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Protein suspension array for detecting tularaemia antibody in serum sample, preparation method and using method thereof

A suspension chip and detection method technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of lack of models and evaluations, and achieve good consistency results

Inactive Publication Date: 2014-01-29
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 Tura 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 array for detecting tularaemia antibody in serum sample, preparation method and using method thereof
  • Protein suspension array for detecting tularaemia antibody in serum sample, preparation method and using method thereof
  • Protein suspension array for detecting tularaemia antibody in serum sample, preparation method and using method thereof

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Experimental program
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Effect test

preparation example Construction

[0045] 2. Preparation of samples to be tested

[0046] 1. Preparation of target analyte samples

[0047] The target analyte is rabbit anti-Tura 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 concentration of rabbit anti-Tura IgG is 0.1768mg / mL. In the comparison experiment, the same sample was used for the detection of ELISA and suspension chip.

[0048] Dilute the sample diluent of rabbit anti-Turra IgG 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 sensi...

Embodiment 1

[0051] Embodiment 1, the preparation of the protein suspension chip that detects Tula antibody

[0052] 1. Capturing antigen-coated encoded microspheres

[0053] The No. 028 coded microsphere used in the present invention was purchased from Bio-Rad Company of the United States. The coded microsphere was used to label the Tura fopA protein antigen capable of capturing the Tura antibody, that is, the Tura fopA protein was used to coat the microsphere.

[0054] A. Activation of encoded microspheres

[0055] 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...

Embodiment 2

[0065] Embodiment 2, optimization of suspension chip preparation method conditions

[0066] 1. Selection of the amount of antigen coating on microspheres

[0067] 100 μL of microspheres coded No. 028 were coated with 1 μg, 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg, 20 μg, and 25 μg, respectively. The tested effect is compared 1~25μg / 1.25×10 6 A coded microsphere or 0.2-100ng / 2500-5000 microspheres / test coating has a good coating effect. 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. 028 microspheres coated with Tula fopA 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 Tula antibody detection.

[0068] 2. Optimization of biotinylated detection substances

[0069] The present invention respectively uses amin...

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Abstract

The invention discloses a protein suspension array for detecting a tularaemia antibody in a serum sample, a preparation method and a using method thereof. The invention has the advantages of high detectability, high sensitivity, high specificity and wide dynamic range; moreover, an opening detection modular platform for detecting a tularaemia antibody-based bacterial antibody by using the protein suspension array is established.

Description

technical field [0001] The invention relates to a protein suspension chip for detecting Tura antibody in a serum sample, a preparation method and a use method thereof. Background technique [0002] Tularenmia is a zoonotic disease caused by Francisellat tularensis, also known as tularemia. Clinically, it is characterized by elevated body temperature, enlarged lymph nodes, and necrosis of the spleen and other internal organs. Tularemia can be used as a pathogenic bacteria in biological warfare. The infected person will have high fever, body pain, enlarged glands, and difficulty in swallowing and eating and other symptoms. [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 ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531
Inventor 王静杨永莉杨宇孙肖红张晓龙景滢滢
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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