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Dengue virus IgG antibody ELISA diagnostic kit

A technology for dengue virus and immunodiagnosis, which is applied in the field of dengue virus antibody enzyme-linked immunoassay diagnostic kits, can solve the problems of high price, difficult to popularize, delayed diagnosis and the like, and achieves the prospect of wide popularization and application, the method is simple and the sensitivity is high Effect

Inactive Publication Date: 2008-11-19
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 IgG antibody ELISA diagnostic kit
  • Dengue virus IgG antibody ELISA diagnostic kit
  • Dengue virus IgG 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 BL21 Star for expression TM (DE3) was purchased from Invitrogen and preserved by the Institute of Microbiology, Guangdong Provincial Center for Disea...

Embodiment 2

[0102] Embodiment two: the mensuration of recombinant antigen to dengue virus IgG antibody reactivity

[0103] The purified extracted protein was serologically tested by indirect ELISA to determine its reactivity to dengue virus antibody-positive sera.

[0104] The antigen obtained in Example 1 was diluted 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 immune activity of dengue antigen is 1:1000.

[0105] The recombinant antigen was coated with selected concentration, and the serum IgG antibody of dengue fever patients was detected by indirect method ELISA, and the reactivity of the recombinant antigen to dengue virus IgG antibody was determined.

[0106] 1. Reactivity of recombinant antigens to serum samples from the 2004 Zhongshan dengue fever outbreak

[0107] Type 1-4 E protein recombinant antigens were coated at a selected concentration, and the IgG antibodies in the serum...

Embodiment 3

[0121] Embodiment three: the production technology of dengue virus IgG antibody ELISA kit according to the present invention

[0122] (1) Materials

[0123] 1. Dengue antigen: see Example 1 for preparation. When the antigen is coated at a ratio of 1:1000, the positive coincidence rate to the in-factory reference product reaches 98%, and the specificity reaches 97%.

[0124] 2. Goat anti-human IgG-HRP: product of Shenzhen Ruisang Company.

[0125] 3.TMB: German Bollinger product.

[0126] 4. Positive control serum: the positive control serum is made by adding the positive serum of a certain concentration in the PBS containing 20% ​​goat serum and 0.02% sodium azide 10mM pH7.4, and the A value of the positive control should be Greater than 1.5.

[0127] 5. Negative control serum: Negative control serum was prepared by adding 5% of the mixed negative serum to 10 mM pH 7.4 PBS containing 20% ​​goat serum and 0.02% sodium azide. The A value of the negative control should be le...

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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 IgG-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. Early diagnoses can be made to patients infected with dengue fever for the second time or more than two times and serum epidemiological investigation to healthy people can be conducted through detecting dengue virus IgG antibodies in serum. Final diagnoses can be made to patients infected with dengue fever for the first time.

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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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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