ELISA (Enzyme-linked Immuno Sorbent Assay) kit and detection method for avian influenza H7N9 hemagglutinin HA antigen

A technology for antigen detection and hemagglutinin, which is applied in biological testing, measuring devices, immunoassays, etc., can solve the problems of in-depth research, low specificity, and long detection window period, and achieve the effect of avoiding economic burden and high sensitivity

Inactive Publication Date: 2017-05-31
剑药(苏州)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the outbreak of H7N9 highly pathogenic avian influenza virus was quickly quelled with the joint efforts of governments at all levels and the whole people, due to the presence of H7N9 avian influenza virus in the natural environment, the risk of sporadic human infection with H7N9 is extremely high. In 2016 In the first half of this year, a total of 24 cases of human infection with H7N9 bird flu occurred in Jiangsu, and 9 people died. Beijing has also reported a total of 3 confirmed cases of human infection with H7N9 bird flu. Experts generally believe that the threat of a comeback of H7N9 highly pathogenic bird flu still exists , so the virology and epidemiology of H7N9 need to be further studied
However, the current H7N9 detection methods have problems such as long detection window period, easy missed detection, high false positive rate, and low specificity.

Method used

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  • ELISA (Enzyme-linked Immuno Sorbent Assay) kit and detection method for avian influenza H7N9 hemagglutinin HA antigen
  • ELISA (Enzyme-linked Immuno Sorbent Assay) kit and detection method for avian influenza H7N9 hemagglutinin HA antigen
  • ELISA (Enzyme-linked Immuno Sorbent Assay) kit and detection method for avian influenza H7N9 hemagglutinin HA antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Obtaining the H7N9 hemagglutinin HA gene

[0049] (1) The avian influenza H7N9 gene sequence HA(ΔTM)(H7N9)(A / Shanghai / 1 / 2013)(a.a.19-524)(GISAID#:EPI439486) was searched from the gene bank Genebank, according to the selected expression system ( Invitrogen pcDNA TM 3.1 (+) eukaryotic cell expression system) carry out codon-optimized design on cDNA and synthesize the full-length gene, and synthesize the designed gene sequence through PCR cycle amplification. The PCR product is electrophoresed in 1% agarose gel, and the band Compatible with the target fragment size.

[0050] (2) Gel cutting recovery: Cut the target gene band on the gel with a knife in a UV light box; weigh the weight of the empty EP tube, put the cut gel with the target gene into the weighed EP tube, and weigh out The weight of the glue; add Buding Buffer according to 1g / 1ml of BudingBuffer (that is, add 1ml of Buding Buffer per 1g of glue); put it in a preheated water bath at 55-60°C for 7 mi...

Embodiment 2

[0051] Example 2: Construction of mammalian cell expression vector and transfection

[0052] The H7N9 hemagglutinin HA gene fragment obtained in Example 1 was subcloned into the eukaryotic expression vector pcDNA3.1(+), Invitrogen pcDNA TM 3.1 (+) eukaryotic cell expression system, amplify and extract the carrier plasmid containing the target gene, sequence and verify the accuracy of the constructed plasmid, obtain a medium amount of recombinant plasmid DNA through medium extraction, and use liposomes to transfer the recombinant plasmid into In HEK293 cells, samples were taken every 12 hours from 24 to 72 hours after transfection, and the expression of the target protein was detected by SDS-PAGE and Western-blot. The test results are shown in the attached figure 1 shown.

Embodiment 3

[0053] Example 3: Mass expression and purification of target protein

[0054] Culture HEK293 cells in large quantities, then transfect the recombinant plasmid constructed in Example 2 into the cells, produce the target protein by transient expression, collect the part (cell or culture supernatant) containing the target protein, and purify the expressed protein by affinity chromatography The recombinant protein is the standard protein. The results of each step are detected by SDS-PAGE electrophoresis and the quality of the final product is controlled. The correctness of the target protein is verified by Western-blot. The purity of the collected target protein is greater than 95%.

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Abstract

The invention relates to an ELISA (Enzyme-linked Immuno Sorbent Assay) kit and detection method for an avian influenza H7N9 hemagglutinin HA antigen. The kit comprises an H7N9 hemagglutinin HA specific antibody pre-coated ELISA plate, a standard protein, a detection antibody, a horseradish peroxidase enzyme-labeled rabbit anti-mouse IgG antibody, an antibody diluting solution, a color developing solution A, a color developing solution B, a stopping solution and a washing solution, wherein the standard protein is a purified recombinant H7N9 hemagglutinin HA; and the detection antibody is a biotin-labeled H7N9 hemagglutinin HA specific antibody. The kit provided by the invention can be used for rapidly and effectively distinguishing H7N9 infection patients from common seasonal influenza patients at the early period of virus infection and the kit is low in price, simple and high in sensitivity; and the kit can be widely applied to front-line hospitals and is used for detecting the H7N9 antigen in a blood serum sample of the influenza infected patients and isolating H7N9 antigen positive patients as soon as possible so as to effectively control epidemic situations.

Description

technical field [0001] The invention specifically relates to an ELISA kit and a detection method for detecting the avian influenza H7N9 hemagglutinin HA antigen. Background technique [0002] H7N9 avian influenza is a new type of avian influenza that was first discovered in Shanghai and Anhui at the end of March 2013. H7N9 avian influenza is a new subtype of influenza virus discovered for the first time in the world. It has not yet been included in my country's legally notifiable infectious disease monitoring and reporting system, and no vaccine has been released until early April 2013. Those who were infected by this virus all had fever and other symptoms in the early stage. As of April 2013, it has not been confirmed whether this type of virus has the characteristics of human-to-human transmission. According to investigations in April 2013, the gene of the H7N9 avian influenza virus came from the reassortment of wild birds in East Asia and chicken flocks in Shanghai, Zhej...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68
CPCG01N33/56983G01N33/6854G01N2469/10
Inventor 徐涵张东
Owner 剑药(苏州)生物技术有限公司
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