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Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof

A technology for suspending chips and detecting antibodies, applied in the fields of resisting vector-borne diseases, measuring devices, instruments, etc., can solve problems such as lack of models and evaluations

Inactive Publication Date: 2010-12-15
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the suspension chip can detect dengue fever antibodies in human serum, and its quantitative detection ability, there is still a lack of models and evaluations.

Method used

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  • Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof
  • Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof
  • Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof

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Experimental program
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Effect test

preparation example Construction

[0043] 2. Preparation of samples to be tested

[0044] 1. Preparation of target analyte samples

[0045] The target analyte is mouse anti-dengue fever IgG, and the interfering samples or samples used as method-specific tests are other antibodies or other proteins other than the target detection object, including rabbit anti-Tura serum, rabbit anti-avian influenza H5 serum, rabbit anti-West Nile Antibodies, BSA, casein, tryptone, etc. All the above-mentioned samples to be analyzed were dissolved in the sample diluent and stored at 4°C. The stock solution concentration of mouse anti-dengue E2 IgG was 1.06 μg / mL.

[0046] Dilute the mouse anti-dengue IgG sample diluent to be analyzed into samples of different concentrations in a 4-fold ratio to draw a standard curve of sample detection dose-response. Among them, the concentration of several samples is lower than the sensitivity of detection, and the concentration of high concentration samples The binding sites of the encoded m...

Embodiment 1

[0049] Embodiment 1, the preparation of the protein suspension chip that detects dengue fever antibody

[0050] 1. Capturing antigen-coated encoded microspheres

[0051] The coded microsphere No. 031 used in the present invention was purchased from Bio-Rad Company of the United States. The coded microsphere is used to label the dengue E2 protein antigen that can capture the dengue antibody, that is, the dengue E2 protein is used to coat the microsphere.

[0052] A. Activation of encoded microspheres

[0053] Take 100μL (1.25×10 6 pcs) encoded microspheres into a 1.5mL centrifuge tube, centrifuge at 14000×g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere washing buffer to suspend, shake and sonicate, centrifuge at 14000×g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere activation buffer, then add 10 μL of freshly prepared EDC (50 mg / mL), then add 10 μL of freshly prepared 50 mg / mL Sulfo-NHS, shake at high speed for 30 sec...

Embodiment 2

[0063] Embodiment 2, optimization of suspension chip preparation method conditions

[0064] 1. Selection of the amount of antigen coating on microspheres

[0065] 100 μL of microspheres coded as No. 031 were coated with 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, and 50 μg, respectively. After testing the effect comparison, with 1~50μg / 1.25×10 6 A coded microsphere, that is, 0.2-200ng / 2500-5000 microspheres / coating effect is the best. After counting under a microscope, store it in a dark place and refrigerate it for later use. Such as figure 1 As shown, the No. 031 microspheres coated with dengue E2 protein antigen all fell in the correct detection area, and obtained high signal-to-noise ratio results (MFI value was much greater than 2000), indicating that the optimized suspension chip detection system can be successfully used for dengue fever Antibody detection.

[0066] 2. Optimization of biotinylated antibodies

[0067] The present invention res...

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Abstract

The invention relates to a protein suspension chip for detecting a dengue antibody in a serum sample and a preparation method and a using method thereof. The methods of the invention have high detection capability, high sensitivity, strong specificity and wide dynamic range and establish an open detection modelization platform for viral antibody, represented by the dengue antibody, protein suspension chip detection.

Description

technical field [0001] The invention relates to a protein suspension chip for detecting dengue fever virus antibody in a serum sample, a preparation method and a use method thereof. Background technique [0002] Dengue fever is an acute infectious disease caused by dengue virus and transmitted by Aedes mosquitoes. The clinical features are sudden onset, high fever, muscle, bone marrow and joint pain all over the body, extreme fatigue, and some patients may have rash, bleeding tendency and enlarged lymph nodes. Dengue virus belongs to group B arboviruses, now classified in the genus flavivirus of the family togaviridae. Virus particles are dumbbell-shaped (700×20-40nm), rod-shaped or spherical (20-50nm in diameter). The nucleus pulposus is a single strand of linear ribonucleic acid (RNA). Virus particles are similar to Japanese encephalitis virus. The outermost layer is an envelope composed of two glycoproteins. The envelope contains type and group-specific antigens. The t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
CPCY02A50/30
Inventor 王静杨永莉杨宇孙肖红胡孔新曹晓梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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