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Method for determining active proteins in pertussis toxin product and diphtheria-pertussis-tetanus vaccine

A technology for pertussis and diphtheria pertussis, applied in the field of vaccine quality evaluation, which can solve the problems of lack of content determination methods and poor repeatability of vaccine toxins

Active Publication Date: 2020-01-10
SHIMADZU (CHINA) CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to improve pertussis toxin (PT, pertussis toxin), filamentous hemagglutinin (FAH, Filamentous hemagglutinin), adhesin (PRN, pertactin), pili protein (FIM, fimbrial) and adenylate cyclization The current situation of lack of determination method of adenylate cyclase toxin (ACT) content, overcome the problem of poor repeatability of ELISA method for testing vaccine toxin, provide the content determination method of the above components, and use it for the content of the above proteins in pertussis toxin products and DTP vaccine Assay and assessment of pertussis toxin subunit integrity

Method used

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  • Method for determining active proteins in pertussis toxin product and diphtheria-pertussis-tetanus vaccine
  • Method for determining active proteins in pertussis toxin product and diphtheria-pertussis-tetanus vaccine
  • Method for determining active proteins in pertussis toxin product and diphtheria-pertussis-tetanus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] 1. Experimental instruments and equipment: high-pressure binary pump, degasser, autosampler, column thermostat and triple quadrupole mass spectrometer.

[0119]2. Experimental reagents: Chinese pertussis toxin (PT) standard, filamentous hemagglutinin (FAH, Filamentoushemagglutinin) standard, adhesin (PRN, pertactin) standard, pili protein (FIM, fimbrial) standard, glandular Adenylate cyclase toxin (ACT) standard, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate, RapiGest TM , trypsin, formic acid, ultrapure water, and acetonitrile.

[0120] 3. Detection conditions: Chromatographic column: Bi℃18 chromatographic column;

[0121] Mobile phase: A-formic acid: water (1:1000, v / v); B-formic acid: acetonitrile (1:1000, v / v);

[0122] Gradient: 0-8min 5% B-40% B, 8-8.1min 40%-100% B, 8.1-10min 100% B, 10-10.1min 100%-5% B, 10.1-15min 5% B; column temperature : 35°C; flow rate: 0.2-0.5mL / min; injection volume: 10μL.

[0123] Mass spectrometry conditions: ion s...

Embodiment 2

[0156] Example 2: Characterization of EU pertussis toxin standard

[0157] Stationary phase: with stationary phase described in embodiment 1;

[0158] A-formic acid: water (1:1000, v / v); B-formic acid: acetonitrile (1:1000, v / v);

[0159] Gradient: 0-8min 5% B-40% B, 8-8.1min 40%-100% B, 8.1-10min 100% B, 10-10.1min 100%-5% B, 10.1-15min 5% B; column temperature : 35°C; flow rate: 0.2-0.5mL / min; injection volume: 10μL.

[0160] LC-MS conditions: ES+ mode; mass spectrometer: triple quadrupole mass spectrometer; nebulizer flow rate: 3L / min; heater flow rate: 10L / min; interface temperature: 200°C; DL temperature: 235°C; heating module temperature : 400° C.; drying gas flow rate: 10 L / min; interface voltage: 3 kV; the detection mode of the mass spectrometer is multiple ion selective monitoring (MRM), and other detection conditions are the same as in Example 1.

[0161] The enzymatic hydrolysis process of the standard product is as follows: take 100 μL each of the EU pertussis t...

Embodiment example 3

[0163] Implementation Case 3: Characterization of the WHO First Generation Pertussis Toxin (PT) Standard

[0164] Stationary phase: with stationary phase described in embodiment 1;

[0165] A-formic acid: water (1:1000, v / v); B-formic acid: acetonitrile (1:1000, v / v);

[0166] Gradient: 0-8min 5% B-40% B, 8-8.1min 40%-100% B, 8.1-10min 100% B, 10-10.1min 100%-5% B, 10.1-15min 5% B; column temperature : 35°C; flow rate: 0.2-0.5mL / min; injection volume: 10μL.

[0167] LC-MS conditions: ES+ mode; mass spectrometer: triple quadrupole mass spectrometer; nebulizer flow rate: 3L / min; heater flow rate: 10L / min; interface temperature: 200°C; DL temperature: 235°C; heating module temperature : 400° C.; drying gas flow rate: 10 L / min; interface voltage: 3 kV; the detection mode of the mass spectrometer is multiple ion selective monitoring (MRM), and other detection conditions are the same as in Example 1.

[0168] The enzymatic hydrolysis process of the standard product is as follows:...

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Abstract

The invention discloses a qualitative and quantitative determination method for active proteins in a pertussis toxin product and a diphtheria-pertussis-tetanus vaccine. A high-performance liquid chromatography-tandem mass spectrometry method is adopted for establishing the high-flux high-selectivity high-sensitivity quantitative method for active proteins including pertussis toxin, filamentous hemagglutinin, adhesin, mycoprotein and adenylate cyclase toxin in the pertussis toxin product and the diphtheria-pertussis-tetanus vaccine. According to the method, characteristic peptide fragments which can be used for qualitative and quantitative analysis of various active vaccine proteins are screened out from a complex vaccine matrix for the first time, and the peptide fragments are different from other reported protein peptide fragments, cannot be obtained through a protein search library and cannot also be simply obtained through amino acid sequences of the vaccine proteins. Simultaneous quantification of five pertussis toxin subunits, filamentous hemagglutinin, adhesin, mycoprotein and adenylate cyclase toxin in pertussis toxin products and diphtheria-pertussis-tetanus vaccines from different manufacturers and of different batches can be realized.

Description

technical field [0001] The present invention specifically relates to a pertussis toxin product and active protein pertussis toxin (PT) five subunits (S1-S5), filamentous hemagglutinin (FAH), adhesin (PRN) and pili protein in DPT vaccine (FIM) and adenylyl cyclase toxin (ACT) qualitative and quantitative determination methods. The invention belongs to the technical field of vaccine quality evaluation. Background technique [0002] Pertussis is an acute respiratory infectious disease caused by Bacillus pertussis. Its clinical feature is that the cough gradually worsens, showing a typical paroxysmal and spasmodic cough, accompanied by a deep and long cock-like inspiratory roar. 2 to 3 months, so it is called whooping cough. In order to prevent whooping cough, China and the World Health Organization have successively developed pertussis toxin products and pertussis-tetanus-diphtheria (pertussis) combined vaccine. The incidence of the disease has decreased substantially since ...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 龙珍卫辰李月琪马霄姚劲挺冀峰李长坤骆鹏王丽婵黄涛宏
Owner SHIMADZU (CHINA) CO LTD
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