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Dengue virus IgM antibody ELISA diagnostic kit

A technique for dengue virus and immunodiagnosis, which is applied in the field of dengue virus antibody enzyme-linked immunodiagnostic kits, can solve problems such as expensive, difficult to popularize, and delayed diagnosis, and achieve the effects of easy operation, simple method, and high sensitivity

Inactive Publication Date: 2012-09-19
广东省疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the laboratory diagnosis of dengue fever in my country mainly relies on imported enzyme-linked immunosorbent reagents, which are expensive and difficult to popularize in grass-roots hospitals and city and county-level disease control centers. Get the reagents in time, but delay the diagnosis
The first patient is often misdiagnosed and effective measures are not taken in time, which leads to the spread of dengue fever
Each epidemic has fallen into a very tense and passive situation when it is discovered, and then spends huge manpower and material resources to control it

Method used

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  • Dengue virus IgM antibody ELISA diagnostic kit
  • Dengue virus IgM antibody ELISA diagnostic kit
  • Dengue virus IgM antibody ELISA diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Cloning, expression and purification of dengue antigen

[0021] (1) Materials

[0022] 1. Standard strains: Dengue type 1 (Hawaii strain), referred to as D1; ​​Dengue type 2 (NGC strain), referred to as D2; Dengue type 3 (H87 strain), referred to as D3; Dengue type 4 (H241 strain) , referred to as D4; purchased from the National Institute for the Control of Pharmaceutical and Biological Products, and preserved by the Guangdong Provincial Center for Disease Control and Prevention.

[0023] 2. Plasmid: The cloning vector BM13 plasmid was purchased from TaKaRa Company, and the expression plasmid pET22b(+) was a product of Novagen. Preserved by the Microbiology Laboratory of the Guangdong Provincial Center for Disease Control and Prevention.

[0024] 3. Bacterial strains: E. coli TOP10F′ for recipient and E. coli BL21Star for expression TM (DE3) was purchased from Invitrogen and preserved by the Institute of Microbiology, Guangdong Provincial Center for Diseas...

Embodiment 2

[0082] Embodiment two: the mensuration of reactivity of recombinant antigen to dengue virus IgM antibody

[0083] The purified extracted protein was serologically tested by indirect ELISA to determine its reactivity to dengue virus antibody-positive sera.

[0084] The antigen obtained in Example 1 was diluted at 1:500, 1:1000, 1:2000, coated on an ELISA plate, and reacted with dengue positive serum to determine the concentration of the coated antigen. The antigenic activity of dengue antigen is 1:1000.

[0085] The recombinant antigen was coated at a selected concentration, and the dengue virus isolation positive and dengue fever epidemic serum IgM antibody were detected by indirect ELISA, and the reactivity of the recombinant antigen to the dengue virus IgM antibody was determined.

[0086] 1. Reactivity of recombinant antigens to virus isolation positive sera

[0087] Recombinant antigens were used to react with 16 positive samples of dengue virus isolates (13 samples of t...

Embodiment 3

[0096] Example Three: Sensitivity and Specificity of Recombinant Antigen Detection of Dengue Virus IgM Antibody

[0097] The recombinant antigen obtained in Example 1 was prepared into a dengue virus IgM antibody detection reagent (hereinafter referred to as self-developed reagent), and a large number of samples were detected with this reagent to evaluate the sensitivity and specificity of the recombinant antigen.

[0098] 1 Analysis of comparative results with imported reagents (Australia PanBio)

[0099] The positive samples D010044, D010046, D010047, D010050, D010052, D010073, D010074 in Table 1 and the 2004 dengue fever epidemic samples A040124, A040134, A040135, A040137 were all positive with the self-developed reagents, and the results were positive with the Australian PanBio dengue reagent (Dengue IgM Capture ELISA) test results were all negative, and the self-developed reagent was more sensitive than the dengue reagent of PanBio Australia for testing the serum of patie...

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Abstract

The invention relates to a dengue virus antibody enzyme-linked immunity diagnostic kit. The kit includes an enzyme labeling board, a negative control serum, a positive control serum, an enzyme marker, a sample diluent, a concentrated washing liquid, a substrate chromogenic liquid, a stop solution and a sealing plate glue, wherein the enzyme marker contains a goat anti-human IgM-HRP; an envelope antigen on the enzyme labeling board includes: a recombinant dengue virus type 1 envelope protein specific antigen with the amino acid sequence of SEQ ID No.1; a recombinant dengue virus type 2 envelope protein specific antigen with the amino acid sequence of SEQ ID No.3; a recombinant dengue virus type 3 envelope protein specific antigen with the amino acid sequence of SEQ ID No.5; and a recombinant dengue virus type 4 envelope protein specific antigen with the amino acid sequences of SEQ ID No .7. An early diagnosis can be made to patients infected with dengue fever for the first time through detecting dengue virus IgM antibodies in serum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an ELISA diagnostic kit, in particular to a dengue virus antibody ELISA diagnostic kit. Background technique [0002] Dengue fever (Dengue Fever, DF) is an acute mosquito-borne infectious disease caused by four serotypes of Dengue Virus (DV), mainly transmitted through the bite of Aedes aegypti or Aedes albopictus. Dengue virus belongs to the family Flaviviridae and belongs to the genus Flavivirus. DF is an arboviral disease with the widest distribution, the most incidence, and great harm. It is widely prevalent in more than 100 countries and regions in tropical and subtropical Africa, America, Southeast Asia and the Western Pacific region. According to WHO estimates, the health of about 2.5 billion people in the world is threatened, and 50 million people are infected with dengue virus every year. With the increasing global warming, the geographical distribution of dengue fever tends...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/53G01N33/531
CPCY02A50/30
Inventor 江立敏周惠琼郑夔柯昌文
Owner 广东省疾病预防控制中心
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