New method and product for detecting avian influenza antibody in serum sample

A technology for detecting antibodies and avian influenza, applied in measuring devices, instruments, fluorescence/phosphorescence, etc., can solve the problems of lack of models and evaluations, and achieve good consistency results

Inactive Publication Date: 2009-09-16
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Whether the suspension chip can detect the avian influenza antibody in human serum, a

Method used

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  • New method and product for detecting avian influenza antibody in serum sample
  • New method and product for detecting avian influenza antibody in serum sample
  • New method and product for detecting avian influenza antibody in serum sample

Examples

Experimental program
Comparison scheme
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-avian influenza H5N1 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 rabbit anti-tuberculosis serum, rabbit anti-SARSIgG, rabbit anti-avian influenza H5N1 serum, Rabbit anti-plague IgG, 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-avian influenza H5N1 IgG is 0.2mg / mL. In the comparison experiment, the same sample was used for the detection of ELISA and suspension chip.

[0047] Dilute the rabbit anti-avian influenza H5N1 IgG sample diluent to be analyzed into samples of different concentrations in a 4-fold ratio serially to draw a standard curve for sample detection dose-response. The concent...

Embodiment 1

[0050] Embodiment 1, the preparation of the protein suspension chip that detects bird flu antibody

[0051] 1. Capturing antigen-coated encoded microspheres

[0052] The coded microsphere No. 032 used in the present invention was purchased from BIO-RAD Company of the United States. The coded microsphere was used to label the avian influenza H5 protein antigen capable of capturing the avian influenza antibody, that is, the avian influenza H5 protein was used to coat the microsphere.

[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, s...

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 as No. 027 were coated with 1 μg, 3 μg, 6 μg, 9 μg, 12 μg, 15 μg, and 20 μg, respectively. After testing the effect comparison, with 6μg / 1.25×10 6 A microsphere, that is, 12-24ng / 2500-5000 microspheres / test coating, has the best 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. 032 microspheres coated with avian influenza H5 protein antigens 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 Detection of antibodies against avian influenza.

[0067] 2. Optimization of biotinylated antibodies

[0068] The present invention resp...

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Abstract

The invention relates to a new quantitative detection method and a product for detecting avian influenza antibody in a serum sample, and a preparation method thereof. The method of the invention has good detection capability, high sensitivity, strong specificity and wide dynamic range. The invention establishes an open detection modularised platform of pathogenic bacteria antibody protein suspension chip detection with the avian influenza as a representative.

Description

technical field [0001] The invention relates to a novel quantitative detection method and product for detecting bird flu antibodies in serum samples and a preparation method thereof. Background technique [0002] Avian influenza virus belongs to type A influenza virus, and type A influenza virus mainly occurs in birds. Generally speaking, there are receptor-specific differences between avian influenza virus and human influenza virus, and avian influenza virus is not easy to infect humans. Individual avian influenza viruses that cause human infection may be mutated viruses, but in 1997, the avian influenza virus in Hong Kong broke through the interspecies barrier and directly infected humans for the first time. A total of 18 Hong Kong residents were infected with the H5N1 subtype influenza virus, and 6 of them died. The occurrence of the virus has broken people's traditional understanding of avian influenza viruses. Studies have shown that the subtypes of avian influenza viru...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/546G01N21/64
Inventor 王静杨永莉杨宇孙肖红胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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