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ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting Japanese encephalitis virus antigens in swine, human and mosquitoes and application

A technology of Japanese encephalitis virus, Japanese encephalitis, applied in the field of animal virology and immunology

Inactive Publication Date: 2013-10-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]Although the ELISA detection method for Japanese encephalitis antigen and antibody has been carried out in China, there is still no A mature method that can be used not only for the detection of Japanese encephalitis antigen in humans, but also for the detection of Japanese encephalitis antigen in pigs, mosquitoes and other animal samples

Method used

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  • ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting Japanese encephalitis virus antigens in swine, human and mosquitoes and application
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting Japanese encephalitis virus antigens in swine, human and mosquitoes and application
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting Japanese encephalitis virus antigens in swine, human and mosquitoes and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1) Cloning of Japanese encephalitis virus E gene and construction of prokaryotic expression vector

[0053] According to the method reported by Xu Gaoyuan (Xu Gaoyuan et al., Cloning, sequencing and expression of E gene of Japanese encephalitis virus SA14-14-2 strain, Chinese Journal of Zoonoses. 2004, 20(1): 22-25) , using the recombinant plasmid pKG-E containing the Japanese encephalitis virus E gene (gene accession number: AF315119.1) (see the plasmid construction diagram image 3 ) to transform Escherichia coli BL21 competent cells, the coating concentration is 60ug / mL LB / ampicillin (Amp) plate, select multiple single colonies and put them into LB liquid medium, cultivate them at 37°C for 12 hours, and then use isopropyl sulfide Dai-β-D-galactoside (IPTG) induces the expression of Japanese encephalitis virus E protein, the nucleotide sequence of which is shown in SEQ ID: 1.

[0054] 2) Preparation of immune spleen cells and myeloma cells

[0055] After purificatio...

Embodiment 2

[0070] 1. Determination of the optimal reaction conditions of the Japanese encephalitis double antibody sandwich ELISA antigen detection kit

[0071] The monoclonal antibody-coated ELISA plate prepared by the present invention is used as the first antibody, the rabbit-derived polyclonal antibody is used as the second antibody, and the HRP-labeled goat anti-rabbit antibody is combined with the rabbit-derived polyclonal antibody to catalyze the color development of the TMB substrate. The optimal coating concentration of monoclonal antibody was determined to be 5ug / ml by square array titration, the optimal dilution of polyclonal antibody was 1:10000, and the optimal dilution of enzyme-labeled antibody was 1:30000.

[0072] 2. Determination of Yin-Yang Boundary of Japanese Encephalitis Double Antibody Sandwich ELISA Antigen Detection Kit

[0073] Detect samples negative for Japanese encephalitis virus infection confirmed by RT-PCR, including: 46 pig brain samples, 20 human cerebro...

Embodiment 3

[0105] 3.1 ELISA kit assembly:

[0106] (1) 96-well ELISA plate, coated with E protein monoclonal antibody;

[0107] (2) Standard positive control: Japanese encephalitis E protein;

[0108] (3) Standard negative control: Escherichia coli BL-21 bacterial protein;

[0109] (4) Rabbit polyclonal antibody;

[0110] (5) horseradish peroxidase-labeled goat anti-rabbit enzyme-labeled antibody;

[0111] (6) Washing liquid (10×concentration): NaCl 80.0g, KH 2 PO 4 2.7g, Na 2 HPO 4 12H 2 O 14.2g, KCl 2.0g, Tween20 5ml, add double distilled water to 1000ml;

[0112] (7) Sample diluent (blocking solution): NaCl 0.8g, KH 2 PO 4 0.27g, Na 2 HPO 4 12H 2 O 1.42g, KCl 0.2g, BSA 10g, add double distilled water to 1000ml;

[0113] (8) Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100ml, add double distilled water to 1000ml;

[0114] (9) Substrate solution B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4ml, add...

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Abstract

The invention belongs to the technical field of animal virology and immunology, in particular to a double-antibody sandwich ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting Japanese encephalitis virus antigens in swine, human and mosquitoes. Core reagents of the kit comprise a Japanese encephalitis virus E protein monoclonal antibody serving as a primary antibody and a rabbit polyclonal antibody serving as a secondary antibody. The invention discloses a preparation and purification method of the Japanese encephalitis virus E protein monoclonal antibody and the rabbit polyclonal antibody. The invention also discloses a detection method of the double-antibody sandwich ELISA. A hybridoma cell strain secreting the monoclonal antibody is preserved in China Center for Type Culture Collection with CCTCC NO. C2010114.

Description

technical field [0001] The invention relates to the technical field of animal virology and immunology. It specifically relates to a double-antibody sandwich ELISA kit for detecting Japanese encephalitis antigen established by using Japanese encephalitis virus mouse-derived monoclonal antibody and rabbit-derived polyclonal antibody, and its application. The kit can be used for rapid detection of pigs, humans, Japanese encephalitis virus antigen in various clinical samples such as mosquitoes. The present invention provides the preparation and purification of Japanese encephalitis virus E protein monoclonal antibody and rabbit-derived polyclonal antibody, as well as the double-antibody sandwich ELISA detection that can detect Japanese encephalitis antigen established by using the monoclonal antibody and polyclonal antibody method. Background technique [0002] Japanese encephalitis (JE, hereinafter referred to as JE) is a severe zoonotic arboviral disease caused by Japanese e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/569G01N33/543C12R1/91
Inventor 曹胜波梅力李么明陈龙邵林朱碧波吴兵陈焕春
Owner HUAZHONG AGRI UNIV
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