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Preparation and application of an ELISA kit for detecting neobunia virus antigen

A new bunya virus, virus antigen technology, applied in the direction of biological testing, measuring devices, material inspection products, etc.

Active Publication Date: 2014-10-22
无锡鑫连鑫生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Because the virus is a new infectious disease, the initial clinical symptoms are the same as common flu, and most of the patients are concentrated in rural areas with weak health care; At the same time, grassroots medical practitioners lack relevant differential diagnosis training, and there is an urgent need for rapid detection methods that can adapt to the operation of grassroots clinical laboratory personnel to facilitate timely treatment of patients
In addition, the epidemiological characteristics, transmission media, and animal infection of the disease are currently blank, and there is an urgent need to develop simple, convenient and fast detection reagents to meet the needs of the disease control system for monitoring people and animals in epidemic areas , and currently there is no relevant product supply in the market

Method used

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  • Preparation and application of an ELISA kit for detecting neobunia virus antigen
  • Preparation and application of an ELISA kit for detecting neobunia virus antigen
  • Preparation and application of an ELISA kit for detecting neobunia virus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: New bunya virus (JS-2007-001 virus strain) immunization rabbit and serum antibody preparation

[0018] Seven New Bunia virus strains were isolated from Jiangsu and Anhui epidemic areas, 6 strains were directly isolated from patient serum, and 1 strain was from a suspected sick dog. All cells were VERO cells when isolated (Table 1).

[0019] Table 1: Isolation time and source of 7 New Bunyavirus strains

[0020]

[0021] Adaptive culture, immunogenicity identification and immunoprotection identification of 7 New Bunia virus strains in VERO cells found that the JS-2007-001 virus strain has the best growth characteristics and adaptability characteristics. When the growth peak is reached, a higher titer virus can be obtained stably, and the antibody level of the immunized animals is higher. The immune serum neutralization test found that it has a good protective effect on all 7 strains of the virus. So choose the JS-2007-001 virus seed as the prepared vir...

Embodiment 2

[0025] Example 2: New Bunia virus (JS-2007-001 strain) immunization of mice and serum antibody preparation and HRP labeling

[0026] Dilute the purified and inactivated New Bunyavirus antigen with normal saline in an appropriate amount at 0.1-200 μg / mL, add Freund's complete adjuvant (Sigma) for mixing, select 15-25 g mice, and then Inject 1 to 5 mg of BCG in the legs for sensitization. Two weeks later, the mice were intraperitoneally injected with the above-mentioned adjuvant antigen, and boosted with the same dose of different immunized parts at an interval of 2 to 4 weeks. A total of 3 to 5 immunizations were performed. The mouse serum was collected 10 days after the last immunization for antibody For titer determination, whole blood was collected after reaching a ratio greater than 1:20000, and serum was routinely separated. Pure antibody was purified by Protein G conventional method.

[0027] Preparation of HRP-labeled mouse anti-New Bunia virus antibody. The antibody ...

Embodiment 3

[0028] Example 3: Antibody-coated plate preparation.

[0029]The purified antibody was appropriately diluted (1-10 μg / mL) with 0.05M pH 9 carbonate coating buffer. Add the diluted antibody liquid into the wells of the enzyme-linked plate, 100 μL per well, and coat at 4°C for 18-20 hours, discard the liquid in the wells, wash the plate several times, and add enzyme stabilizer (Shandong, Taitianhe Biotech) In each well of the plate, 150-200 μL per well. React at 4°C for 4-6 hours. Discard the protective agent in the well, dry the coated plate by freeze-drying method, seal the enzyme-linked plate and store it at 2-8°C for later use.

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Abstract

The invention relates to preparation and application of an enzyme-linked immunosorbent assay (ELISA) kit for detecting novel bunyavirus antigen of feverwith thrombocytopenia associated syndrome. According to the kit, a domestic rabbit and a mouse are immunized by using severe febrile and thrombocytopenicyndrome virus, called novel bunyavirus JS-2007-001 for short, to obtain antiserum. An enzyme-linked reaction plate is coated by using rabbit antibody, mouse antibody is coupled with horse radish peroxidase (HRP), and auxiliary reagents such as an antigen quantitative reference and an enzyme substrate TMB (Tetramethylbenzidine) color developing agent are matched to form the kit.

Description

technical field [0001] The invention belongs to the field of immune analysis and detection, and in particular relates to the preparation and application of an ELISA kit for detecting neobunia virus antigens. Background technique [0002] Novel Bunyavirus (Novel Bunyavirus, SFTS Bunyavirus), is the pathogen of fever with thrombocytopenia syndrome (Fever with Thrombocytopenia Associated Syndrome, SFTS), a new type of virus belonging to the genus Phleboviridae of the Bunyaviridae family, which was released in 2009. It was first discovered in China in 1999, and it was also the first new virus discovered in China. Patients develop severe acute infectious diseases characterized by high fever, thrombocytopenia, leukopenia, multiple organ dysfunction, and bleeding, which seriously affect people's health and life safety. In 2010, a number of public health incidents caused by tick bites broke out in central and eastern China, including Anhui, Jiangsu, Hubei, Henan, Shandong and othe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/543
Inventor 万里明焦永军曾晓燕
Owner 无锡鑫连鑫生物医药科技有限公司
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