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

An African swine fever virus and detection test paper technology, which is applied in the field of virus disease diagnosis technology and animal quarantine, can solve the problems of poor specificity of indirect detection methods, unsuitable antigens of competing methods, and poor sensitivity, etc., and achieves fast detection speed and batch-to-batch detection. The effect of good stability and low cost

Active Publication Date: 2022-04-01
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 its preparation method and application
  • African swine fever virus antibody rapid detection test strip and its preparation method and application
  • African swine fever virus antibody rapid detection test strip and its preparation method and application

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 a test strip for rapid detection of African swine fever virus antibody. The test strip is composed of two joint inspection test strips and a treatment solution. The sample pad, latex microsphere pad, nitrocellulose membrane and absorbent paper are fixed in sequence; the latex microsphere pads of the two test strips are latex microspheres labeled African swine fever virus P30 protein; the nitrocellulose of the two test strips There are detection lines and quality control lines drawn on the surface of the plain film, and the quality control lines are monoclonal antibodies against African swine fever virus P30 protein. One of the detection lines is African swine fever virus P30 protein, and the other detection line is sheep Anti-pig secondary antibody. The test strip provided by the invention is suitable for the detection of pig serum, whole blood and interstitial fluid. It has strong specificity, high sensitivity, good stability and fast detection speed, and can be used for early screening of African swine fever virus, especially suitable for on-site Diagnosis of African swine fever infection, epidemiological investigation and quarantine inspection of pigs for international trade, etc.

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 Patents(China)
IPC IPC(8): C07K16/08
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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