A rapid qualitative and quantitative detection method for oil adjuvant vaccines

A quantitative detection method and oil adjuvant technology, which is applied in the direction of material inspection products, test pharmaceutical preparations, test samples preparation, etc., can solve the problems affecting the detection process, reducing the antigen signal intensity, vaccine demulsification detection obstacles, etc., to achieve The antigen recovery rate is high and the effect of improving the antigen recovery rate

Active Publication Date: 2020-07-10
SHANGHAI SHEN LIAN BIOMEDICAL CORP
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AI Technical Summary

Problems solved by technology

As we all know, vaccines are emulsified by antigens and adjuvants in a certain ratio and according to a specific procedure. However, adjuvants have brought huge obstacles to the detection of vaccine demulsification.
[0004] Due to the complex composition of the oil adjuvant used in the vaccine emulsification process, including surfactants, immune enhancers and other substances, the water phase after demulsification often contains the above-mentioned impurities and demulsifiers, and the industry has not been able to remove the above-mentioned Impurities and demulsifiers, and impurities and demulsifiers will greatly affect the subsequent detection process, causing signal masking or interference, lowering the intensity of the antigen signal, and even unable to effectively detect the antigen in it, due to the randomness of the impurities Distributability, resulting in poor repeatability of the detection method, while increasing the cost of instrument maintenance
In the vaccine industry, how to demulsify the oil adjuvant vaccine and detect and identify the ingredients in the vaccine is a recognized technical difficulty in the industry. Because the oil adjuvant vaccine has complex components and contains a large amount of surfactants and immune enhancers Such substances will interfere with all detection methods, or even fail to detect, and cannot reflect the true state of the antigen in the vaccine
[0005] Although there are demulsification methods in the industry, their efficiency and effect are not good. The traditional demulsification methods will have various problems after demulsification, such as incomplete demulsification, unclear boundary between water phase and oil phase, and poor detection in water phase. Issues such as antigens have been plaguing the industry
There is no report that this method is used for antigen purification in the prior art

Method used

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  • A rapid qualitative and quantitative detection method for oil adjuvant vaccines
  • A rapid qualitative and quantitative detection method for oil adjuvant vaccines
  • A rapid qualitative and quantitative detection method for oil adjuvant vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] This embodiment provides a rapid qualitative and quantitative detection method for oil adjuvant vaccines, using the following steps:

[0033] 1) The demulsification of the sample will be demulsified according to the following method:

[0034] Take 10ml of the vaccine to be tested (commercially available synthetic peptide vaccine for porcine foot-and-mouth disease, the concentration is 75ug / ml) mixed with n-butanol at a volume ratio of 1:1, add 50mg of histidine, shake and mix well, at 4°C, with Centrifuge at 3000r / min for 15 minutes, and carefully extract the lower aqueous phase with a 10ml syringe after centrifugation to obtain the aqueous phase antigen sample.

[0035] 2) ZIPTIP purification of samples

[0036] 2.1 Freeze-dry or concentrate the aqueous phase antigen sample prepared in step 1), and use ZIPTIP to purify, the steps are as follows:

[0037] 1. Use 50% ACN to activate ZIPTIP;

[0038] 2. Draw the sample for several times;

[0039] 3. Use 0.05% TFA wate...

Embodiment 2

[0049] This embodiment provides a rapid qualitative and quantitative detection method for oil adjuvant vaccines, using the following steps:

[0050] 1) The demulsification of the sample will be demulsified according to the following method:

[0051] Take 10ml of the vaccine to be tested (commercially available synthetic peptide vaccine for porcine foot-and-mouth disease, the concentration is 75ug / ml) mixed with n-butanol at a volume ratio of 1:1, add 10mg of phenylalanine, shake and mix well, at 4°C, Centrifuge at 3000r / min for 15 minutes, and carefully extract the lower aqueous phase with a 10ml syringe after centrifugation to obtain the aqueous phase antigen sample.

[0052] The foot-and-mouth disease vaccine was demulsified by the method of this example, and the demulsification efficiency was 87.6%.

[0053] 2) ZIPTIP purification of samples

[0054] 2.1 Freeze-dry or concentrate the aqueous phase antigen sample prepared in step 1), and use ZIPTIP to purify, the steps are...

Embodiment 3

[0062] This embodiment provides a rapid qualitative and quantitative detection method for oil adjuvant vaccines, using the following steps:

[0063] 1) The demulsification of the sample will be demulsified according to the following method:

[0064] Take 10ml of the vaccine to be tested (commercially available synthetic peptide vaccine for porcine foot-and-mouth disease, the concentration is 75ug / ml) and n-butanol at a volume ratio of 1:1, add 200mg of proline to each tube, oscillate and mix well, and store at 4°C , centrifuge at 3000r / min for 15 minutes, and carefully extract the lower aqueous phase with a 10ml syringe after centrifugation to obtain the aqueous phase antigen sample.

[0065] The foot-and-mouth disease vaccine was demulsified by the method of this example, and the demulsification efficiency was 94.8%.

[0066] 2) ZIPTIP purification of samples

[0067] 2.1 Freeze-dry or concentrate the aqueous phase antigen sample prepared in step 1), and use ZIPTIP to purif...

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Abstract

The invention provides a fast qualitative and quantitative detection method of an oil adjuvant vaccine. The method is characterized by comprising the following steps: carrying out demulsification on the oil adjuvant vaccine, purifying an obtained water phase antigen sample by using ZIPTIP, and carrying out qualitative and quantitative detection on the purified sample, wherein a demulsification method comprises the steps of mixing the oil adjuvant vaccine with n-butyl alcohol, then adding a competitive agent into the mixture, oscillating and evenly mixing, and centrifuging to obtain the water phase antigen sample. According to the method, an antigen in the oil adjuvant vaccine is released into a water phase by adding the competitive agent and a surface active agent to compete for an antigen binding site, so that the recovery rate of the antigen in the water phase is greatly increased; the sample subjected to the demulsification is then purified by using ZIPTIP, so that impurities in the sample are effectively removed, and the purity of the sample is greatly improved; after that, the sample is qualitatively and quantitatively analyzed, so that the accuracy and reliability of detection results are improved.

Description

technical field [0001] The invention relates to the technical field of foot-and-mouth disease vaccine detection, in particular to a rapid qualitative and quantitative detection method for oil adjuvant vaccines. Background technique [0002] The existing vaccine quality standards stipulate that the efficacy test must use this animal for testing. Since the country implements a 100% enhanced immunization policy, it is difficult to select susceptible animals for testing, and animal challenge has high requirements for experimental facilities (BSL3 level laboratory), time-consuming (more than one month), and large capital expenditure. If the serum neutralization test is used to select susceptible animals, it is technically difficult to exclude non-susceptible animals with cellular immunity, and the problem of irregular test data often occurs in practice, which affects the accuracy of the test. Therefore, the quality inspection of foot-and-mouth disease vaccines, especially vaccin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/15G01N1/34G01N1/30
CPCG01N1/30G01N1/34G01N33/15
Inventor 马贵军俞爱敏石海芳刘健
Owner SHANGHAI SHEN LIAN BIOMEDICAL CORP
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