African swine fever virus test paper, kit and preparation method of test paper

A technology for detecting African swine fever virus and test paper, which is applied in the field of detection test paper for African swine fever virus, kits and their preparation, can solve the problems of insufficient sensitivity, short reaction time, poor specificity, etc., and achieve high detection sensitivity and overall The effect of short time consumption and small batch variance

Active Publication Date: 2020-01-07
北京纳百生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the binding reaction of antigen and antibody, the part where the antigen participates in the binding is called the epitope of the antigen. The epitope is the basis of the antigenicity of the protein. The epitope peptide commonly used in the current technology is still weak in immunogenicity and low in specificity. Therefore, there is an urgent need for a recombinant antigenic protein of African swine fever virus p72 with strong immunogenicity and good specificity, and the recombinant antigenic protein can be used to prepare monoclonal or polyclonal antibodies combined with it, and can be further utilized to obtain Antibodies for detection of African swine fever virus
[0004] In addition, the traditional methods for diagnosis and detection of African swine fever virus include molecular biology methods and enzyme-linked immunoimmunoassay kit methods. The molecular biology methods are represented by polymerase chain reaction (PCR), which confirms whether the animal is infected by detecting virus molecules. Infected, the operation is cumbersome, the reagents and equipment are expensive, and it is difficult to operate for pig farms; ELISA uses 96-well ELISA plate as the carrier, and uses the principle of antigen-a

Method used

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  • African swine fever virus test paper, kit and preparation method of test paper
  • African swine fever virus test paper, kit and preparation method of test paper
  • African swine fever virus test paper, kit and preparation method of test paper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of African swine fever virus p72 recombinant protein monoclonal antibody

[0056] 1. African swine fever virus p72 recombinant protein gene cloning and recombinant expression vector construction

[0057] According to the African swine fever virus p72 gene sequence published by Genbank (sequence number: AAT84439.1) as a reference, the optimized gene sequence was designed to make the N / C terminal epitope of the antigen more easily exposed and show more advantages. Thus, a relatively suitable monoclonal antibody is screened. The present invention analyzes the dominant epitope region of the protein sequence through DNAstar and IEDB databases, and uses GGGGS as a linker to express the two major epitope regions in tandem. The two epitope regions The amino acid sequences of the regions are respectively SEQ ID No.1 and SEQ ID No.2, which minimize the influence of steric hindrance while retaining more amino acids. The nucleotide sequence of the optimized A...

Embodiment 2

[0098] Example 2 Preparation of Latex Microsphere-labeled African Swine Fever Virus p72 Recombinant Protein Monoclonal Antibody

[0099] Dilute the prepared African swine fever virus p72 recombinant protein monoclonal antibody (7A7) with diluent and label it with latex microspheres. The specific operation is as follows:

[0100] 1. Cleaning of latex microspheres: Measure a certain volume of latex microspheres with a particle size of 300nm (purchased from Suzhou Weidu Biotechnology Co., Ltd., item number DR05C), pour them into a clean centrifuge tube, and add Proportional to 900 µL of labeling buffer (50 mM MES, pH 6.0) was added to the labeling buffer. Centrifuge at 17000 r / min for 10 min, remove the supernatant, then add 1000 μL of labeling buffer, centrifuge at 17000 r / min for 10 min, remove the supernatant, add 1000 μL of labeling buffer to resuspend the microspheres.

[0101] 2. Activation of latex microspheres: Weigh 20 mg of NHS and EDC each, dissolve in 1 mL of labelin...

Embodiment 3

[0104] The preparation of embodiment 3 latex microsphere cushion

[0105] 1. Take out the latex microsphere-labeled African swine fever virus p72 recombinant protein monoclonal antibody solution from the 4°C refrigerator and return to room temperature.

[0106] 2. Take a 20cm×30cm piece of glass fiber and cut it into 0.6cm×30cm size with an auxiliary material cutting machine.

[0107] 3. Spread a layer of plastic wrap on the table, place the cut glass fibers on the plastic wrap, and use a pipette to absorb 1 mL of latex microsphere-labeled African swine fever virus p72 recombinant protein monoclonal antibody solution to evenly wet A sheet of cut fiberglass. Dry at room temperature for 12-16 hours, then transfer to a 40°C oven for 1 hour, then place in a sealed bag with a desiccant, seal it, and set it aside.

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Abstract

The invention provides African swine fever virus test paper, a kit and a preparation method of the test paper. The African swine fever virus test paper comprises a back plate; a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate; the latex microsphere pad is coated with a labeled antibody labeled by latex microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a capture antibody, and the detection line is arranged close to one sideof the latex microsphere pad; the quality control line is coated with a goat-anti-mouse anti-antibody, and the quality control line is arranged close to one side of the water absorption pad; and thelabeled antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with a preservation number of CGMCC No.18540 or a monoclonal antibody secreted by a hybridoma cell 3E5 with a preservation number of CGMCC No.18539, and the capture antibody is an African swine fever virus antibody. The African swine fever virus test paper can realize rapid, high-specificity and high-sensitivity detection of the African swine fever virus.

Description

technical field [0001] The invention belongs to the technical field of animal epidemic detection, and in particular relates to a test paper for detection of African swine fever virus, a kit and a preparation method thereof. Background technique [0002] African swine fever (ASF) is a hemorrhagic, highly fatal, viral infectious disease of pigs caused by African swine fever virus (ASFV). Pigs of all ages are susceptible. The incubation period of natural infection of ASF is long, ranging from 4 to 19 days. The average death time after infection is 2-10 days. The clinical manifestations include high fever, loss of appetite, skin and internal organ bleeding, etc. The clinical symptoms are similar to swine fever and swine danqing virus. After pigs or wild boars are infected with African swine fever virus, the incubation period is usually 3-15 days, and the mortality rate of virulent strains can reach 100%. For investigation and diagnosis in the early stage, serological method i...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/54313G01N33/56983G01N2333/01
Inventor 杨春江杨先富赵荣茂袁志波杨晓霞吴佳兴于在江马孝斌朱琳
Owner 北京纳百生物科技有限公司
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