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African swine fever virus (ASFV) antigen detection fluorescent microsphere immunoassay test strip, preparation method and application

An African swine fever virus and fluorescent microsphere technology, applied in the field of virus disease diagnosis technology and animal quarantine, can solve the problem that the detection sensitivity needs to be further improved, and achieve the effects of avoiding human factor interference, convenient use and high accuracy.

Pending Publication Date: 2019-09-03
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunofluorescence and western blotting tests require professionals to use professional equipment to operate, which cannot be carried out in general grassroots laboratories, farms, farmers, etc.; colloidal gold test paper has the advantages of convenience and speed in detecting ASFV antigens, but the detection sensitivity needs to be further improved

Method used

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  • African swine fever virus (ASFV) antigen detection fluorescent microsphere immunoassay test strip, preparation method and application
  • African swine fever virus (ASFV) antigen detection fluorescent microsphere immunoassay test strip, preparation method and application
  • African swine fever virus (ASFV) antigen detection fluorescent microsphere immunoassay test strip, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0050] 1. Preparation of anti-ASFV p30 monoclonal antibody

[0051] (1) Mab1, Mab2 ascites preparation

[0052]Eight-week-old BALB / c mice (provided by the Comparative Medicine Center of Yangzhou University) were intraperitoneally injected with sterile liquid paraffin, 0.5 mL / mouse; one week later, the mice were injected intraperitoneally with hybridoma cells secreting Mab1 and Mab2, 10 6 cells / only, collect ascites after 6 days, and centrifuge at 12,000 rpm to remove cells and fat.

[0053] (2) Add 3 times the volume of 0.06M CH to the ascites 3 COONa buffer solution (pH=5.0), and then add 1M hydrochloric acid to adjust the pH to 4.5; add 1 / 3 volume of caprylic acid drop by drop, stir while dripping, continue stirring for 30min after the dripping, and stand at 4°C for 1.5h Centrifuge at 10000rpm at 4°C for 30min, suck out the supernatant; add 1M NaOH solution to the supernatant to adjust the pH to 7.4, add an equal volume of saturated (NH 4 ) 2 SO 4 The solution was stirr...

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Abstract

The invention discloses an African swine fever virus (ASFV) antigen detection fluorescent microsphere immunoassay test strip, which comprises a PVC bottom plate. A sample pad, a fluorescent pad, a nitrocellulose membrane and an absorbent pad are sequentially fixed on the PVC bottom plate; the fluorescent pad contains a monoclonal antibody Mab1 fluorescently labeled with an epitope of an anti-ASFVp30 antigen; the surface of the nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is a monoclonal antibody Mab2 for another epitope of the ASFV p30antigen, and the quality control line is a goat anti-mouse IgG antibody. The test strip is suitable for detection on the ASFV p30 antigen in peripheral blood, serum, saliva and tissue (lung and spleen) polishing slurry of infected pigs, ASFV infection is diagnosed in a short time, and the test strip is particularly suitable for on-site ASFV infection diagnosis, epidemiological investigation and international trade quarantine inspection of pigs; and a result is directly displayed in collaboration with an immunofluorescence analyzer, the operation is quick and simple, the accuracy is high, andthe practicability is strong.

Description

technical field [0001] The invention relates to a fluorescent microsphere immune test paper card for detecting African swine fever virus antigen, in particular to a fluorescent microsphere immune test paper card for detecting African swine fever virus antigen and its preparation method and application, belonging to the technical field of virus epidemic diagnosis technology and animal quarantine technology . Background technique [0002] African swine fever (ASF) is a highly contagious infectious disease of pigs. The highly pathogenic ASFV infection of domestic pigs can lead to 100% mortality. The disease has been endemic in many African countries and has now spread to Gruzi Neighboring countries such as Asia, Armenia, Azerbaijan and Russia, the disease entered my country in August 2018, causing huge losses to my country's pig industry. [0003] Since there is currently no vaccine for ASF prevention, rapid and accurate diagnosis is very important to prevent the spread and epi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/58G01N33/533G01N33/544
CPCG01N33/56983G01N33/582G01N33/585G01N33/533G01N33/544G01N2333/183
Inventor 张鑫宇刘潇羽党红军熊新灿孙怀昌
Owner YANGZHOU UNIV
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