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Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen

A new bunya virus and virus antigen technology, applied in the field of immunodiagnosis in the field of biotechnology, can solve problems such as lack of training in differential diagnosis for primary medical practitioners, lack of verification methods for virus infection, and inability to assess the protective effect of vaccines on previous infection levels.

Active Publication Date: 2012-12-05
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

Through the joint research of several units of the National Center for Disease Control and relevant provincial CDCs, it has been found that the pathogen causing the infectious disease is a new type of hemorrhagic fever virus, which belongs to the Bunyaviridae family (Bunyaviridae) in taxonomy. White Phlebovirus genus, currently named as Severe Febrile and Thrombocytopenicyndrome Virus (SFTSV), the sequence of the virus has been elucidated, and the detection standard based on RT-PCR has been established, but the immunological detection of SFTSV Reagents have not yet been established, making the evaluation of the virus infection lack a validation method for PCR results
It is also impossible to assess the level of past infection in the population and the protective effect of the vaccine
[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 ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen
  • Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen
  • Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen

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Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: New bunya virus (JS-2007-001 virus strain) immunization rabbit and serum antibody preparation

[0017] 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).

[0018] Table 1: Isolation time and source of 7 New Bunyavirus strains

[0019]

[0020] 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

[0024] Example 2: New Bunia virus (JS-2007-001 strain) immunization of mice and serum antibody preparation and HRP labeling

[0025] Dilute the purified and inactivated Neobunia virus antigen with normal saline in an appropriate amount to a content of 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 intervals of 2 to 4 weeks. A total of 3 to 5 immunizations were carried out. 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.

[0026] Preparation of HRP-labeled mouse anti-New Bunia virus antibody. ...

Embodiment 3

[0027] Example 3: Antibody-coated plate preparation.

[0028] The purified antibody was appropriately diluted (1-10 μg / mL) with 0.05M pH 9 carbonate coating buffer. Add the diluted antibody solution 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 Bio) In each plate well, 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

1. Field of invention [0001] The invention belongs to the category of immunodiagnosis in the field of biotechnology. According to different target classifications, the current diagnosis of infectious diseases can be divided into two categories: molecular biology and immunology diagnosis. Molecular biology diagnosis has the advantages of rapidity and low detection limit, but the dependence on cutting-edge instruments, easy cross-contamination, and high-quality requirements for operators limit its application and promotion at the grassroots level. Immunological diagnosis (also known as serological diagnosis) is based on the specific combination between antigens and antibodies, such as the detection of antibodies by pathogen-specific antigens, and vice versa. Traditional immunological diagnostic methods include virus microneutralization, immunofluorescence, plaque reduction, etc., but the above experiments all involve problems such as long time, need to manipulate live viruses, ...

Claims

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Application Information

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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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