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Immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and detection kit thereof

A bovine parainfluenza virus and immunofluorescence technology, applied in the biological field, can solve the problems of low specificity, low sensitivity, and long testing time, and achieve the effect of strong specificity, high sensitivity, and fast diagnosis speed

Pending Publication Date: 2020-02-07
华威特(江苏)生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For the detection of bovine parainfluenza virus type 3, most of them are serological detection methods, including virus neutralization test, indirect hemagglutination test, enzyme-linked immunosorbent assay, etc. These methods have the disadvantages of low sensitivity and long testing time ; As a molecular biological detection method, PCR has the characteristics of sensitivity and simplicity, but it has the disadvantages of weak specificity and high false positive rate.

Method used

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  • Immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and detection kit thereof
  • Immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Preparation of bovine parainfluenza virus type 3 antigen

[0016] Filter the virus liquid with a 0.45 μm filter membrane to remove cell debris; slowly add solid PEG and NaCl to the virus liquid and stir until the final concentrations are 70 g / L and 23 g / L respectively; stir overnight at 2-8°C; Centrifuge at 8,000r / min for 50 minutes at 2-8°C, discard the supernatant, and resuspend the pellet with 1% of the original volume of NTE buffer; use NTE buffer to prepare concentrations of 10%, 20%, and 30% respectively , 40% and 50% sucrose solutions, prepare a 10%-50% sucrose density gradient, and equilibrate overnight at 2-8°C; add PEG to concentrate the virus sample, and centrifuge at a high speed for 12 hours at 2-8°C; draw The virus sample located in the centrifuge zone of 40% sucrose was dissolved in an appropriate amount of PBS; after purification by Sepharose 4Fast Flow (4FF) column chromatography system, it was sterilized by filtration with a 0.22 μm filter membrane and...

Embodiment 2

[0018] Preparation of monoclonal antibody against bovine parainfluenza virus type 3

[0019] 1 Animal immunization: Take the concentrated and purified viral protein, dilute it with an appropriate amount of PBS, emulsify it with an equal volume of Freund's complete adjuvant, and inoculate BALB / c mice intraperitoneally, 50ug / only; on the 14th day and 28th day after the first immunization, respectively After emulsification with an equal volume of Freund's incomplete adjuvant, the second and third immunizations were carried out; on the 14th day after the third immunization, the blood was collected by docking the tail to separate the serum, and the antibody titer of the serum was detected by indirect ELISA method. When the serum antibody titer was greater than 1:10000 3 days before cell fusion, the virus solution without adjuvant was used to boost immunization once through the tail vein, 100ug / cause.

[0020] 2. Cell fusion: mix SP2 / 0 cells and splenocytes of immunized mice at a ra...

Embodiment 3

[0033] Preparation of immunofluorescence reagents

[0034] 1 Monoclonal antibody dilution The purified BPIV3 monoclonal antibody was diluted with carbonate buffer (0.1 mol / L, pH 9.0) to a protein concentration of 5 mg / ml.

[0035] 2 Preparation of fluorescein solution Weigh fluorescein isothiocyanate (FITC), and prepare 1 mg / ml with carbonate buffer (0.1 mol / L, pH 9.0) under dark conditions.

[0036] 3 Stirring method labeling According to the ratio of 1ml monoclonal antibody solution (5mg / ml) to 250ul FITC solution (1mg / ml), add FITC solution (1mg / ml) dropwise to the purified monoclonal antibody solution, and stir while adding , at room temperature, gently mix for 2 hours in the dark.

[0037] 4 Removal of free fluorescein Use a Sephadex G-25M column to remove free fluorescein, and collect the eluate in separate tubes. Use a UV spectrophotometer to detect the absorption value of each eluate at a wavelength of 280nm. It can be seen that there are 2 elution peaks, and the con...

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Abstract

The invention belongs to the technical field of biology and particularly relates to an immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and a detection kit of the immunofluorescence reagent. The immunofluorescence reagent is an anti-bovine parainfluenza virus type 3 monoclonal antibody marked by fluorescent dye, and the monoclonal antibody is prepared by taking virus particles obtained by concentrating and purifying the bovine parainfluenza virus type 3 as an antigen and adopting a hybridoma cell monoclonal antibody technology. The fluorescent dye is fluorescein isothiocyanate (FITC). The subtype of the monoclonal antibody is IgG2. An immune reagent in an immunofluorescence kit for detecting the bovine parainfluenza virus type 3 is an immunofluorescence reagent for detecting the bovine parainfluenza virus type 3. The invention has the advantages of high sensitivity, strong specificity, good stability, high diagnosis speed and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and a detection kit thereof, which are suitable for the diagnosis of bovine parainfluenza virus type 3 and parainfluenza virus type 3 in tissues, serum or cells detection and identification. Background technique [0002] Bovine parainfluenza virus type 3 (BPIV3) is a member of the Respirovirus genus in the Paramyxoviridae family of the order Unimolecular Negative-Strand RNA Viruses. Cows infected with bovine parainfluenza virus type 3 often show clinical symptoms such as depression, loss of appetite, runny nose, tears, cough, fever and dyspnea, and at the same time cause immune suppression of the body, and then secondary infection with other bacteria, viruses and mycoplasma, Causing bovine respiratory disease syndrome, causing great harm to the cattle industry. In 1959, researchers in the Unite...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/577G01N33/58
CPCG01N33/56983G01N33/577G01N33/582G01N2333/115
Inventor 陈超阳李真光和彦良夏铭崎孟庆森朱勇俊武文慧闫喜军武华
Owner 华威特(江苏)生物制药有限公司
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