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African swine fever virus antibody rapid detection test strip and preparation method and application thereof

A technology for detection of African swine fever virus and test strips, which is applied in the field of virus epidemic diagnosis technology and animal quarantine, can solve the problems of poor specificity of indirect detection methods, unsuitable antigens for competitive methods, poor sensitivity, etc., and achieve fast detection speed and batch-to-batch Good stability and low cost

Active Publication Date: 2021-11-12
杭州恒奥科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for African swine fever virus antibodies, the competition method is not applicable (the antigen is large and has more than one antigenic site, and the competition method is prone to false negatives)
However, the sensitivity of the double-antigen sandwich method is poor, and the specificity of the indirect detection method is poor.

Method used

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  • African swine fever virus antibody rapid detection test strip and preparation method and application thereof
  • African swine fever virus antibody rapid detection test strip and preparation method and application thereof
  • African swine fever virus antibody rapid detection test strip and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preparation of embodiment 1 African swine fever virus P30 protein

[0026] The codon-optimized nucleotide sequence of the African swine fever virus P30 protein (the specific sequence is shown in SEQ ID NO.1) is cloned into a eukaryotic expression vector (such as ). The nucleotide sequence of the codon-optimized African swine fever virus P30 protein (the specific sequence is shown in SEQ ID NO.1) was used as a template, and EcoRI and HindⅢ double restriction sites were used to connect to construct pEE12.4-P30 -6His expression plasmid. The recombinant plasmids identified as positive were sent to Huada Biological Company for sequence determination, and software was used to analyze and compare the determined nucleotide sequence and encoded amino acid sequence to check the correctness of the reading frame.

[0027]The correctly identified pEE12.4-P30-6His expression plasmid was transfected into CHO-K1 cells. Pressurized screening started 24 hours after transfection: Ta...

Embodiment 2

[0029] Embodiment 2 Preparation of Anti-African Swine Fever Virus P30 Protein Monoclonal Antibody

[0030] The African swine fever virus P30 recombinant protein prepared in Example 1 was used to immunize 8-week-old BALB / c mice. During the first immunization, the African swine fever virus P30 recombinant protein was emulsified with an equal volume of Freund's complete adjuvant, and the mice were inoculated intraperitoneally. 100 μg protein / mouse; 7 days later, African swine fever virus P30 recombinant protein was emulsified with an equal volume of Freund’s incomplete adjuvant, and the mice were immunized by the second intraperitoneal inoculation route, 100 μg protein / mouse; 7 days later, the third mouse peritoneal route was directly Immune African swine fever virus P30 recombinant protein, 100 μg / monkey; on the third day after immunization, take mouse splenocytes and mouse myeloma cell SP2 / 0 fusion, and culture in HAT selective medium; 10 days later, African swine fever virus T...

Embodiment 3

[0034] Embodiment 3 African swine fever virus antibody rapid detection test strip preparation

[0035] 1Materials Latex microspheres (300nm) for manufacturing were purchased from Shanghai Huizhi Biotechnology Co., Ltd.; rabbit anti-pig IgG was purchased from Beijing Suolaibao Company; PVC floor was purchased from Hangzhou Ruijian; sample mat was purchased from Tongcheng Paper; Pads were purchased from Tongcheng Paper; absorbent paper was purchased from Shanghai Jiening Biotechnology Co., Ltd.

[0036] 2 quality control line antibody (anti-African swine fever virus P30 protein monoclonal antibody) preparation (test strip 1 and test strip 2 general) anti-African swine fever virus P30 protein monoclonal antibody is the monoclonal antibody prepared in Example 2, Dilute to 1mg / mL with PBS and set aside.

[0037] 3 Preparation of nitrocellulose membrane

[0038] 3.1 Preparation of coating buffer Add 1 g of sucrose to 100 mL of PBS (0.01 mol / L, pH 7.2) to prepare a coating buffer, ...

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Abstract

The invention discloses an African swine fever virus antibody rapid detection test strip, the test strip is composed of two joint detection test strips and a treating fluid, each test strip comprises a PVC base plate, and a sample pad, a latex microsphere pad, a nitrocellulose membrane and absorbent paper are sequentially fixed on the PVC base plate; the latex microsphere pads of the two test strips are latex microsphere labeled African swine fever virus P30 protein; detection lines and quality control lines are marked on the surfaces of the nitrocellulose membranes of the two test strips, the quality control lines are anti-African swine fever virus P30 protein monoclonal antibodies, one detection line is African swine fever virus P30 protein, and the other detection line is a goat anti-pig secondary antibody. The test strip provided by the invention is suitable for detection of porcine serum, whole blood and tissue fluid, has the advantages of strong specificity, high sensitivity, good stability and fast detection speed, can be used for early screening of African swine fever viruses, and is especially suitable for on-site African swine fever infection diagnosis, epidemiological investigation, live pig international trade quarantine inspection and the like.

Description

technical field [0001] The invention belongs to the field of virus epidemic disease diagnosis technology and animal quarantine, and in particular relates to a test strip for rapid detection of African swine fever virus antibody and its preparation method and application. Background technique [0002] African swine fever (English name: African Swine fever, referred to as ASF) is caused by African swine fever virus (English name: African Swine fevervirus, referred to as ASFV) infecting domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.) An acute, hemorrhagic, severe infectious disease. The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease, the most acute and acute infection mortality rate is as high as 100%, clinical manifestations are fever (up to 40-42 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/569G01N33/577G01N33/543C07K16/08C12N15/85C07K14/01
CPCG01N33/558G01N33/56983G01N33/577G01N33/54306C07K16/081C12N15/85C07K14/005G01N2333/01G01N2469/20C07K2317/56C12N2800/22C12N2800/107C12N2710/12022
Inventor 孙统威徐慧珍周晶晶查银河
Owner 杭州恒奥科技有限公司
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