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Xinjiang hemorrhagic fever virus immunochromatography fast detection test paper

A technology of hemorrhagic fever virus and immunochromatography, which is applied in the field of immunological detection to achieve the effect of rapid detection

Inactive Publication Date: 2009-08-05
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the application of the monoclonal antibody to the nucleocapsid protein of Xinjiang hemorrhagic fever virus to immunochromatographic test paper to detect Xinjiang hemorrhagic fever virus

Method used

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  • Xinjiang hemorrhagic fever virus immunochromatography fast detection test paper
  • Xinjiang hemorrhagic fever virus immunochromatography fast detection test paper
  • Xinjiang hemorrhagic fever virus immunochromatography fast detection test paper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation of Monoclonal Antibody to Nucleocapsid Protein of Xinjiang Hemorrhagic Fever Virus

[0028] (1) Construction of a recombinant plasmid containing the nucleocapsid protein gene of Xinjiang hemorrhagic fever virus:

[0029] A full-length 1458bp S gene fragment was synthesized according to the nucleotide sequence of the CCHFV strain (GenBank accession number NC_005302), and BamH1 and XhoI restriction sites were added to the 5' end and 3' of the gene, respectively. The fragment was cloned into the pET30a(+) vector recovered by the same digestion to obtain the recombinant plasmid pENP.

[0030] (2) Induced expression and purification of recombinant nucleocapsid protein:

[0031] Transform the recombinant plasmid pENP into BL21(DE3) competent cells, pick a single colony in 5ml LB medium (25g / ml kanamycin resistance), culture at 37°C until the OD600 is about 0.6, add IPTG to make the final Concentration to 1mmol / L, 25 ℃ induced culture for 3h, collected...

Embodiment 2

[0036] Example 2: Western blot characteristic identification of monoclonal antibody to nucleocapsid protein of Xinjiang hemorrhagic fever virus

[0037] After SDS-PAGE electrophoresis, the CCHFV recombinant NP protein was transferred to a nitrocellulose membrane, and the diluted 10B12 and 3G5 ascites monoclonal antibodies were used as the primary antibody, and HRP-goat anti-mouse IgG was used as the secondary antibody for Western blot identification , and set pET30a (+) empty vector bacterial liquid as a control. image 3 It is the result of Western blot analysis of 3G5 monoclonal antibody and recombinant NP protein. The first lane to the third lane are respectively 1:200 dilution of monoclonal antibody, 1:1000 dilution of monoclonal antibody and 1:2000 dilution of monoclonal antibody The detection band of Western blot; the result shows that both 10B12 and 3G5 can specifically recognize the 59kDa NP recombinant protein.

Embodiment 3

[0038] Example 3: Indirect immunofluorescence test characteristic identification of monoclonal antibody to nucleocapsid protein of Xinjiang hemorrhagic fever virus

[0039] Prepare insect cell antigen sheets infected with recombinant baculovirus expressing NP protein, add 10B12 and 3G5 monoclonal antibodies as primary antibodies (1:50 dilution) to the detection wells, react at 37°C for 30 minutes, wash with PBS 3 times, add FITC-labeled goat anti-mouse IgG was reacted at 37°C for 60 minutes, washed three times with PBS, and observed under a fluorescent microscope. As a result, both 10B12 and 3G5 monoclonal antibodies reacted with Ac-NP-infected cells, and obvious green fluorescence appeared, while the negative control had no green fluorescence.

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Abstract

The invention provides a Xinjiang hemorrhagic fever virus immunochromatography rapid detection test paper comprising a supporting and fixing adhering lining layer on which a sample absorbing layer, a gold-marking binding pad, a fibrin film layer and a water absorbing layer are sequentially adhered to the supporting and fixing adhering lining layer, wherein the fibrin film layer takes a monoclonal antibody which is printed with Xinjiang hemorrhagic fever virus nucleocapsid protein as a detection blot zone. The fibrin film layer of the Xinjiang hemorrhagic fever virus immunochromatography rapid detection test paper is printed with sheep-anti-mouse IgG antibody solution as a contrapositive blot zone. A monoclonal antibody of the Xinjiang hemorrhagic fever virus nucleocapsid protein which is marked by colloidal gold is absorbed on the gold-marking binding pad of the test paper. The Xinjiang hemorrhagic fever virus immunochromatography rapid detection test paper can effectively and rapidly detect the Xinjiang hemorrhagic fever virus.

Description

technical field [0001] The invention relates to an immunological detection method, in particular to a rapid detection test paper for Xinjiang hemorrhagic fever virus immunochromatography. Background technique [0002] Crimean-Congo hemorrhagic fever virus (CCHFV) is an arbovirus transmitted by ticks and belongs to the genus Nairovirus in the Bunyaviridae family. The genome of CCHFV is a segmented, single-stranded, negative-strand RNA consisting of three segments, small (S), medium (M), and large (L), encoding nucleoprotein (NP), glycoprotein (GP) and RNA polymerase (RNA-dependent polymerase). Crimean-Congo hemorrhagic fever (CCHF) caused by CCHFV is a highly contagious disease prevalent in southern Xinjiang of China, northern Russia, the Middle East, southern Eurasia and Sahara Africa, with an average case fatality rate of 10% to 10%. 50%. China's CCHF was first diagnosed in southern Xinjiang in 1965, and antibodies were also detected in northern Xinjiang, so it was calle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 相大鹏师永霞黄吉城郑夔洪烨李小波幸芦琴郭波旋钟玉清
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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