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Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S

A foot-and-mouth disease vaccine and antigen technology, applied in the field of quantitative analysis of the foot-and-mouth disease vaccine antigen 146S, can solve the problems of undetectable antigens, and achieve the effects of improving quality control, accurate test results, and wide practicability

Inactive Publication Date: 2015-05-20
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent CN 102998378 B discloses a method for quantifying the content of 146S in FMD antigen by using a liquid chromatography system. However, since this method only uses the detection system of the liquid chromatography system, it can only detect the purified 146S and cannot Detection of Antigens Containing Impurities

Method used

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  • Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S
  • Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S
  • Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 foot-and-mouth disease antigen 146S standard item

[0030] 1. Materials

[0031] (1) Test sample: Type O foot-and-mouth disease inactivated virus liquid comes from Lanzhou Veterinary Research Institute

[0032] (2) Instruments and reagents: The ultracentrifuge was Sorvall WX Ultra, the rotor was Surespin 630 horizontal rotor, both were purchased from Thermo Company, the sucrose was purchased from Xilong Chemical Co., Ltd., and the ultrafiltration concentration tube was purchased from Millipore Company

[0033] 2. Experimental steps:

[0034] (1) Concentrate virus by ultrafiltration: Use an ultrafiltration concentration tube with a molecular weight cut-off of 30kDa, centrifuge at 5000g for 25min at 4°C, and concentrate 100ml of virus liquid to 2ml.

[0035] (2) Sucrose density gradient centrifugation: prepare 35ml of 15%-45% (w / v) uniform sucrose density gradient solution in a 38ml ultracentrifuge tube, take 2ml of the virus concentrate ...

Embodiment 2

[0043] Example 2 Sensitivity detection of 146S antigen detected by high performance liquid chromatography and standard curve drawing

[0044] The high performance liquid chromatography system was purchased from Agilent, and the size exclusion high performance liquid chromatography column TSK G4000 SW XL Purchased from Tosohaas Company,

[0045] (1) Dilution of pure 146S antigen: the pure 146S antigen obtained in Example 1 was diluted with 20mM phosphate buffer (pH7.0-pH7.5) by 1, 2, 4, 10, 30, 150, 250 times;

[0046] (2) Size-exclusion high-performance liquid chromatography analysis: a high-performance liquid chromatography system and a size-exclusion high-performance liquid chromatography column were used for analysis. Mobile phase: Phosphate buffer (pH7.2) containing 0.1M sodium sulfate, flow rate: 0.6ml / min, column: TSK G4000 SW XL , the injection volume is 100μl, and the detection wavelength is 259nm.

[0047] (3) Establish a standard curve: after HPLC gel filtration ...

Embodiment 3

[0050] Example 3 Determination of 146S antigen in different serotypes of foot-and-mouth disease vaccine finished products.

[0051] With the method of the invention, the content of 146S antigen in the finished products of foot-and-mouth disease vaccines of different serotypes can be determined. The following vaccines were used as samples for determination: a. O-type and Asian type I bivalent inactivated vaccine (ONXC strain + JSL strain) b. O-type inactivated porcine foot-and-mouth disease vaccine (O / Mya98 / xj / 2010 strain + O / GX / 09-7 strain) c. swine foot-and-mouth disease type O inactivated vaccine (O / GX / 09-7 strain+O / XJ / 10-11 strain).

[0052] (1) Pretreatment of finished vaccine: Since adjuvants are added to the finished vaccine, demulsification is required before measurement. Add n-butanol according to 1 / 9 of the volume of the vaccine stock solution, mix well, let stand at 4°C for 1 hour, centrifuge at 5000rpm for 2 minutes, and collect the bottom solution containing the ...

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Abstract

The invention discloses a method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S. A size exclusion high-efficiency liquid-phase chromatographic column in a molecular weight separation range of 2*10<4> to 1*10<7>Da is adopted to carry out the chromatographic separation on a detected sample on a high-efficiency liquid-phase chromatography. The operation pressure of the chromatography is 1.0MPa to 2.5MPa, the flow rate in the chromatographic column is 0.5 to 1.0 ml / min, a flow phase is phosphate buffer (pH 7.0 to 7.5) containing 0.1M sodium sulfate, and the column temperature is 15 to 25 DEG C. An ultraviolet and laser detector is used for detecting an optical signal of effluent at an outlet of the size exclusion high-efficiency liquid-phase chromatographic column, and a peak area of a sample can be analyzed by virtue of a computer software system of the high-efficiency liquid-phase chromatography. A standard curve of the absorption peak area and 146S concentration is established by virtue of a relation between the ultraviolet absorption peak and the concentration of different 146S standard products of different concentrations. Chromatograph is carried out on the detected sample through the size exclusion high-efficiency liquid-phase chromatographic column. The ultraviolet absorption peak area is measured, and the concentration of 146S in the detected sample can be acquired according to the standard curve.

Description

technical field [0001] The invention relates to the quantitative analysis of the foot-and-mouth disease vaccine antigen 146S, in particular to a method for quantifying the content of the foot-and-mouth disease antigen 146S in the virus liquid by using a size-exclusion high-performance liquid chromatography column, a high-performance liquid chromatography system and a method. Background technique [0002] In the foot-and-mouth disease virus sample, it is mainly intact virus particles that are related to stimulating the body to produce antibodies and sensitizing lymphocytes. Its sedimentation coefficient is 146S, which is considered to have a parallel relationship with the efficiency of the foot-and-mouth disease vaccine. Therefore, the content of 146S is an important index to evaluate the quality of foot-and-mouth disease vaccine. 146S has weak stability, for example, it will be cleaved under mild heating or pH below 6.0, and the immunogenicity of the cleaved product is extre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 苏志国张松平马光辉杨延丽张焱
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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