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New method and product for detecting influenza antibody in serum sample

A technology for detecting antibodies and influenza, applied in the field of new detection of influenza antibodies and products in serum samples, which can solve problems such as lack of models and evaluations

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

AI Technical Summary

Problems solved by technology

[0005] The currently developed suspension chip is mainly aimed at the detection of cytokines, whether it can detect plague antibodies in human serum, and how its quantitative detection ability is, there is still a lack of models and evaluations

Method used

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

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0042] 2. Preparation of samples to be tested

[0043] 1. Preparation of target analyte samples

[0044] The target analyte is mouse anti-influenza NP 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 IgG, rabbit anti-SARS IgG, 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 mouse anti-influenza NP IgG was 0.5 mg / mL.

[0045] Dilute the mouse anti-influenza NP 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 sample should saturate the binding sites of the encoded microspheres.

[0046] 2. Processing of human serum samples

[0047] Dilute the...

Embodiment 1

[0048] Embodiment 1, the preparation of the protein suspension chip that detects influenza antibody

[0049] 1. Capturing antigen-coated encoded microspheres

[0050] 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 influenza NP protein antigen that can capture influenza antibodies, that is, the influenza NP protein is used to coat the microsphere.

[0051] A. Activation of encoded microspheres

[0052] 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...

Embodiment 2

[0062] Embodiment 2, optimization of suspension chip preparation method conditions

[0063] 1. Selection of the amount of antigen coating on microspheres

[0064] 100 μL of microspheres coded as No. 031 were coated with 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, and 30 μg, respectively. After testing the effect comparison, with 10μg / 1.25×10 6 A microsphere, that is, 20-40ng / 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. 031 microspheres coated with influenza NP 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 for influenza Antibody detection.

[0065] 2. Optimization of biotinylated antibodies

[0066] The present invention respectively uses amino a...

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Abstract

The invention relates to a new quantitative detection method and a product for detecting influenza antibody in a serum sample. 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 influenza antibody as a representative.

Description

technical field [0001] The invention relates to a novel quantitative detection method and product for detecting influenza antibodies in serum samples and a preparation method thereof. Background technique [0002] Influenza virus, referred to as influenza virus, is an RNA virus that causes influenza in humans and animals. In taxonomy, influenza virus belongs to the Orthomyxoviridae family. It can cause acute upper respiratory tract infections and rapidly spread through the air. There are often periodic pandemics around the world. Influenza virus can cause more serious symptoms such as pneumonia or heart and lung failure in the elderly or children with weakened immunity and some patients with immune disorders. Influenza virus consists of three layers, the inner layer is the viral nucleocapsid, which contains nucleoprotein (NP), P protein and PNA. NP is a soluble antigen (S antigen), with type specificity and stable antigenicity. P protein (P1, P2, P3) may be a polymerase r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N21/64G01N33/545
Inventor 王静杨永莉孙肖红杨宇胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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