Method for quantification of 146S content in foot-and-mouth disease antigen by using liquid chromatography detection system

A liquid chromatography and detection system technology, applied in the field of biological detection, can solve the problems of insufficient removal of impurity proteins and nucleic acids, complex sample processing processes, and lack of detection patterns, and achieve accurate and fast detection, wide versatility, and quantitative results. accurate effect

Active Publication Date: 2014-05-07
内蒙古必威安泰生物科技有限公司
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Problems solved by technology

Chinese Patent Publication No. CN101655452A, the publication date is February 24, 2010, and the invention name is "Method for Quantitative Detection of Foot-and-Mouth Disease Antigen 146S Content with Sucrose Density Gradient Ultraviolet Light". The method for quantitatively detecting the content of FMD antigen 146S has the disadvantages that firstly, the pre-sample treatment process of the invention is complicated, and the impurity protein and nucleic acid in the sample are not fully removed, which affects the accuracy of the next step of quantification; secondly, the calculation formula requires The parameters are complicated, there is no detection spectrum to support it, and the continuous flow ultraviolet photometer at home and abroad is difficult to meet the required parameters of the formula, and the application is limited

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  • Method for quantification of 146S content in foot-and-mouth disease antigen by using liquid chromatography detection system
  • Method for quantification of 146S content in foot-and-mouth disease antigen by using liquid chromatography detection system
  • Method for quantification of 146S content in foot-and-mouth disease antigen by using liquid chromatography detection system

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Experimental program
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Effect test

Embodiment 1

[0049] Quantitative detection of 146S antigen content in the sample of embodiment 1

[0050] 1. Materials

[0051] 1.1 Test samples: Bovine Asia I / JSL / GSZY / 06 virus, bovine O-type ONXC / 92 virus, and pig O-type O / ZK / 93-08 virus were purchased from Lanzhou Veterinary Research Institute.

[0052] 1.2 Instruments and reagents: Liquid chromatography detection system was purchased from Labtech, sucrose and protamine sulfate were purchased from sigma, and PEG6000 was purchased from AMRESCO.

[0053] The phase chromatography detection system is composed of a liquid chromatography detector, a chromatography workstation and a constant flow pump. The model of the liquid chromatography detector is UV600 ultraviolet-visible variable wavelength detector, and the model of the constant flow pump is P600 high pressure constant flow pump. The light source of the liquid chromatography detector is a deuterium lamp, the spectral bandwidth is 8nm, the wavelength accuracy is ±0.2nm, and the wavelen...

Embodiment 2

[0088] The 146S content recorded by the method of the present invention is based on the preparation of the vaccine, and the corresponding 146S content in the prepared bivalent vaccine is equal to the foot-and-mouth disease O type and the Asia I type antigen liquid. (Foot-and-mouth disease O type, Asia I type bivalent inactivated vaccine manufacture and inspection trial procedure " carry out, result is as shown in table 5, 146S content described in the table is foot-and-mouth disease O type or Asia I type antigen 146S content, the two are equal, For example: in bovine O type ONXC / 92-Asia I type bivalent inactivated antigen solution 3, the 146S content of FMD O type is 17.17 μg / ml, and the Asian I type 146S content is also 17.17 μg / ml, each half of the protection (PD 50 ) value is 15.59 for Asian I type and 12.51 for O type.

[0089] Table 5 The half protection value (PD) corresponding to the antigen solution with different 146S content (μg / ml) 50 )

[0090]

[0091]

[...

Embodiment 3

[0094] The optimization of embodiment 3 foot-and-mouth disease virus purification concentration conditions

[0095] 1 material

[0096] 1.1 Vaccine strain: Asia I / JSL / GSZY / 06 live virus was purchased from Lanzhou Veterinary Research Institute.

[0097] 1.2 Chemical reagents: protamine sulfate, a product of sigma; PEG6000 was purchased from AMRESCO.

[0098] 2 methods

[0099] According to the literature data and the results of preliminary experiments, the main factors affecting the purification and concentration of FMD were selected: the concentration, temperature and time of protamine sulfate, the concentration and stirring time of PEG6000 were optimized.

[0100] 2.1 Optimization of concentration of protamine sulfate

[0101] Take the Asia I / JSL / GSZY / 06 virus antigen of suspension culture in large-scale production, add protamine sulfate to different final concentrations, successively 0.5mg / ml, 1.0mg / ml, 1.5mg / ml, 2.0mg / ml and Five concentration gradients of 2.5mg / ml, aft...

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Abstract

The present invention discloses a method for quantification of 146s content in foot-and-mouth disease antigen by using a liquid chromatography detection system. The method comprises: adopting protamine sulfate to purify virus, adopting PEG6000 to concentrate the virus, carrying out sucrose density gradient ultracentrifugation on the concentrated virus, adopting a liquid chromatography detection system to detect an absorption peak of the centrifugated sample at a wavelength of 259 nm, and calculating 146S content according to the following formula: C=FRS / 76Wb. The method can be used for determination of the 146S content in various types of foot-and-mouth disease antigens. With quantification of the 146S content in the foot-and-mouth disease antigen, foot-and-mouth disease vaccine preparation can be guided, vaccine efficacy can be indirectly evaluated, and major guiding significances are provided for enhancing quality of the foot-and-mouth disease vaccine in our country.

Description

technical field [0001] The invention relates to a detection method for quantifying the 146S content in the foot-and-mouth disease antigen, in particular to a method for quantifying the 146S content in the foot-and-mouth disease antigen in a sucrose density gradient centrifuged sample using a liquid chromatography detection system. It belongs to the field of biological detection. Background technique [0002] In the foot-and-mouth disease virus sample, there are 4 kinds of virus particles with different sizes, the largest one is the complete virus particle with a sedimentation coefficient of 146S, the slightly smaller particle is an empty capsid with a sedimentation coefficient of 75S, and the other is a capsid protein The subunit has a sedimentation coefficient of 12S, and the smallest one is VIA antigen with a sedimentation coefficient of 3-4.5S. In animal immunity, the FMD 146S antigen is mainly related to stimulating the body to produce antibodies and sensitizing lymphoc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/74
Inventor 倪春霞徐树兰李少英赵炳武辛俊宝张澍王钦富武华
Owner 内蒙古必威安泰生物科技有限公司
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