Preparation and application of enzyme-linked immuno sorbent assay (ELISA) kit for detecting virus antibody IgM of fever with throbocytopenia associated syndrome

A kit and antibody technology, applied in the field of preparation of ELISA kits, can solve problems such as the inability to evaluate the protective effect of vaccines on the level of past infection in groups
CN102809649AInactive Publication Date: 2012-12-05万里明
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
万里明
Publication Date
2012-12-05
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention relates to preparation and application of an enzyme-linked immuno sorbent assay (ELISA) kit for detecting a virus-specific antibody of fever with throbocytopenia associated syndrome. The kit comprises an anti-human-IgM-enveloped ELISA reaction plate, horse radish peroxidase (HRP)-coupled nucleocapsid protein (NP) and a tetramethylbenzidine (TMB) color development agent. A preparation method for a ribonucleoprotein (rNP)-enveloped reaction plate comprises the following steps of: amplifying an open reading frame sequence of nucleocapsid protein of a virus strain of a novel bunyavirus JS-2007-001 by a reverse transcription polymerase chain reaction (PCR) method, inserting the sequence into pQE30 to construct a pQE30-NP expression plasmid, transfecting an M15 strain by using pQE30-NP, performing inducible expression of 6His-NP recombinant protein by using isopropyl-beta-d-thiogalactoside (IPTG), purifying the 6His-NP recombinant protein by using Hitrap, and marking to obtain HRP-coupled NP(HRP-rNP). The reaction plate can be combined with a novel bunyavirus-resistant antibody (IgM) in a sample when incubated with the sample to be tested conventionally, and the HRP-rNP incubation is used in the subsequent detection, so that an 'anti-human IgM-antibody-enzyme-labeled antigen' capture detection system is formed; the color reaction after an enzyme substrate, namely the TMB color development agent is added is analyzed by an enzyme-labeled instrument; and the content of the specific antibody (IgM) of the novel bunyavirus is obtained according to the color development degree.
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Description

1. Field of invention

[0001] The invention belongs to the field of biotechnology, and in particular relates to the preparation and application of an ELISA kit for detecting neobunia virus antibody IgM. 2. Background of the invention

[0002] New Bunyavirus (Novel Bunyavirus, SFTS Bunyavirus), is the pathogen of fever with thrombocytopenia syndrome (Fever with Throbocytopenia Associated Syndrome, SFTS), and is a member of the Bunyaviridae family. It is a new type of virus of the genus Pyrivirus, first discovered in China in 2009, and it is also the first new type of 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. So far, more than 300 cases have been found in 16 provinces across the country, including Henan, Jiangsu, Hubei, Shandong, Anhui and Liaoning, resulting in more than 40 deaths. The...

Claims

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