Preparation method and using method of protein suspension chip for detecting tick borne encephalitis antibody in serum sample

A tick-borne encephalitis and suspension chip technology, which is applied to the preparation and use of protein suspension chips for detecting tick-borne encephalitis antibodies in serum samples, can solve the problems of lack of models and evaluations, and achieve good detection results

Inactive Publication Date: 2011-01-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
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  • Application Information

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Problems solved by technology

However, whether the suspension chip method can detect tick-borne encephalitis antibodies in ...

Method used

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  • Preparation method and using method of protein suspension chip for detecting tick borne encephalitis antibody in serum sample
  • Preparation method and using method of protein suspension chip for detecting tick borne encephalitis antibody in serum sample
  • Preparation method and using method of protein suspension chip for detecting tick borne encephalitis antibody in serum sample

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0041] 2. Preparation of samples to be tested

[0042] 1. Preparation of target analyte samples

[0043] The target analyte is rabbit anti-tick-borne encephalitis IgG, and the interfering sample or the sample used as method-specific test is 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-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-tick-borne encephalitis IgG was 0.01. In the comparison experiment, the same sample was used for the detection of ELISA and suspension chip.

[0044] The rabbit anti-tick-borne encephalitis IgG sample diluent to be analyzed was diluted into samples of different concentrations in a 4-fold ratio serially to draw a standard curve for sample detection dose-response, and the concentrati...

Embodiment 1

[0047] Example 1. Preparation of a protein suspension chip for detecting tick-borne encephalitis antibodies

[0048] 1. Capturing antigen-coated encoded microspheres

[0049] The No. 027 coded microspheres used in the present invention were purchased from Bio-Rad Company of the United States. The coded microspheres are used to label the tick-borne encephalitis E-D3 protein antigen that can capture tick-borne encephalitis antibodies, that is, to use the tick-borne encephalitis E-D3 Protein-coated microspheres.

[0050] A. Activation of encoded microspheres

[0051] 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 p...

Embodiment 2

[0061] Embodiment 2, optimization of suspension chip preparation method conditions

[0062] 1. Selection of the amount of antigen coating on microspheres

[0063] 100 μL of microspheres coded No. 027 were coated in amounts of 1 μg, 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg, 20 μg, 25 μg, and 30 μg, respectively. After testing the effect comparison, 1~30μg / 1.25×10 6 A coded microsphere is 0.2-120ng / 2500-5000 microspheres / test, and the coating effect is good. After counting under a microscope, store in dark and refrigerated until use. Such as figure 1 As shown, the No. 027 microspheres coated with tick-borne encephalitis E protein antigen all fell in the correct detection area, and obtained high signal-to-noise ratio results (MFI value is much greater than 2000), indicating that the optimized suspension chip detection system can be successful For the detection of tick-borne encephalitis antibodies.

[0064] 2. Optimization of biotinylated detection substances

[0065] The...

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Abstract

The invention discloses a preparation method and a using method of a protein suspension chip for detecting a tick borne encephalitis antibody in a serum sample. The using method of the invention has the advantages of high detection capacity, high flexibility, high specificity and wide dynamic range; and an open detection modularized platform for the detection of the protein suspension chip on a viral antibody represented by the tick borne encephalitis antibody is established.

Description

technical field [0001] The invention relates to a method for preparing and using a protein suspension chip for detecting antibodies to tick-borne encephalitis in serum samples. Background technique [0002] Tick-borne encephalitis (TBE), a common viral central nervous system (CNS) disease with hundreds of hospitalizations each year, is caused by tick-borne encephalitis virus (TBEV), a flavivirus transmitted by ticks Clinically, it is characterized by sudden high fever, meningeal irritation, disturbance of consciousness and paralysis, abnormal changes in cerebrospinal fluid, and often sequelae. [0003] Immunological methods Enzyme-linked immunoassay (ELISA) uses the specific reaction between antigen and antibody to detect antigen or antibody, mainly including double-antigen sandwich detection of antibody, double-antibody sandwich detection of antigen, competition method, indirect method, etc. The principle of indirect antibody detection is that the specific antigen is bound...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/64
Inventor 王静杨永莉杨宇胡孔新张晓龙曹晓梅
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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