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Monoclone antibody and application thereof

A monoclonal antibody and antibody technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulins, etc., can solve the problems of cumbersome operation and low monosensitivity, so as to improve accuracy and make up for prevention and/or treatment. Effect

Active Publication Date: 2016-08-10
LUOYANG PULIKE WANTAI BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection methods of swine fever antibody at home and abroad include in vitro virus neutralization test and in vivo virus neutralization test, which are cumbersome to operate and require specialized laboratories and technical personnel; ), the test results need to be judged by the naked eye and are greatly affected by subjective and human factors; agar diffusion test (AGP), but its single sensitivity is low

Method used

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  • Monoclone antibody and application thereof
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  • Monoclone antibody and application thereof

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Experimental program
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Embodiment approach

[0023] As an embodiment of the present invention, the antibody is monoclonal antibody 15A9, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 15A9 is SEQ ID No. 2, and the amino acid sequence of the light chain variable region is SEQ ID No. 2. ID No.4.

[0024] As an embodiment of the present invention, the antibody is monoclonal antibody 15A9, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 15A9 is the nucleotide sequence shown in SEQ ID No.1 or its degenerate sequence The coding, and the amino acid sequence of the light chain variable region are coded by the nucleotide sequence shown in SEQ ID No.3 or its degenerate sequence.

[0025] Described monoclonal antibody 15A9 is anti-swine fever virus monoclonal antibody 15A9, and its neutralizing activity titer is 1:6400, has good neutralizing activity; IPMA (immunoperoxidase monolayer cell test) titer is 1:6400 12800, has good reactivity with classical...

Embodiment 1

[0060] Example 1 Preparation, purification and identification of monoclonal antibody against classical swine fever virus

[0061] 1.1 Preparation of CSFV antigen and determination of its content

[0062] Refer to He Yan (He Yan. The expression of classical swine fever virus E2 protein in insect cells and the establishment of indirect ELISA antibody detection method. 2008, Nanjing Agricultural University master's degree thesis) literature operation method to prepare the classical swine fever virus antigen that is E2 protein; BCA Protein Concentration Determination Kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was used to determine the concentration of E2 protein, and the concentration was 1.1 mg / ml.

[0063] 1.2 Preparation and purification of monoclonal antibody against CSFV

[0064] The E2 protein of classical swine fever virus was immunized into mice according to the amount of 100 μg / 200 μl, according to the operation of Harlow E et al. (Harlow E, Lane D. ...

Embodiment 2

[0090] Example 2 Preparation, detection method and application of swine fever virus ELISA antibody detection kit (blocking method)

[0091] 2.1 Preparation and content determination of enzyme-labeled monoclonal antibody 15A9

[0092] Under dark conditions, use the modified sodium periodate method to label the monoclonal antibody with HRP: Weigh 20mg of horseradish peroxidase (HRP) and dissolve it in 1ml of ultrapure water, add 1ml of freshly prepared NaIO 4 Solution (specifically 20mg NaIO 4 Dissolve in 1ml ultrapure water), mix well, and protect from light at 4°C for 30 minutes; add 20 μl of ethylene glycol solution to the above solution, and react at 4°C for 30 minutes; Add 100 μl of the above mixture, mix the two, add to the dialysis bag and mix, and dialyze with carbonic acid buffer for 6 hours; transfer the dialyzed mixture to a 1.5ml EP tube, add 10 μl of freshly prepared NaBH 4 Solution (specifically 10mg NaBH 4 Dissolve in 1ml ultrapure water), act for 2 hours at ro...

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Abstract

The invention provides a monoclone antibody and application thereof. The monoclone antibody comprises a heavy chain as shown in SEQ ID No. 2 and a light chain as shown in SEQ ID No. 4. An enzyme-linked immunosorbent assay (ELISA) antibody detection kit (a blocking method) prepared with the monoclone antibody is good in sensitivity and high in specificity, and pigs infected with hog cholera virus can be detected more accurately. An ELISA antibody detection kit (a competition method) prepared with the monoclone antibody is short in detection time and can be used for quickly preliminarily screening pigs infected with the hog cholera virus and support further purification against the hog cholera virus. The monoclone antibody further has neutralizing activity and can further be used for preparing medicines for preventing and / or treating hog cholera relevant diseases.

Description

technical field [0001] The invention relates to an anti-swine fever virus monoclonal antibody, an antibody detection kit and a pharmaceutical composition prepared from the monoclonal antibody, and belongs to the field of biotechnology. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease caused by classical swine fever virus (CSFV), characterized by acute onset, high fever retention, and extensive hemorrhage and necrosis of tissues and organs; The International Veterinary Bureau identified it as a category A disease, and it was listed as a first-class animal infectious disease in my country. The disease is widely prevalent in many countries and regions, causing huge economic losses. Even so, although my country has controlled the occurrence and prevalence of severe swine fever, it still persists in my country with new characteristics and forms, which seriously threatens my country's pig industry (Huang Wenfeng, Yang Huichao. Epidemic chara...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/577G01N33/569A61K39/42A61P31/14
CPCA61K2039/505C07K16/1081
Inventor 田克恭郝丽影
Owner LUOYANG PULIKE WANTAI BIOTECH
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