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

Inactive Publication Date: 2012-12-05
万里明
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the virus sequence has been elucidated, and the detection standard based on RT-PCR has been established, but the immunological detection reagents for SFTS have not yet been perfected, so...

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Example 1: Expression and purification of rNP

[0015] The inactivated preservation number is CCTCC V201211 New Bunia virus JS-2007-001 strain, and a conventional QIAamp RNA viral RNA mini kit (Qiagen, Germany) is used to prepare a viral RNA template; the following primers are used as amplification primers:

[0016] SEQ ID No. 1:

[0017] Primer1 5`-ggatccgcatgcATGTCAGAGTGGTCCAGGATTG-3`;

[0018] SEQ ID No.2:

[0019] Primer2 5`-gccaagcTTACAGATTCCTGTAAGCAGCAGCAG-3`;

[0020] Invitrogen One step RT-PCR kit (Invitrogen, USA) was used to amplify the open reading frame of the New Bunia virus NP protein; the reading frame was inserted into the site SphI of the bacterial expression plasmid pQE30 (Qiagen, Germany) using conventional molecular biology methods and HindIII, the bacterial expression plasmid pQE30-NP was obtained. The plasmid was conventionally transformed into E. Coli M15 strain (Qiagen, Germany), and the rNP recombinant protein was induced and expressed accor...

Embodiment 2

[0021] Example 2: Preparation of HRP-rNP.

[0022] rNP was labeled by modified sodium periodate method (Basic Medical Immunology Experiment, Jin Boquan, Li Enshan. Beijing World Book Publishing House, 1990). Dissolve 5mg of HRP in 0.5mL of distilled water, add 0.5mL of freshly prepared 0.06M NaIO4 aqueous solution, mix well and place in the refrigerator at 4°C for 30 minutes, take out and add 0.5mL of 0.16M ethylene glycol aqueous solution, place at room temperature for 30 minutes, then add Purified rNP aqueous solution 1mL, mix well and put in a dialysis bag, slowly stir and dialyze with 0.05M, pH9.5 carbonate buffer solution in a 4°C refrigerator for 6 hours to combine, then suck it out, add NaBH4 solution (5mg / mL) 0.2mL, put in a 4°C refrigerator for 2 hours, add the above-mentioned conjugate mixture into an equal volume of saturated ammonium sulfate solution, place in a 4°C refrigerator for 30 minutes, and centrifuge, and dissolve the obtained precipitate in a little 0.02...

Embodiment 3

[0023] Example 3: Preparation of anti-human IgM coated ELISA reaction substrate.

[0024] Anti-human IgM antibody was diluted to concentration (1-10 μg / mL) with 0.05M pH 9.6 carbonate coating buffer. Add the diluted antibody solution into the wells of the reaction plate, 100 microliters per well, coat at 4°C for 18-20 hours, discard the liquid in the wells, wash the plate several times, and use enzyme stabilizer (Shandong, Taitianhe Biological ) into the wells of each plate, 150-200 microliters per well. React at 4°C for 4-6 hours. Discard the enzyme stabilizer in the well, dry the coated plate by freeze-drying method, seal the reaction substrate with a sealing agent, 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 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.

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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Application Information

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IPC IPC(8): G01N33/543G01N33/53
Inventor 承静张晨炜许定花张欢欢王建梅吴亭亭
Owner 万里明
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