Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and application

A technology for porcine blue ear virus and test strips, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problem of long maintenance time of N protein antibody, and achieve the effect of saving manpower and material resources, easy to popularize, and clear and easy to distinguish the results.

Active Publication Date: 2013-03-06
辽宁迪浩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the immune response stimulated by the N protein is the strongest, and the PRRSV antibody produced by the body is mainly directed against the N protein. After a pig is infected with PRRSV, the N protein antibody can be detected after one week, and the N protein antibody lasts for a long time

Method used

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  • Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and application
  • Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and application
  • Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning and expression of the N protein of porcine PRRS virus

[0048] (1) Obtaining the target gene

[0049] The genome of porcine PRRS virus was provided by Beijing Institute of Animal Husbandry and Veterinary Medicine. The pET-32a(+) expression vector and PMD-18T were purchased from Novagen. According to the characteristics of the pET-32a(+) expression vector, design primers with restriction enzymes BamHI and XhoI cleavage sites at both ends:

[0050] Upstream is 5′C GGA TCC ATG CCA AAT AAC AAC G 3′

[0051] Downstream is 5'G CTC GAG TCA TGC TGA GGG TGA T 3'

[0052] The target fragment N was amplified, and the amplification conditions were: denaturation at 94°C for 3 minutes, 45s at 94°C, annealing at 56°C for 90s, extension at 72°C for 90s, 30 cycles, and finally extension at 72°C for 10 minutes.

[0053] (2) Cloning of the target gene and screening of positive recombinants

[0054] After electrophoresis, the PCR amplified product was recovered by ge...

Embodiment 2

[0061] Embodiment 2: the development of the monoclonal antibody cell line of porcine blue ear disease virus N protein

[0062] (1) Myeloma cells

[0063] SP2 / 0 myeloma cells: resuscitate SP2 / 0 cells stored in a liquid nitrogen tank, and culture them in DMEM medium containing 10% calf serum for 48-72 hours. Uniform, neatly arranged, logarithmically split, ready for fusion.

[0064] (2) Immunized mice

[0065] After the prepared genetically engineered antigen was taken out from the -20°C low-temperature refrigerator and dissolved, it was injected subcutaneously into BALB / C mice at multiple points (0.2ml / mouse) at intervals of 10 days. A total of 3 times of immunization. Three days before the fusion, mice were challenged with 0.15 ml of antigen in the peritoneal cavity.

[0066] (3) cell fusion

[0067] Fusion agent PEG (molecular weight 1500, produced in Japan); culture medium: 10% calf serum DMEM. Lymphocytes of SP2 / 0 cells and immunized BALB / C mice were divided into 2×10...

Embodiment 3

[0073] Embodiment 3: Preparation and assay of the monoclonal antibody of porcine blue ear disease virus N protein

[0074] (1) Monoclonal antibody ascites preparation:

[0075] Mice: SPF grade mice, no rodent virus contamination after inspection, if the animals are found to be unhealthy, bitten or infected during the ascites production process, they should be discarded.

[0076] Expanded culture of cell lines: Take one tube of the production batch of cells to resuscitate, add nutrient solution to expand the culture, and one production cell is only used once, and will not be frozen.

[0077] Inoculation of hybridoma cell lines: preparation of ascites should be carried out under sterile conditions, before injection of hybridoma cells, each mouse was intraperitoneally injected with liquid paraffin 0.5ml. One week later, each mouse was intraperitoneally injected with 1-3×10 hybridoma cells. 6 / 0.2ml.

[0078] Ascites collection: 7 to 10 days after injecting the cell line, or th...

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Abstract

The invention provides a test strip for the detection of a swine blue ear virus. The monoclonal antibody of swine blue ear virus N protein or polyclonal antibody of anti swine blue ear virus N protein, and double-antibody are coated on a nitrate cellulose film (NC film), and a membrane chromatography double antibody sandwich method is adopted to detect the swine blue ear virus in a specimen in combination with a monoclonal antibody of colloidal gold labeled swine blue ear virus N protein. The test strip is simple in operation, convenient, and fast, and has the advantages of no requirements of special instruments and special training, clear and identified result, and easy popularization. The test strip is suitable for base course, large scale site detection of an accident and epidemiological investigation, and has auxiliary effect on the diagnosis of swine blue ear virus infection.

Description

technical field [0001] The invention relates to a colloidal gold detection test strip for porcine blue ear virus, a preparation method and application thereof, and belongs to the field of biological detection. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), with high fever, high morbidity and mortality, adult pig reproductive disorders, premature birth, abortion and stillbirth, piglet respiratory Characterized by abnormalities, it is a contagious disease. The incidence of piglets can reach 100%, and the mortality rate is about 50%. The abortion and stillbirth of sows can reach more than 30%, which is very harmful to the pig industry. The disease occurs in almost all pig-raising countries in the world, and was introduced into my country in the mid-1990s. Because of its serious threat to the pig industry, the Internationa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558G01N33/545
Inventor 刘明
Owner 辽宁迪浩生物科技有限公司
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