Immunologic diagnosis kit for detecting dengue virus NS1 antigen and application thereof

A technology for dengue virus and immunodiagnosis, applied in the field of medicine, can solve the problems of unfavorable clinical practice application, difficult to repeat standardization, low virus content, etc., and achieve the effect of overcoming difficulty in repeating, reducing missed detection, and high sensitivity

Active Publication Date: 2010-06-09
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

Several polyclonal antibody-based antigen capture ELISA methods for detecting dengue virus NS1 have been reported, but these methods are all established on the basis of polyclonal antibodies, and there are great differences in different batches of antisera. It is difficult to repeat and achieve laboratory standardization; and there are only two commercial kits using anti-NS1 monoclonal antibodies to detect dengue virus: Pan-EDengue Early ELISA test kit, Panbio, Queensland, Australia; PLATELIATM DENGUE NS1 AG, Bio-Rad, France, this kit contains monoclonal antibodies against dengue virus NS1 of types I~IV, and the early diagnosis of dengue virus infection can be realized by ELISA method, and its sensitivity, specificity and stability are relatively good
However, the inventors have further studied and found that the sensitivity of the kit (Pan-E Dengue Early ELISA test kit, Panbio, Queensland, Australia) to dengue viruses of types I to IV is different, especially to types III and type IV dengue viruses The sensitivity of the detection is very low, and the virus content in the blood samples of patients with early dengue virus infection is relatively low. Using this kit to detect the infection of type III and type IV dengue virus may be missed, which is not conducive to clinical practice application

Method used

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  • Immunologic diagnosis kit for detecting dengue virus NS1 antigen and application thereof
  • Immunologic diagnosis kit for detecting dengue virus NS1 antigen and application thereof
  • Immunologic diagnosis kit for detecting dengue virus NS1 antigen and application thereof

Examples

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preparation example Construction

[0016] (1) Preparation of immune antigen

[0017] The immunogen used to prepare the monoclonal antibody in the present invention is gene recombinant DV2NS1, DV3NS1, DV4NS1 protein and inactivated natural virus antigen. The genetically recombinant DVNS1 protein is prepared by an engineering strain carrying the DVNS1 gene, and its preparation is carried out according to a conventional method, and the NS1 antigen is obtained by purifying it with nickel primary nitrogen triacetate metal affinity chromatography, and the detailed preparation method Refer to the user manual. After NS1 protein was purified, it was quantified with Coomassie protein assay reagent (PIERCE, Cat, No. ED62976). The results of Western blot identification of the purified recombinant protein showed that the mouse anti-his mAb had a specific reaction band at a molecular weight of about 45KDa, which was consistent with the predicted molecular weight of DVNS1. The inactivated natural viral antigens are obtained...

example 1

[0043] (1) Preparation of reagents used

[0044] a. Sample treatment solution: composed of sample treatment solution A and sample treatment solution B, wherein A is a 1.5M glycine solution (PH2.8), and B is a 1.5M Tris-Hcl solution (PH9.7);

[0045] b. Concentrated lotion: 20×PBS containing 2% Tween-20, that is, 1L solution contains 4.56g NaH2PO4, 58.02gNa2HPO4.12H 2 O, 175.3g NaCl, 15 pounds of 20min after autoclaving, add 20ml Tween-20 and stir well, dilute 20 times when used;

[0046] c. Positive control: 1:1000 dilution of recombinant DVNS1 protein;

[0047]d. Negative control: 10mM PH7.4PBS containing 0.1% Tween-2, its preparation method is: 4.56g NaH 2 PO4, 58.02gNa 2 HPO4·12H 2 O, 175.3g NaCl dissolved in water and quantified to 1L, autoclaved at 15 lbs pressure for 20min, 0.1% Tween-20 was added after 20-fold dilution;

[0048] e. Chromogenic solution: It is composed of chromogenic solution A and B. When using, take the two equal amounts and mix them well. Wherei...

example 2

[0088] 1. The kit of the present invention consists of the following reagents:

[0089] (1) Microwell reaction plates coated with antibodies 2E8A5, DV2-M6 and 4B14A1;

[0090] (2) Sample treatment solution: composed of sample treatment solution A and sample treatment solution B, wherein A is a 1.5M glycine solution (PH2.8), and B is a 1.5M Tris-Hcl solution (PH9.7);

[0091] (3) Biotin-labeled monoclonal antibody 3B1A15;

[0092] (4) horseradish peroxidase-labeled avidin, purchased from Zymed Company;

[0093] (5) Concentrated lotion: 20×PBS containing 2% Tween-20, that is, 4.56g NaH in 1L solution 2 PO 4 , 58.02gNa 2 HPO 4 .12H 2 O, 175.3g NaCl, 15 pounds of 20min after autoclaving, add 20ml Tween-20 and stir well, dilute 20 times when used;

[0094] (6) Positive control: 1:1000 dilution of DVNS1 antigen;

[0095] (7) Negative control: 10mM PH7.4PBS containing 0.1% Tween-20, that is, 4.56g NaH in 1L solution 2 PO 4 , 58.02gNa 2 HPO 4 .12H 2 O, 175.3g NaCl, 15 pound...

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Abstract

The invention discloses an immunologic diagnosis kit for detecting dengue virus NS1 antigen and application thereof. The kit comprises a capturing antibody and a detecting body combined with a marker, and the kit is characterized in that the capturing antibody consists of a monoclonal antibody 2E8A5, a monoclonal antibody DV2M6 and a monoclonal antibody 4B14A1, the detecting antibody is a monoclonal antibody 3B1A15, and the marker can be biotin, horseradish peroxidase, alkaline phosphatase, colloidal gold or fluorescein. The kit can specifically detect NS1 proteins of four serotype dengue viruses and does not have cross reaction with other viruses of flavivirus system, such as flavivirus and encephalitis B virus, the sensitivity of the kit is at least four times higher than that of detection of foreign commodity kits, and the kit can efficiently reduce the omission factor of the clinical application.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a medical preparation, in particular to a kit for diagnosing dengue virus. Background technique [0002] Dengue fever is currently an important vector-borne infectious disease second only to malaria, and is mainly prevalent in tropical and subtropical regions; due to the development of tourism and international trade, changes in the ecological environment, global population growth and concentration in cities, and global climate change With the change of factors such as warming, the spread of dengue virus is no longer limited to some areas, but spread rapidly in most parts of the world with the flow of population. According to WHO statistics, more than 100 million people are infected with dengue virus every year, 500,000 of them develop into dengue hemorrhagic fever, and the annual death toll is as high as 30,000. At present, nearly 2.5 billion people in the world live in dengue fever-end...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/531
CPCY02A50/30
Inventor 车小燕丁细霞潘玉先丘立文
Owner SOUTHERN MEDICAL UNIVERSITY
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