Antibody m19 of O-type foot-and-mouth disease virus structural protein as well as preparation method and application of antibody m19

A foot-and-mouth disease virus and structural protein technology, which is applied in the field of antibodies to the O-type foot-and-mouth disease virus structural protein, can solve problems such as complex antigen structure, scarcity of monoclonal antibodies, and obstacles to detection methods, and achieve the effect of improving sensitivity and specificity

Active Publication Date: 2022-04-12
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex structure of the FMDV antigen, the development of a broad-spectrum and specific antibody detection kit for type O foot-and-mouth disease virus requires screening for specific monoclonal antibodies tha

Method used

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  • Antibody m19 of O-type foot-and-mouth disease virus structural protein as well as preparation method and application of antibody m19
  • Antibody m19 of O-type foot-and-mouth disease virus structural protein as well as preparation method and application of antibody m19
  • Antibody m19 of O-type foot-and-mouth disease virus structural protein as well as preparation method and application of antibody m19

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 monoclonal antibody m19

[0038] 1. Preparation of hybridoma cells

[0039] 1.1 Immunization of mice

[0040] 5-6 week-old Balb / c mice were immunized with type O foot-and-mouth disease virus (O / Mya98) 146s as the antigen. The immunization method was multi-point subcutaneous injection on the back, and the injection volume was 100 μg per mouse. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. The interval between each immunization was two weeks. The mouse serum was collected one week after the third immunization to detect the antibody titer. When the antibody titer is not low At 1:1440, impulse immunization was performed, that is, the antigen was injected intraperitoneally, and three days after the impulse immunization, the spleen of the mouse was taken for fusion with myeloma cells.

[0041] 1.2 Cell Fusion

[0042] Spleen cells of immunized m...

Embodiment 2

[0061] Embodiment 2 monoclonal antibody m19 reacts with different lineage foot-and-mouth disease virus antigens

[0062] 1. Indirect immunofluorescence test:

[0063] Representative strains of different lineages of type O foot-and-mouth disease (O / ZK / 93, O / Mya / 98 and O / HK / 93 strains) and representative strains of different lineages of type A foot-and-mouth disease (A / AF72, A / GDMM / 2013 and A / WH / CHA / 09) (both strains are preserved by the National Reference Laboratory for Foot-and-Mouth Disease) to infect BHK-21 cells, and the reaction between the prepared monoclonal antibody m19 and the FMDV antigen was determined by the fluorescence intensity under the fluorescence microscope. The specific operation steps are as follows:

[0064] (1) Cell culture: spread BHK-21 cells evenly in a 12-well cell culture plate, and inoculate the cells after the cells grow to about 70%;

[0065] (2) Inoculation: inoculate 3 lineages of type O virus strains and 3 lineages of type A virus strains in...

Embodiment 3

[0072] Example 3 Establishment and application of solid-phase competitive ELISA detection method for O-type foot-and-mouth disease virus structural protein

[0073] 1. Determination of the optimal reaction conditions for solid-phase competitive ELISA detection method of O-type foot-and-mouth disease virus structural protein

[0074] 1.1 Determination of the optimal concentration of rabbit antiserum and O-type FMD virus inactivated antigen for structural protein of O-type foot-and-mouth disease virus:

[0075] The optimal concentration of rabbit antiserum and inactivated antigen of type O foot-and-mouth disease virus (preserved in our laboratory) was determined by square array titration. First, use Na 2 CO 3 / NaHCO 3 The coating solution (pH=9.6) was serially diluted from 1:250 in the dilution plate to the O-type foot-and-mouth disease virus structural protein rabbit antiserum (horizontal), and correspondingly transferred into the enzyme plate (50 μ L / well), 4 Overnight at ...

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Abstract

The invention relates to the technical field of biology, in particular to an O-type foot and mouth disease virus structural protein antibody, a preparation method and application. The O-type foot-and-mouth disease virus 146s is used as an immunogen to immunize a Balb/c mouse, a hybridoma cell line capable of efficiently secreting the monoclonal antibody is obtained through cell fusion, and the mouse monoclonal antibody m19 is obtained. Indirect immunofluorescence tests show that the monoclonal antibody m19 has good reactivity to O-type strains of different lineages and does not react with A-type strains of different lineages. The O-type foot-and-mouth disease virus structural protein solid-phase competitive ELISA detection kit is established by utilizing the monoclonal antibody m19 and O-type structural protein rabbit antiserum, compared with an existing antibody, the sensitivity and specificity of a detection result are remarkably improved, the kit is suitable for detection of structural protein antibodies after O-type foot-and-mouth disease infection or inactivated vaccine immunization, and meanwhile, the kit can be used for detecting the structural protein antibodies after O-type foot-and-mouth disease infection or inactivated vaccine immunization. And the method is a novel method for monitoring the infection condition of the non-immune area of the foot-and-mouth disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody m19 to the structural protein of O-type foot-and-mouth disease virus, a preparation method and application. Background technique [0002] Foot-and-mouth disease is an infectious disease common to cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV) infection. FMDV is a member of the genus Aphthavirus of the picornaviridae family, and currently has O, A, C, SAT1, SAT2, SAT3 (namely, South African foot-and-mouth disease virus types 1, 2, and 3) and Asia1 (Asian type 1). ) 7 serotypes. There is almost no immune protection between the various types, and animals infected with one type of FMD virus can still be infected with another type of FMD virus. [0003] Because the antigenicity of different subtypes within the same serotype is different in different degrees, the degree of serological cross-reactivity is also different, which makes the diagnosis and contr...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N5/20G01N33/569G01N33/577G01N33/543G01N33/58
Inventor 郑海学杨洋秦晓东茹毅张伟杨帆曹伟军朱紫祥卢炳州赵东梅任蕊芳吴秀萍郝荣增刘华南李亚军吴国华李丹田宏张克山毛箬青
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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