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

A technology of suspension chip and sine Ni, applied to the protein suspension chip for detecting West Nile antibody in serum samples and the fields of its preparation and use, can solve the problems of lack of models and evaluation, and achieve the effect of good consistency

Inactive Publication Date: 2011-01-05
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 method can detect West Nile antibody 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 west nile antibody in serum sample and preparation method and using method thereof
  • Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof
  • Protein suspension chip for detecting west nile 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

[0044] 2. Preparation of samples to be tested

[0045] 1. Preparation of target analyte samples

[0046] The target analyte is rabbit anti-West Nile 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 mouse anti-dengue fever IgG, rabbit anti-avian influenza H5 serum, rabbit anti-Tura Antibody, rabbit anti-dengue fever NS3 antibody, 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 titer of rabbit anti-West Nile IgG was 0.08. In the comparison experiment, the same sample was used for the detection of ELISA and suspension chip.

[0047] Dilute the rabbit anti-West Nile IgG sample diluent to be analyzed into samples of different concentrations in a 4-fold ratio serially to draw a standard curve of sample detection dose-response, among which several sample concentration...

Embodiment 1

[0050] Embodiment 1, the preparation of the protein suspension chip that detects West Nile antibody

[0051] 1. Capturing antigen-coated encoded microspheres

[0052] The coded microsphere No. 025 used in the present invention was purchased from Bio-Rad, USA. The coded microsphere was used to label the West Nile E protein antigen capable of capturing the West Nile antibody, that is, the microsphere was coated with the West Nile E protein.

[0053] A. Activation of encoded microspheres

[0054] 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 seconds, wrap in a...

Embodiment 2

[0064] Embodiment 2, optimization of suspension chip preparation method conditions

[0065] 1. Selection of the amount of antigen coating on microspheres

[0066] 100 μL of microspheres coded No. 025 were coated with 1 μg, 2 μg, 4 μg, 6 μg, 8 μg, 10 μg, 12 μg, 16 μg, 20 μg, and 24 μg, respectively. After testing the effect comparison, with 1~24μg / 1.25×10 6 coded microspheres or 0.2-96ng / 2500-5000 microspheres / test, the coating effect is the best, counted under the microscope and stored in dark and refrigerated until use. Such as figure 1 As shown, the No. 025 microspheres coated with West Nile E 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 in the detection of West Nile antibodies.

[0067] 2. Optimization of biotinylated detection substances

[0068] The present invention respectively use...

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Abstract

The invention relates to a protein suspension chip for detecting a west nile antibody in a serum sample and a preparation method and a using method thereof. The method has the advantages of high detectability, high sensitivity, high specificity and wide dynamic range; and an open detection modular platform for the protein suspension chip to detect viral antibodies represented by the west nile antibody is established.

Description

technical field [0001] The invention relates to a protein suspension chip for detecting West Nile antibody in a serum sample, a preparation method and a use method thereof. Background technique [0002] West Nile virus disease is an infectious disease caused by West Nile Virus (WNV), and it is a zoonotic disease. Humans do not spread each other after being infected with West Nile virus, and it is usually a recessive infection. The incubation period is 3 to 15 days, and most infected people have mild symptoms, accompanied by fever, headache, sore throat, back pain, muscle pain, joint pain, Fatigue, conjunctivitis, rash, swollen lymph nodes, anorexia, abdominal pain, diarrhea and respiratory symptoms, etc. For the elderly and children, it may cause high fever (≥40°C), severe headache and symptoms and signs of the central nervous system, such as neck stiffness, drowsiness, coma, convulsions, paralysis, etc., and even death. [0003] Immunological methods Enzyme-linked immunoa...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/64
Inventor 王静杨永莉孙肖红杨宇胡孔新姚李四
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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