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Qualitative and quantitative foot and mouth disease virus antigen detection method

A detection method and quantitative detection technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inability to detect qualitatively, poor stability, and limited detection concentration range, so as to improve the range of detection capabilities, improve stability, and have no background interference effect

Inactive Publication Date: 2015-11-11
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the detection of inactivated antigen of foot-and-mouth disease virus mainly relies on the quantitative detection of 146S virus by the sucrose density gradient method. The essence of 146S detection is the complete virus, and the complete virus sample is easily broken during processing, which leads to large detection errors; The relative physical content of FMDV antigen, which lacks specificity and cannot be qualitatively detected
[0003] The existing ELISA method for detecting the content of FMD virus antigen has the defect of background interference such as cell debris, which is prone to false positive and false negative results, and has poor stability
[0004] The existing immunoblotting method can only detect antigens qualitatively but not quantitatively, and the detection concentration range is very limited. The existing immunoblotting methods have significant defects and have not been applied to the quantitative detection of FMD antigens

Method used

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  • Qualitative and quantitative foot and mouth disease virus antigen detection method
  • Qualitative and quantitative foot and mouth disease virus antigen detection method
  • Qualitative and quantitative foot and mouth disease virus antigen detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 immunoblotting luminescence combined with gray value method to determine foot-and-mouth disease virus antigen-specific band

[0039] Include the following steps:

[0040] (1) Processing virus antigen, SDS-PAGE electrophoresis:

[0041] Sample pretreatment: Centrifuge 10 mL of the sample to be tested at 10,000 rpm for 10 minutes, take the supernatant, add it to a 100KD regenerated fiber membrane ultrafiltration centrifuge tube and centrifuge at 4,000 rpm to obtain the pretreated sample to be tested .

[0042] Processing samples: take the pretreated foot-and-mouth disease test samples, negative control, negative control and loading buffer, mix them evenly, treat them in a boiling water bath for 5 minutes, and cool them down to 25°C for later use. Prepare 12% separating gel and 5% stacking gel. The amount of sample loaded in each well was 20 μL. After loading the sample, electrophoresis was performed, and the voltage was controlled at 60 volts. After 30 min...

Embodiment 2

[0050] Example 2 Establishment of immunoblotting luminescence combined with gray value detection method for foot-and-mouth disease virus antigen

[0051] (1) Processing virus antigen, SDS-PAGE electrophoresis:

[0052] Sample pretreatment: Mix 10mL of the sample to be tested with 0.2925gNaCl to make the final concentration of NaCl 0.5mol / L, mix with 0.8g polyethylene glycol to obtain a mixture; let the mixture stand at 4°C for 12 hours and then rotate it at 10000 rpm Centrifuge at a speed of 10 minutes for 30 minutes, collect the precipitate, and add PBS to the precipitate to fully dissolve to obtain the pretreated foot-and-mouth disease sample to be tested.

[0053] Processed samples: Take the pretreated foot-and-mouth disease test samples, negative control samples and sample buffer, mix them evenly, treat them in a boiling water bath for 5 minutes, cool to room temperature and set aside. Prepare 12% separating gel and 5% stacking gel. For sample loading electrophoresis, th...

Embodiment 3

[0065] Example 3 Western blot luminescence combined with gray value method for qualitative detection of foot-and-mouth disease virus antigen

[0066] Include the following steps:

[0067] (1) Processing virus antigen, SDS-PAGE electrophoresis:

[0068] Sample pretreatment: Mix 10mL of the sample to be tested with 0.2925gNaCl to make the final concentration of NaCl 0.5mol / L, mix with 0.8g polyethylene glycol to obtain a mixture; let the mixture stand at 4°C for 12 hours and then rotate it at 10000 rpm Centrifuge at a speed of 10 minutes for 30 minutes, collect the precipitate, and add PBS to the precipitate to fully dissolve to obtain the pretreated foot-and-mouth disease sample to be tested.

[0069] Centrifuge the above 10 mL sample to be tested at a speed of 10000 rpm for 10 minutes, take the supernatant, add it to a 100KD regenerated fiber membrane ultrafiltration centrifuge tube and centrifuge at a speed of 4000 rpm to obtain a pretreated sample to be tested.

[0070] Pr...

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Abstract

The invention provides a western blotting luminescence and grey level combined foot and mouth disease virus antigen detection method. The method comprises the following steps: (1) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis); (2) blotting transfer; (3) immune reaction; (4) developing or luminous reaction; (5) judgment of the property of an antigen to be detected according to a specific antigen band; (6) calculation of the content according to the luminous grey level of the antigen. The method can not only qualitatively but also quantitatively detect the foot and mouth disease virus antigen, has the characteristics of being safe, quick, simple, convenient, accurate and specific, realizes specific quantitative detection of various foot and mouth disease virus antigens, originates the mode of adopting western blotting luminescence to quantitatively detect the foot and mouth disease virus antigen, and has an important guiding meaning for vaccine production.

Description

technical field [0001] The invention relates to the technical field of biological product detection, in particular to a method for qualitative and quantitative detection of foot-and-mouth disease virus antigen. Background technique [0002] At present, the detection of inactivated antigen of foot-and-mouth disease virus mainly relies on the quantitative detection of 146S virus by the sucrose density gradient method. The essence of 146S detection is the complete virus, and the complete virus sample is easily broken during processing, which leads to large detection errors; The relative physical content of FMDV antigen lacks specificity and cannot be qualitatively detected. [0003] The existing ELISA method for detecting the content of FMD virus antigen has the defects of background interference such as cell debris, which is prone to false positive and false negative results, and has poor stability. [0004] The existing immunoblotting method can only detect antigens qualitat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/545
CPCG01N33/577G01N33/545G01N33/56983
Inventor 潘春刚杨小蓉黄文强
Owner CHINA ANIMAL HUSBANDRY IND
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