Forest encephalitis virus IgM antibody ELISA detection kit and preparation method thereof

A forest encephalitis virus and detection kit technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that the influence of the detection results cannot be completely eliminated, the specificity of the measurement is reduced, and the operation steps are complicated, etc. Interfering ability, good specificity, cost reduction effect

Inactive Publication Date: 2019-04-12
长春生物制品研究所有限责任公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Moreover, the virus strains used in the preparation process of the existing forest encephalitis virus IgM antibody detection kits are all Western European subtypes, while the popular virus strains in my country are Far East subtypes. In theory, existing foreign products are used for domestic forest encephalitis virus. The clinical determination of inflammatory viruses will reduce the specificity of determination; in addition, in the preparation process of the existing forest encephalitis virus IgM antibody detection kit, in order to reduce the influence of IgG class antibodies in the sample on the measurement results, all in the sample diluent Adding rheumatoid factors or anti-human IgG antibodies, although theoretically reducing the impact of IgG in the sample on the test results, it cannot completely eliminate its impact on the test results, and the addition of rheumatoid factors or anti-human IgG antibodies also It will lead to an increase in production cost and complicated operation steps

Method used

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  • Forest encephalitis virus IgM antibody ELISA detection kit and preparation method thereof
  • Forest encephalitis virus IgM antibody ELISA detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the preparation of forest encephalitis virus antigen

[0037] Take the Vero cells that have grown into a dense monolayer, discard the cell growth medium, add Versene solution to wash, discard, add cell digestion solution with a pH value of 7.4 and a final concentration of 0.1% for digestion, and wait until the cells appear rounded and ground-glass-like After the change, the cell digestion solution was discarded, and the cell growth solution was prepared (newborn bovine serum with a final concentration of 10% was added to the 199 solution, and 7.5% NaHCO 3 Adjust the pH value of the solution to 6.8-7.6), and use the cell growth solution to disperse the cells, and the seeding concentration of the cells is 1.0×10 4 / ml. Cells are cultured in cell culture flasks at a temperature of 36-38°C. After 48 hours of culture, they can grow into dense monolayer cells. Abandon the cell growth liquid in the cell culture flask, discard after washing the cells with Earle's ...

Embodiment 2

[0041] Embodiment 2: the preparation of anti-sylvan encephalitis virus antibody

[0042] The inactivated antigen of the forest encephalitis virus "Senzhang" strain prepared in Example 1 was used as an immunogen, and Freund's complete adjuvant was added and emulsified at a volume of 1:1, and 9-week-old female healthy BALB / C mice were immunized , through intraperitoneal, intramuscular and subcutaneous multi-point injection, the immunization program is three immunizations on the 0th day, the 7th day and the 14th day. NS-1 cells were fused, and BHK-21 cells were infected with forest encephalitis virus "Senzhang" strain and then used indirect immunofluorescence to screen and obtain a total of 5 hybridoma cell lines stably expressing monoclonal antibodies against forest encephalitis virus. The secreted monoclonal antibodies were collected respectively, and numbered as monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, monoclonal antibody 4 and monoclonal antibody 5...

Embodiment 3

[0047] Embodiment 3: Composition and preparation of forest encephalitis virus IgM antibody ELISA detection kit

[0048] The kit of the present invention is based on the principle of capture enzyme-linked immunosorbent assay, that is, anti-human IgM antibody (anti-μ chain) is pre-coated on the microwell strip, which can specifically bind to the IgM antibody in the sample, and the unbound antibody can be removed by washing. Sample and IgM antibody, and then add antigen reagent and enzyme-labeled monoclonal antibody for secondary incubation. When IgM antibody exists in the sample, a complex of "anti-IgM antibody-IgM antibody-antigen-enzyme-labeled monoclonal antibody" is formed. The horseradish peroxidase connected to the object catalyzes the reaction of the chromogenic agent to generate a blue product, which turns yellow after the reaction is terminated. Whether there is IgM antibody in the sample can be judged by whether the color is displayed or the OD value is measured.

[00...

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Abstract

The invention relates to the field of biological detection, in particular to a forest encephalitis virus IgM antibody ELISA detection kit and a preparation method thereof. The kit is particularly suitable for early auxiliary diagnosis of forest encephalitis viruses in China, has the advantages of high specificity, high sensitivity, fast detection, convenient operation, low cost and the like, and is suitable for clinical promotion.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a medical detection kit, more specifically to an ELISA detection kit for forest encephalitis virus IgM antibody and a preparation method thereof. Background technique [0002] Forest encephalitis virus, also known as tick-borne encephalitis virus (TBEV), belongs to the Flavivirus genus. Forest encephalitis virus is a spherical particle, and its genome is a single-stranded positive-strand RNA with a length of about 11kb, encoding three structures in total Proteins are capsid protein (C), membrane protein (M), envelope protein (E) and 7 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5), among the 3 structural proteins, The E protein is the most important structural protein of the virus, which contains a variety of T cell epitopes and B cell epitopes, which determine the tissue tropism of the virus, mediate the binding of the virus to the cell membrane receptor, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 常军亮邹勇孙宏亮曹玉峰张秀霞唐剑光吴月韩慧利李雨桐严永男
Owner 长春生物制品研究所有限责任公司
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