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Swine Hemagglutinating encephalomyelitis virus colloidal gold antigen detecting test paper and preparing method thereof

A technology of encephalomyelitis virus and colloidal gold, which is applied in the field of disease detection, can solve problems affecting rapid detection, achieve the effects of easy observation and judgment, avoid operation, and improve prevention and control capabilities

Inactive Publication Date: 2009-11-25
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, conventional HEV detection techniques mainly include virus isolation and identification, RT-PCR, neutralization test, complement fixation test, agar diffusion test, etc. The above methods play a certain role in the detection of HEV, but they are used for rapid and accurate clinical detection. There are varying degrees of deficiencies in the diagnosis of the disease, and the lag in the study of these diagnostic methods and quarantine procedures has affected the rapid detection of the disease when it is prevalent

Method used

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  • Swine Hemagglutinating encephalomyelitis virus colloidal gold antigen detecting test paper and preparing method thereof
  • Swine Hemagglutinating encephalomyelitis virus colloidal gold antigen detecting test paper and preparing method thereof
  • Swine Hemagglutinating encephalomyelitis virus colloidal gold antigen detecting test paper and preparing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Preparation of Porcine Hemagglutination Encephalomyelitis Virus Antigen

[0016] The porcine hemagglutination encephalomyelitis virus was inoculated into PK-15 cells, and the cytopathic effect (CPE) reached more than 80%. , centrifuged for 30 min, and the supernatant was centrifuged at 24,000 r / min for 3 h, and the precipitate was fully suspended with a small amount of PBS to obtain crude virus. Carry out 25%, 35%, 45%, 55% discontinuous sucrose density gradient centrifugation on the crude virus preparation, centrifuge at 20,000r / min for 2h, collect the virus band, then add an appropriate amount of PBS buffer to remove the sucrose by ultracentrifugation again, and finally use PBS buffer Dilute the precipitate to obtain purified virus.

Embodiment 2

[0018] Preparation and purification of porcine hemagglutination encephalomyelitis virus monoclonal antibody

[0019] The hybridoma cell line secreting anti-HEV monoclonal antibody prepared by the applicant was injected into the peritoneal cavity of Balb / C mice to induce ascites and produce monoclonal antibody. Take 5ml of the obtained mouse ascites and mix it with an appropriate amount of silicon dioxide, add an equal volume of barbiturate buffer, shake at room temperature for 30 minutes, let it stand at 4°C for 1 hour, and centrifuge at 3000r / m for 10 minutes; take the supernatant and add twice the volume of For acetic acid buffer, adjust the pH value to 4.6 with 0.1M hydrochloric acid, add 33μl octanoic acid per ml of ascites under a magnetic stirrer, stir for 30min, let stand at 4°C for 2h, centrifuge at 15,000r / m for 30min at 4°C, and obtain 25ml of supernatant , add 2.5ml of 0.1M phosphate buffer solution, adjust the pH value to 7.6 with 0.1M NaOH, slowly add an equal vol...

Embodiment 3

[0021] Preparation and Purification of Rabbit Anti-HEV Polyclonal Antibody

[0022] Take 2 healthy rabbits, and use the purified HEV antigen plus the same amount of Freund's complete adjuvant for the first subcutaneous immunization, each 400 μg (2ml), two weeks later the same dose of antigen plus Freund's incomplete adjuvant booster immunization, two weeks Then repeat once, 7 days later, detect the titer of HEV polyclonal antibody in rabbit serum by indirect ELISA (wavelength: 490nm), and wait until the serum antibody titer is greater than 1:6×10 4 At that time, blood was extracted from the jugular vein of rabbits to extract serum. The serum is purified by the octanoic acid saturated ammonium sulfate method, and used on the detection line on the NC membrane of the test strip, which can be combined with the gold-labeled antibody-HEV antigen-antibody complex to form a sandwich antigen-antibody complex.

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Abstract

The present invention relates to a novel technique-colloidal gold immunochromatography for quickly detecting the Swine Hemagglutinating encephalomyelitis virus (HEV), and to a test paper which is prepared by the technique for preparing the Swine Hemagglutinating encephalomyelitis virus. The invention develops a monoclonal antibody (Ab1) of the Swine Hemagglutinating encephalomyelitis virus and a monoclonal antibody (Ab2) of rabbit anti- Swine Hemagglutinating encephalomyelitis virus. The Ab1 labeled colloidal gold is taken as the gold-labeled antibody and the Ab2 is taken as the catching antibody. The normal anti-mouse IgG antibody is used as a quality control antibody. The materials of antibody, cellulose nitrate film (NC film), combining pad, sample pad, back lining, plastic card, etc. are processed and assembled to the test paper. The test paper can detect the Swine Hemagglutinating encephalomyelitis virus in the sickened pig brain tissue and the feces. The technique of the invention has the characteristics of simple operation, high speed, excellent sensitivity, excellent specificity, etc. Furthermore the technique of the invention is especially suitable for the clinic sample detection of disease appearance field, clinic, places with no available experiment condition, etc.

Description

Technical field: [0001] The invention relates to a detection method for diseases caused by hemagglutination encephalomyelitis virus, especially provides a colloidal gold antigen detection test strip of porcine hemagglutination encephalomyelitis virus, and also discloses its preparation method, which belongs to the disease detection technology field. Background technique: [0002] Porcine hemagglutination encephalomyelitis is an acute, highly contagious infectious disease of piglets caused by hemagglutination encephalomyelitis virus (abbreviation: HEV). HEV mainly affects piglets aged 1-3 weeks. Clinically, piglets are mainly characterized by vomiting, exhaustion and obvious neurological symptoms, and the mortality rate is as high as 20-100%. Due to the worldwide distribution of the disease, its threat or potential impact on the swine industry is relatively large. At present, conventional HEV detection techniques mainly include virus isolation and identification, RT-PCR, ne...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N33/577G01N33/532
Inventor 陈克研高丰贺文琦陆慧君宋德光岳占碰高巍王丽
Owner JILIN UNIV
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