Method applying AlphaLISA to detect dengue virus and corresponding combination and kit

A dengue virus and detection method technology, applied in the field of biomedicine, can solve the problems of long time consumption, difficulty in adapting to large sample detection, cumbersome operation steps, etc., and achieve good specificity and sensitivity

Inactive Publication Date: 2019-03-08
珠海国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method is cumbersome in operation steps, takes a long time, requires multi-step washing, and is difficult to adapt to large sample detection

Method used

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  • Method applying AlphaLISA to detect dengue virus and corresponding combination and kit
  • Method applying AlphaLISA to detect dengue virus and corresponding combination and kit
  • Method applying AlphaLISA to detect dengue virus and corresponding combination and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of AlphaLISA kit for detecting dengue virus.

[0032] The buffers of the capture antibody and the detection antibody, which are paired antibodies, were exchanged to desalt and purify and remove sodium azide (NaN 3 ). Zeba from Thermo Fisher Scientific TM Put the centrifugal desalting column into the corresponding collection tube, and centrifuge at 1500g for 1min to remove the storage solution. Wash 2 more times with 1×PBS. Add the antibody stock solution and put on a new collection tube, centrifuge at 1500g for 1min, keep the antibody solution in the collection tube, and prepare the antibody concentration at 1mg / ml.

[0033] Detection antibody conjugated to biotin. Add the purified 1mg / mL detection antibody with pH ≥ 7.0 to 2mg / mL NHS-ChromaLink-biotin biotin labeling reagent solution and adjust the volume to 200uL, incubate at 23°C for 2 hours, and then use Zeba TM The biotin-labeled antibody was collected after desalting spin column purific...

Embodiment 2

[0038] Example 2: Evaluation test of the AlphaLISA kit for detecting dengue virus.

[0039] Detection reagents: Prepare relevant reagents according to the method described in Example 1.

[0040] Detection steps: add the sample to be tested or positive quality control or negative quality control to a 384-well microplate, then add receptor beads coated with capture antibody, then add biotin-labeled detection antibody, and incubate at 23°C After 30-60 minutes, add the donor microbeads coated with streptavidin, incubate for another 20-30 minutes in a dark environment, and then detect the fluorescence value.

[0041] 1. Linear correlation analysis

[0042] The positive quality control product and the negative quality control product of gradient dilution are detected simultaneously with the method of the present invention, take the logarithm of the sample concentration as the abscissa, the fluorescent signal value is the ordinate, and fit a linear curve to obtain dengue virus NS1 ...

Embodiment 3

[0049] Embodiment 3: performance evaluation test

[0050] Method 1 is the AlphaLISA method established by the present invention. The second method is a commercial kit (dengue fever NS1 rapid detection reagent from cortez company in the United States), and its detection sensitivity is 6 ng / mL.

[0051] Sample: Randomly select 100 clinical serum samples, including dengue positive cases and normal people, and use the above two methods for detection at the same time.

[0052] After the test was completed, the negative-positive coincidence rate and the percentage of consistency were analyzed. The results are shown in Table 1.

[0053] Table 1 Qualitative detection methodological performance evaluation results

[0054]

[0055]

[0056] According to the test results, the positive coincidence rate is 95.38%, the negative coincidence rate is 94.29%, and the degree of agreement is 95%.

[0057] Conclusion: The AlphaLISA kit for detecting dengue virus provided by the present in...

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Abstract

The invention provides a method applying AlphaLISA to detect a dengue virus and corresponding combination and kit. An AlphaLISA detection composition comprises receptor bead coated with a capture antibody, a detection antibody marked with biotin and donor bead coated with streptavidin, and the capture antibody and the detection antibody are a pair of matched antibodies resistant to dengue virus NS1 protein. The composition serves as antigen for the NS1 protein, thereby having high specificity and sensitivity. The invention further discloses an AlphaLISA kit for detecting the dengue virus. Thekit comprises the AlphaLISA detection composition, a 384-hole microporous plate, a positive quality control product and a negative quality control product, the positive quality control product is matrix serum of the dengue virus NS1 protein, and the negative quality control product is a solution containing basic buffer. The microporous plate with many holes can be used for detection, and quick andqualitative detection of the dengue virus in high-throughput large samples can be realized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, relates to medicine, immunology, microbiology and epidemiology, and specifically relates to a dengue virus NS1 protein AlphaLISA detection method. Background technique [0002] Dengue fever (dengue fever) is an acute insect-borne infectious disease of humans caused by dengue virus transmitted by mosquitoes, and it is widely prevalent in the world. Dengue virus infection can lead to latent infection, dengue fever, and dengue hemorrhagic fever after infecting the human body. The typical clinical manifestations of dengue fever are sudden onset, high fever, headache, severe pain in muscles, bones and joints, rash, bleeding tendency, enlarged lymph nodes, decreased white blood cell count, and thrombocytopenia in some patients. The disease is mainly popular in tropical and subtropical regions, and my country's Guangdong, Hong Kong, Macao and other places are the endemic areas of dengue fever. Bec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/533
CPCG01N33/533G01N33/56983Y02A50/30
Inventor 莫秋华史蕾田绿波汪海波杨泽陈新彬李伟刚苏影涂承宁
Owner 珠海国际旅行卫生保健中心
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