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General foot-and-mouth disease virus structural protein antibody and blocking ELISA detection reagent kit thereof

A technology of foot-and-mouth disease virus and structural protein, which is applied in the field of foot-and-mouth disease virus structural protein antibody and its blocking ELISA detection kit, which can solve the problems of FMDV antibody detection method obstacles, complex antigen structure, and scarcity of monoclonal antibodies, and eliminate non-specificity Reaction, good display of antigen structure, simple effect of reagent composition

Active Publication Date: 2020-01-03
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The antigenic structure of FMDV is complex, the antigenic sites and epitopes of different serotypes, genotypes and isolates are different, there are extensive antigenic variations, and there are also conserved antigenic structures that determine the characteristics of the virus species, and the development of broad-spectrum The FMDV antibody detection kit for different types needs to screen out broad-spectrum specific monoclonal antibodies that recognize the conserved epitopes between the types. Certain obstacles

Method used

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  • General foot-and-mouth disease virus structural protein antibody and blocking ELISA detection reagent kit thereof
  • General foot-and-mouth disease virus structural protein antibody and blocking ELISA detection reagent kit thereof
  • General foot-and-mouth disease virus structural protein antibody and blocking ELISA detection reagent kit thereof

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preparation example Construction

[0030] In the present invention, the preparation method of the monoclonal antibody E32 preferably includes the following steps: combining the coding sequences of the heavy chain variable region and the light chain variable region of the antibody E32 with the bovine IgG2 heavy chain and light chain respectively After the constant region sequences were connected, they were respectively inserted into the pcDNA3.4 eukaryotic expression vector, and the heavy chain and light chain expression plasmids were mixed in proportion to transfect CHO suspension culture cells, and the complete IgG2 subtype antibody was expressed, and the antibody was purified by affinity chromatography. For the preparation method of the antibody E32, please refer to the description in the patent document application number CN201810929067.1.

[0031]The present invention provides a general monoclonal antibody composition of foot-and-mouth disease virus structural protein, comprising said monoclonal antibody E32...

Embodiment 1

[0056] 1. Materials

[0057] Carbonate buffer (Na 2 CO 3 / NaHCO 3 ) and gelatin were purchased from SIGMA.

[0058] BCA protein concentration assay kit was purchased from Beyotime Company.

[0059] EZ-Link Plus Activated Peroxidase Kit was purchased from Thermo Company.

[0060] Horseradish peroxidase-labeled avidin was purchased as a commercial reagent (Genscript product).

[0061] Both FMDV broad-spectrum monoclonal antibodies F104 and E32 were developed and stored in our laboratory using the single B cell antibody preparation technology. The antibody preparation method is described in the patent document CN201810929067.1 (obtained patent number ZL201810929067.1).

[0062] The control serum and the inactivated antigens of type O and A viruses were prepared and preserved by the National Reference Laboratory for Foot-and-Mouth Disease.

[0063] The Asia1 type inactivated antigen of foot-and-mouth disease virus is the reserve inactivated antigen produced in 2015.

[0064...

Embodiment 2

[0093] 1. Serum samples

[0094] Clinically healthy animal serum samples include: 127 healthy bovine serum samples and 30 healthy pig serum samples from clinically healthy cattle, tested negative for O, A, Asia1 type structural protein antibodies and non-structural protein antibodies; 87 sheep serum samples The samples and 88 swine serum samples were from non-immune healthy sheep and pigs in epidemic-free areas. Serum samples from infected animals included: 121 bovine serum samples from 3 to 230 days after infection with type O or A FMDV challenge; 79 sheep serum samples from 7 days to 1 year after challenge with type O FMDV; After 7 days to 6 months, 80 pig serum samples were collected. Quality control sera include: O, A, Asia1 FMDV-infected pigs, cattle, and sheep sera, each of which were tested positive for non-structural protein antibodies.

[0095] 2. Determination of the optimal reaction conditions of the FMD virus structural protein antibody blocking ELISA detection m...

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Abstract

The invention provides a general foot-and-mouth disease virus structural protein antibody and a blocking ELISA detection reagent kit thereof, and belongs to the technical field of virus detection. Thegeneral foot-and-mouth disease virus structural protein antibody and the blocking ELISA detection reagent kit thereof comprise the following composition of an antigen coating elisa plate and a biotinmarker concentrating monoclonal antibody, wherein a monoclonal antibody in the biotin marker concentrating monoclonal antibody is a monoclonal antibody E32, and the antigen coating elisa plate is anFMDV antigen indirectly coated through a monoclonal antibody F104. The E32 antibody and the F104 antibody are specifically combined to between-genotype conservative epitope in FMDV structural proteinVP2 protein, the sensitivity and the specificity are high, the general foot-and-mouth disease virus structural protein antibody and the blocking ELISA detection reagent kit thereof are suitable for detection of structural protein antibodies after O, A and Asia1 type FMDV infected or inactivated vaccine immunity, and are a new method for monitoring FMD non-immune animal community infection conditions, and a complete set of identifying and diagnosing method is provided for a marker vaccine having FMDV structural protein VP2 epitope deletion.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a general-purpose foot-and-mouth disease virus structural protein antibody and a blocking ELISA detection kit. Background technique [0002] Foot-and-mouth disease (FMD) is a severe infectious disease caused by foot-and-mouth disease virus (FMDV) infection in cloven-hoofed animals. Its incidence rate is extremely high, and it has a serious impact on the breeding industry and international trade in livestock products. Therefore, countries attach great importance to the prevention and control of this disease. The key to preventing and controlling FMD is the application of efficient vaccines and accurate diagnostic methods. At present, the prevention and treatment of foot-and-mouth disease in my country mainly relies on inactivated vaccine immunization. Under this background, detection of FMDV nonstructural protein (NSP) antibody is an effective method to distin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/569
CPCC07K16/10C07K2317/56G01N33/56983G01N2333/09
Inventor 付元芳李坤卢曾军曹轶梅刘在新王省包慧芳李平花白兴文孙普马雪青张婧李志勇李冬陈应理
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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