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Monoclonal antibody capable of being specifically combined with nonstructural protein of foot-and-mouth disease and application of monoclonal antibody

A monoclonal antibody and non-structural protein technology, applied in the direction of immunoglobulin, antiviral immunoglobulin, antibody medical components, etc., can solve the problems of identification of immunized animals and naturally infected animals, interference, incomplete purification, etc., and achieve high sensitivity , the effect of widening the linear range

Active Publication Date: 2019-02-12
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inactivated vaccines only use filtration technology to remove limited cell debris, so the purification is not complete, resulting in the production of non-structural protein antibodies after immunization, which interferes with the identification of immunized animals and naturally infected animals.

Method used

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  • Monoclonal antibody capable of being specifically combined with nonstructural protein of foot-and-mouth disease and application of monoclonal antibody
  • Monoclonal antibody capable of being specifically combined with nonstructural protein of foot-and-mouth disease and application of monoclonal antibody
  • Monoclonal antibody capable of being specifically combined with nonstructural protein of foot-and-mouth disease and application of monoclonal antibody

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Experimental program
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Embodiment approach

[0020] As an embodiment of the present invention, the antibody is monoclonal antibody 7D7, the amino acid sequence of its heavy chain variable region is the amino acid sequence shown in SEQ ID No.2, and its light chain variable region amino acid sequence is SEQ ID No. . The amino acid sequence shown in 4.

[0021] The monoclonal antibody 7D7 is the monoclonal antibody 7D7 of the non-structural protein 3ABC of the foot-and-mouth disease virus, is an IgG antibody, has a relative affinity greater than 5.0 ng / ml, and has good reactivity with the non-structural protein of the foot-and-mouth disease virus.

[0022] The term "foot-and-mouth disease virus antigen" refers to an antigen prepared from the non-structural protein of foot-and-mouth disease virus.

[0023] The term "luminol chemiluminescent system" includes luminescent substrate 1: containing 1.0~6.8×10 -3 mol / L Tris-HCl buffer solution of hydrogen peroxide (0.05~0.5mol / L, pH 8.5), and luminescent substrate 2 (protected fro...

Embodiment 1

[0053] Preparation, identification and inspection of embodiment 1 foot-and-mouth disease virus nonstructural protein monoclonal antibody

[0054] 1.1 Preparation and identification of FMDV nonstructural protein

[0055] According to NCBI (http: / / www.ncbi.nlm.nih.gov) reported in the foot-and-mouth disease virus (accession number is DQ478936.1) in the gene sequence design primer pair 3ABC protein upstream primer 1: 5-AGCAGATCTCAATTCCTTCCCAAAAATCTG-3 ', Downstream primer 1: 5'-AATCACTCGTGGTGCGGTTCGGGGTC-3'. According to the method described by Cao Yimei (Cao Yimei, Liu Zaixin, Lu Zengjun, etc. The expression of non-structural protein gene 3ABC of foot-and-mouth disease virus in E. The 3ABC gene was added, and the cDNA reverse-transcribed from the viral RNA was amplified by PCR with the designed primers. It was detected by 1% agarose gel electrophoresis that a band of about 1.3 Kb was produced, which was in line with the expectation.

[0056]The cloned sequence of the FMDV 3ABC...

Embodiment 2

[0079] Preparation and application of embodiment 2 foot-and-mouth disease virus nonstructural protein antibody detection kit

[0080] 2.1 Preparation of the kit

[0081] Coated plate: Dilute the FMDV 3ABC prepared in Example 1.1 with CB buffer (0.02mol / L, pH 9.6) to 2.0μg / ml, coat at 2-8°C overnight, discard the coating solution and press 200μl / well Add PB solution (0.02mol / L, pH 7.4) containing 20% ​​calf serum and block at 37°C for 2 hours, discard the blocking solution, dry and package.

[0082] Enzyme-labeled reagent: Dilute the enzyme-labeled monoclonal antibody 7D7 prepared in Example 1.6 with sample diluent, select a representative concentration to describe the concentration range of the enzyme-labeled reagent in the kit, see Table 1.

[0083] Gold-labeled reagent: Dilute the gold-labeled monoclonal antibody 7D7 prepared in Example 1.7 with sample diluent, select a representative concentration to describe the concentration range of the enzyme-labeled reagent in the kit...

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Abstract

The invention discloses a monoclonal antibody 7D7 of nonstructural protein of a foot-and-mouth disease virus (FMDV), a foot-and-mouth disease virus nonstructural protein antibody detection kit containing the monoclonal antibody and application of the monoclonal antibody. The antibody detection kit provided by the invention has high sensitivity and a linear range of detection can be remarkably widened; in a detection process, the monoclonal antibody is not limited by animal species and is applicable to large-scale screening of foot-and-mouth diseases of cloven hoof type animals. A vaccine prepared from a conjugate containing the monoclonal antibody 7D7 can be used for removing the nonstructural protein; after the animals are immunized, interference to the nonstructural protein antibody of the FMDV can be avoided, so that infected animals and immunized animals are accurately distinguished.

Description

technical field [0001] The invention relates to the monoclonal antibody of non-structural protein of foot-and-mouth disease virus and the application of the detection kit containing the monoclonal antibody, belonging to the field of biomedicine. Background technique [0002] Foot and Mouth Disease (FMD) is an acute, febrile, highly contagious severe infectious disease caused by Foot and Mouth Disease Virus (FMDV). The disease can infect more than 70 kinds of domestic animals and wild animals, mainly cattle, sheep, and pigs. The mortality rate of adult susceptible animals is 5% to 20%, and the mortality rate of young animals is 50% to 80%. Once the disease breaks out, it will spread rapidly. As a severe infectious disease that must be notified by the International Veterinary Bureau OIE and my country, it has caused huge economic losses to countries all over the world. In view of the fact that the main measure to prevent and treat the disease in my country is to implement im...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/577G01N33/569A61K39/135A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552C07K16/10C07K2317/56C12N2770/32134
Inventor 田克恭孙明晋育丹陈西钊
Owner LUOYANG PULIKE WANTAI BIOTECH
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