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Monoclonal Antibody for Detecting Antigen of Foot-and-Mouth Disease Type O Guangxi Strain and Its Application

一种单克隆抗体、口蹄疫的技术，应用在口蹄疫O型广西株抗原的特异,准确检测,酶联免疫检测试剂盒,快速领域，能够解决无法区分、不能口蹄疫病毒区分定量检测、口蹄疫146S抗原定量检测等问题

Active Publication Date: 2021-02-09
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently on the market, most of the ELISA kits used to detect FMD antigens use polyclonal antibodies, such as the O-type FMD 146S antigen quantitative ELISA detection kit developed by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. This kit patent (application number CN103076451A) shows Type O foot-and-mouth disease rabbit antiserum was used as the capture antibody, and horseradish peroxidase-labeled guinea pig antiserum was used as the detection antibody. Type O could be distinguished from type A and Asian type 1, but different strains of type O could not be distinguished Therefore, it is impossible to quantitatively detect the FMD 146S antigen of each strain in the multivalent vaccine. The homology between them is very high, which can exceed 85%. There may be only a few tens or even a few amino acid sequence differences, and the same antigenic epitope may exist. Therefore, the double antibody sandwich ELISA antigen detection reagent established based on polyclonal antibodies The box cannot distinguish and quantitatively detect the foot-and-mouth disease virus of different strains of the same serotype

Method used

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  • Monoclonal Antibody for Detecting Antigen of Foot-and-Mouth Disease Type O Guangxi Strain and Its Application
  • Monoclonal Antibody for Detecting Antigen of Foot-and-Mouth Disease Type O Guangxi Strain and Its Application
  • Monoclonal Antibody for Detecting Antigen of Foot-and-Mouth Disease Type O Guangxi Strain and Its Application

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Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1, the screening of the specific hybridoma cell line of foot-and-mouth disease O type Guangxi strain (O / GX / 09-7 strain) monoclonal antibody

[0057] Include the following steps:

[0058] 1) Obtain the 146S antigen of FMD O-type Guangxi strain (O / GX / 09-7 strain) (provided by China Animal Husbandry Co., Ltd. Lanzhou Biopharmaceutical Factory) by using the sucrose density gradient method, and adjust the concentration of the antigen to 10 μg / ml as Immunogen use;

[0059] 2) The immunized animals were BALB / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), which were immunized four times in a row with an interval of 14 days. The immunization method of intraperitoneal injection, each mouse was injected with 10 μg of 146S antigen;

[0060] 3) Seven days after the last immunization, the tail blood of the mice was taken to separate the serum, and the serum was detected by indirect ELISA. After the titer was >1:50000, the splenocy...

Embodiment 2

[0063] Embodiment 2, pairing test of foot-and-mouth disease O type Guangxi strain (O / GX / 09-7 strain) monoclonal purified antibody

[0064] 1) Prepare coated plate with specific monoclonal antibody of foot-and-mouth disease O-type Guangxi strain (O / GX / 09-7 strain)

[0065]Dilute the purified specific monoclonal antibody with pH 9.6 carbonate solution into 0.5μg / ml, 1μg / ml, 2μg / ml, 4μg / ml coating working solution, then add to 96-well polystyrene enzyme Linked reaction plate, 100μl / well, placed at 2-8°C for 8-12 hours to fully combine the specific monoclonal antibody with the enzyme-linked reaction plate, and then add 10mg / ml bovine serum albumin pH7.4 at 300μl / well PBS buffer solution, 37°C blocking treatment for 2-3 hours, after drying, the enzyme-linked reaction plate was dried and stored in a sealed manner at 2-8°C.

[0066] 2) Preparation of horseradish peroxidase-labeled foot-and-mouth disease type O Guangxi strain (O / GX / 09-7 strain) specific monoclonal antibody

[0067] ...

Embodiment 3

[0083] Embodiment 3, the establishment of the gene sequencing of the specific hybridoma cell line of the monoclonal antibody of foot-and-mouth disease O type Guangxi strain (O / GX / 09-7 strain) and monoclonal antibody recombinant expression system

[0084] Include the following steps:

[0085] 1) Total RNA extraction, reverse transcription, PCR and sequence determination of specific positive clone hybridoma cell lines:

[0086] ①Total RNA extraction: take 250 μl of hybridoma cell suspension, add 750 μl of Trizol, mix up and down, add 200 μl of chloroform, mix well, and centrifuge at 12000 rpm at 4 °C for 15 min. Aspirate the supernatant into a new 1.5ml EP tube, add 600μl of isopropanol, mix well and centrifuge for 10min. The isopropanol was discarded, washed with 75% DEPC ethanol, and centrifuged. Discard the ethanol, dry it, and dissolve the RNA with 20 μl RNase-free water.

[0087] ②Reverse transcription: use the Invitrogen reverse transcription kit to carry out reverse tr...

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Abstract

The invention discloses a monoclonal antibody for specifically quantitatively detecting the antigen of the O-type Guangxi strain of foot-and-mouth disease and its application. The monoclonal antibody, which can specifically bind to the 146S antigen of the Guangxi strain of foot-and-mouth disease O type, is the monoclonal antibody described in any one of the following: 1) containing the heavy chain variable region 1E10-VH and the light chain variable region 1E10 ‑VL; 2) contains a heavy chain variable region 7G2‑VH and a light chain variable region 7G2‑VL. The monoclonal antibody that can specifically bind to the 146S antigen of the foot-and-mouth disease type O Guangxi strain is used as a capture antibody or a detection antibody to prepare an enzyme-linked immunosorbent assay kit, which does not cross-react with other strains of foot-and-mouth disease at present, and has high sensitivity and specificity. Strong, can effectively distinguish and detect the FMD O-type Guangxi strain (O / GX / 09-7 strain) antigen content in the sample.

Description

[0001] This application is a divisional application of a patent application with the application number "201811508548.1" and the title of the invention "Enzyme-linked immunosorbent assay kit for detecting foot-and-mouth disease O-type Guangxi strain antigen and its application" technical field [0002] The invention belongs to the technical field of biological detection. More specifically, the invention relates to an enzyme-linked immunoassay kit for specific and quantitative detection of the antigen of the Guangxi strain of foot-and-mouth disease O-type, which is suitable for the specific, rapid and accurate detection of the antigen of the Guangxi strain of foot-and-mouth disease O-type. . Background technique [0003] Foot-and-mouth disease (FMD) is a severe infectious disease caused by foot-and-mouth disease virus (FMDV) infection in cloven-hoofed animals. The fatality rate of foot and mouth disease is very low, but the infection rate is high. The main symptoms of the di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10G01N33/577G01N33/569
CPCC07K16/1009C07K2317/56C07K2317/565G01N33/535G01N33/56983G01N33/577G01N33/581
Inventor 董春娜张蕾李静肖进王飞蔡薇齐鹏
Owner CHINA ANIMAL HUSBANDRY IND