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Method for quantifying foot-and-mouth disease virus antigen

A foot-and-mouth disease and antigen technology, applied in the field of quantitative analysis of foot-and-mouth disease antigens, can solve the problems of low accuracy of results, deviation of results, poor pressure resistance of glucan gel, etc. The effect of low overall cost

Active Publication Date: 2019-02-26
SHANGHAI SHEN LIAN BIOMEDICAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The deviation of the result can reach 33%, so it cannot be used for accurate determination of FMD antigen content
During the preparation of FMD antigen, the conductance of the solution will change with the processing flow, and the conductance of the product batch will also deviate, which leads to the problem that the accuracy of the results is not high in actual operation.
And the inventor used three brand new TSK-G4000SW in the early stage XL In the repeated detection of the demulsification vaccine by the chromatographic column, its service life was 312 times, 241 times, and 449 times respectively. Adjuvant contamination, difficult to clean

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  • Method for quantifying foot-and-mouth disease virus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 foot-and-mouth disease antigen standard preparation

[0041] 1. Materials

[0042] (1) Test sample: Type O foot-and-mouth disease inactivated vaccine

[0043] (2) Instruments and reagents: centrifuge, PEG6000, n-butanol, chloroform

[0044] 2. Experimental steps

[0045] (1) Demulsification of inactivated vaccines: add n-butanol to the inactivated vaccine according to seedling:n-butanol=9:1, mix well, centrifuge at 6000g, 4°C for 8min, and suck out the lower aqueous phase with a syringe.

[0046] (2) Chloroform treatment of the aqueous phase: add 10% chloroform to the aqueous phase, shake and mix, centrifuge at 6000 g at 4° C. for 5 min, and take the supernatant.

[0047] (3) PEG6000 concentrated virus: add PEG to the aqueous phase treated with chloroform to a final concentration of 7%, let stand at 4°C for 18 hours, then centrifuge at 8000g for 50min at 4°C, discard the supernatant. Resuspend the pellet in PBS to 1 / 10 of its original volume.

[0048...

Embodiment 2

[0050] The standard curve drawing of embodiment 2 high performance liquid chromatography detection 146S

[0051] (1) Dilute the virus standard samples prepared in Example 1 to 2 μg / mL, 5 μg / mL, 10 μg / mL, 20μg / mL, 40μg / mL, a series of different conductivities (87.2mS / cm, 72.9mS / cm, 49.9mS / cm, 26.0mS / cm, 15.0mS / cm, 4.7mS / cm) were obtained at different concentrations Virus standard samples.

[0052] (2) Agilent's high performance liquid chromatography system and Agilent Bio SEC-5 chromatographic column were used to measure the above standard samples. The mobile phase contains 0.1M sodium sulfate phosphate buffer (pH7.2), the flow rate is 1ml / min, the injection volume is 100μl, and the detection wavelength is 259nm.

[0053] (3) The 146S antigen is analyzed as a single absorption peak by high performance liquid chromatography, and the correlation curve of the standard product is obtained by correlating the antigen concentration and the high performance liquid chromatography i...

Embodiment 3

[0056] Example 3 High-performance liquid chromatography detects high-salt and low-salt samples

[0057] (1) Demulsification of inactivated vaccines: Add n-butanol to the inactivated vaccine according to the ratio of seedlings:n-butanol=9:1, mix well, centrifuge at 6000g, 4°C for 8min, and suck out the lower aqueous phase with a syringe.

[0058] (2) Chloroform treatment of the aqueous phase: add 10% chloroform to the aqueous phase, shake and mix, centrifuge at 6000 g at 4° C. for 5 min, and take the supernatant.

[0059] (3) The sample concentration of the demulsified aqueous phase after chloroform treatment was determined to be 4.4 μg / mL by sucrose density gradient centrifugation. Add equal volumes of PBS buffer solution with different NaCl concentrations to the chloroform-treated aqueous phase prepared in step (2) to dilute to a virus concentration of 2.2 μg / mL to obtain mixed solution 1 (NaCl concentration 500 mM) and mixed solution 2 (NaCl concentration 50 mM ).

[006...

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Abstract

The invention provides a method for quantifying a foot-and-mouth disease virus antigen. The method comprises the following steps: preparing standard foot-and-mouth disease virus antigen solution samples with different conductivities and different antigen concentrations, determining the HPLC absorption peak area of each standard solution sample, and acquiring a correlation formula between foot-and-mouth disease virus antigen concentrations and the HPLC absorption peak areas and the conductivities; determining the conductivity and the HPLC absorption peak area of a foot-and-mouth disease virusantigen sample to be measured; calculating the concentration of the foot-and-mouth disease virus antigen sample to be measured based on the determined conductivity and the determined HPLC absorptionpeak area according to the correlation formula so as to obtain the concentration of the foot-and-mouth disease virus antigen. The method of the invention solves the dilemma that HPLC cannot be widelyused in the quantitation of foot-and-mouth disease viruses and allows HPLC to be more extensively and accurately applicable.

Description

technical field [0001] The invention relates to the technical field of quantitative analysis of foot-and-mouth disease antigens, in particular to a quantitative method for foot-and-mouth disease antigens. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious animal disease that can spread quickly and over long distances caused by foot-and-mouth disease virus (FMDV). There are as many as 70 species of susceptible animals and wild artiodactyls. At present, vaccine immunization is one of the most important and successful measures in the prevention and control of foot-and-mouth disease, among which conventional inactivated vaccines are the most important vaccines for the prevention and control of foot-and-mouth disease. [0003] The quantitative analysis method of FMD antigen 146S is currently based on sucrose gradient density, the detection procedure is complicated, and it is not suitable for rapid detection. The development and ut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 马贵军张震石海芳张丽刘自立张新廉姬明放俞爱敏
Owner SHANGHAI SHEN LIAN BIOMEDICAL CORP