098] Mobile phase: A-contain 10mmol / L ammonium formate aqueous acetonitrile (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 95%; B-10mmol / L ammonium formate aqueous solution (adjust pH to 4.3 with formic acid) ;

[0099] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L.

[0100] Gradient: 0-5min 80%A-20%A; Column temperature: 30°C; Flow ra...

Embodiment 2

[0125] The content of Bacillus pertussis tracheal cytotoxin in the pertussis toxin product of manufacturer 1 is detected, and the detection conditions are as follows:

[0126] Stationary phase: with stationary phase described in embodiment 1;

[0127] Mobile phase: A-contain 10mmol / L ammonium formate aqueous acetonitrile (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 95%; B-10mmol / L ammonium formate aqueous solution (adjust pH to 4.3 with formic acid) ;

[0128] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L.

[0129] Gradient: 0-5min 80%A-20%A; Column temperature: 30°C; Flow rate: 0.3mL / min; Injection volume: 10μL. LC-MS conditions: Ion source: ESI positive and negative ion scanning simultaneously, nebulizer flow rate 3L / min, heater flow rate 10L / min, interface temperature 200°C, DL temperature 230°C, heating module temperature 400°C, dryi...

Embodiment 3

[0132] The content of Bacillus pertussis tracheal cytotoxin in the intermediate product of pertussis toxin from manufacturer 1 is tested under the following conditions:

[0133] Stationary phase: stationary phase II;

[0134] Mobile phase: A-contain 10mmol / L ammonium formate aqueous acetonitrile (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 95%; B-10mmol / L ammonium formate aqueous solution (adjust pH to 4.3 with formic acid) ;

[0135] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L.

[0136]Gradient: 0-5min 5%A-30%A; Column temperature: 50°C; Flow rate: 0.3mL / min; Injection volume: 10μL. LC-MS conditions: Ion source: ESI positive and negative ion scanning simultaneously, nebulizer flow rate 3L / min, heater flow rate 10L / min, interface temperature 200°C, DL temperature 230°C, heating module temperature 400°C, drying gas flow rate 10L / min mi...

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Abstract

The invention provides a screening method and content measurement method for bordetella pertussis tracheal cytotoxin in pertussis or diphtheria-pertussis-tetanus vaccine. The content detection methodcomprises the following steps of (1) drawing a standard working curve, particularly weighting an appropriate amount of bordetella pertussis tracheal cytotoxin reference substance, preparing a standardworking solution, analyzing the standard working solution by high performance liquid chromatography tandem mass spectrometry, obtaining a chromatogram of the reference substance, and obtaining the standard working curve according to a relation between solution concentration and the corresponding chromatographic area; and detecting a sample, particularly taking and adding a to-be-measured sample to acetonitrile with the same volume, performing centrifuging after uniform mixing, taking a supernatant, analyzing and detecting the supernatant by the high performance liquid chromatography tandem mass spectrometry, and obtaining content of the bordetella pertussis tracheal cytotoxin of a to-be-measured constituent according to the response peak area of the to-be-measured constituent and the standard working curve. The method has ultrahigh sensitivity, and the limitation of the method is lower than the limitation required by European Pharmacopoeia by 1,409 times.

Description

technical field [0001] The invention specifically relates to a method for determining the content of Bacillus pertussis tracheal cytotoxin in a pertussis toxin product and a diphtheria pertussis vaccine. The invention belongs to the technical field of vaccine quality evaluation. Background technique [0002] B. pertussis tracheal cytotoxin is a glycopeptide with a molecular weight of 921 in the culture supernatant of B. pertussis. B. pertussis tracheal cytotoxin can attach to hamster tracheal epithelial cells, causing damage to tracheal tissue culture, prolonging the recovery of damaged ciliated cells and blocking the division and differentiation of the underlying nascent basal cell population. Bacillus pertussis tracheal cytotoxin can make pertussis patients difficult to relieve clinical symptoms with drugs. When B. pertussis tracheal cytotoxins are present, the cough symptoms of pertussis patients can persist for more than several weeks even if the B. pertussis is no lon...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/86G01N30/06
CPCG01N30/02G01N30/8679G01N30/06
Inventor 龙珍卫辰李月琪马霄姚劲挺冀峰李长坤谭亚军黄涛宏
Owner SHIMADZU (CHINA) CO LTD
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