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A Quantitative Method for Foot-and-Mouth Disease Antigen

A foot-and-mouth disease antigen technology, applied in the field of quantitative analysis of foot-and-mouth disease antigens, can solve the problems affecting the reliability of the measurement results, low virus concentration, and low accuracy of the results, so as to achieve a wide range of applications and accuracy, improve detection efficiency, and shorten processing time Effect

Active Publication Date: 2020-02-21
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%, and it cannot be used for accurate determination of FMD antigen content under different solution conditions.
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.
[0004] 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
[0005] In addition, in the pretreatment process of antigen samples, conventional PEG precipitation method is usually used to precipitate virus samples, but because the virus concentration of the sample to be tested is too low (usually not more than 20 μg / mL), the precipitation cannot be obtained in a short time, Usually it needs to stand overnight to carry out the follow-up steps, which cannot meet the requirements of high efficiency, fastness and accuracy. However, the method of conventional ultrafiltration concentration followed by PEG precipitation will cause the adsorption of viruses on the membrane, and the resulting loss cannot be determined. Reliability of assay results

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 foot-and-mouth disease antigen standard preparation

[0050] 1. Materials

[0051] (1) Test sample: Type O foot-and-mouth disease inactivated vaccine

[0052] (2) Instruments and reagents: centrifuge, PEG6000, n-butanol, chloroform

[0053] 2. Experimental steps

[0054] (1) Inactivated vaccine demulsification: Add n-butanol to the inactivated vaccine according to the volume ratio of seedlings:n-butanol=9:1, mix well, 6000g, 4°C, centrifuge for 8min, suck out the lower aqueous phase with a syringe .

[0055] (2) Chloroform treatment of the water phase: add 10% chloroform to the water phase, shake and mix, centrifuge at 6000g, 4°C for 5 minutes, and take the supernatant.

[0056] (3) PEG6000 concentrated virus: add PEG to the aqueous phase treated with chloroform to a final concentration of 7%, mix well, let stand at 4°C for 18 hours, then centrifuge at 8000g for 50 minutes at 4°C, discard the supernatant. Resuspend the pellet in PBS to 1 / 10 of its ori...

Embodiment 2

[0059] The standard curve drawing of embodiment 2 high performance liquid chromatography detection 146S

[0060] (1) Dilute the virus standard sample prepared in Example 1 with PBS to 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL.

[0061] (2) The high performance liquid chromatography system of Agilent Company and the Agilent Bio SEC-5 chromatographic column were used for HPLC determination of 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.

[0062] (3) The 146S antigen is analyzed by high performance liquid chromatography as a single absorption peak, and the correlation curve of the standard product is obtained by correlating the antigen concentration and the integrated area of ​​high performance liquid chromatography

[0063] C=0.0542PA+0.494

[0064] Among them, C is the concentration of 146S, the unit is μg / mL, PA is the ...

Embodiment 3

[0065] The impact of embodiment 3 sample pretreatment on determination

[0066] (1) Dilute the virus standard sample prepared in Example 1 with PBS to 2 μg / mL, 10 μg / mL, 40 μg / mL.

[0067] (2) Add PEG to 6% of the standard samples with different concentrations, mix well, and centrifuge at 10000g for 15 minutes at 4°C. No precipitation occurred.

[0068] (3) Add BSA to the standard samples with different concentrations to a final concentration of 2 mg / mL, then add PEG to a final concentration of 6%, centrifuge at 10,000 g for 15 minutes at 4° C., and redissolve the precipitate in PBS to its original volume. At the same time, the sample treated with the conventional PEG precipitation method without adding BSA was used as a control sample. The control sample was added with PEG to a final concentration of 6%, left at 4°C for 18 hours, centrifuged at 6000g for 50 minutes, and the obtained precipitate was redissolved in PBS to the original volume.

[0069] (4) Using a high perfo...

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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: A, preparing standard foot-and-mouth disease virus antigen samples with different concentrations and determining the standard foot-and-mouth disease virus antigen samples by using HPLC to obtain the correlation formula between standard antigen concentrations and the absorption peak areas of antigens; and B, pretreating sample to be measured, then determining the antigen absorption peak area of the sample to be measured by using HPLC, and then calculating the antigenconcentration of the sample to be measured according to the correlation formula in the step A. The method of the invention solves the dilemma that HPLC cannot be widely used in the quantitation of foot-and-mouth disease viruses and allows HPLC to be more extensively and accurately applicable. Moreover, the pretreatment of addition of miscellaneous proteins is additionally performed in the invention, so the treatment time of the sample can be greatly shortened and detection accuracy is improved.

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] At present, the quantitative analysis method of FMD is mainly based on sucrose gradient density, and the detection procedure is complicated, which is not suitable for rapid detection. The development and uti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 张新廉马贵军张丽俞爱敏姬明放石海芳刘自立郁莉
Owner SHANGHAI SHEN LIAN BIOMEDICAL CORP