Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip

A liquid phase chip and antibody detection technology, which is applied in the field of biomedical immunochemistry, can solve the problems of consumption of samples and reagents, high detection cost, waste of time and labor, etc.

Active Publication Date: 2013-12-18
NANJING BOMINDA BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the usual allergen-specific IgE antibody detection methods, such as ImmunoCAP, enzyme-linked immunoassay and chemiluminescent method, can only obtain one indicator for each reaction; if multiple indicators are to be detected, multiple reactions are required to complete, not only Waste of time and labor, while consuming a large amount of samples and reagents, the detection cost is relatively high

Method used

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  • Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip
  • Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip
  • Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: Composition of the liquid phase chip for detecting allergen specific antibody according to the present invention

[0071] In the present embodiment, the formula of various solutions is as follows:

[0072] Assay buffer, that is, PBS containing 0.1% BSA, 0.025% Tween-20, 0.05% NaN3, pH7.4.

[0073]

[0074] (1) A liquid phase chip for detecting 12 kinds of allergen-specific antibodies, the composition is as follows

[0075] (1) Probes: See Example 2 for the preparation methods of 12 kinds of allergen probes and total IgE probes; the coatings of various probes and the fluorescent codes of microspheres are as follows,

[0076]

[0077] (2) Detection antibody: a) biotin-labeled goat anti-human IgE antibody, used to detect allergen-specific IgE, IgE standard and total IgE; or b) biotin-labeled mouse anti-human IgG4 antibody, used for Detection of allergen-specific IgG4 antibodies;

[0078] (3) Streptavidin-labeled phycoerythrin (SA-PE); if the resultin...

Embodiment 2

[0082] Embodiment 2: A kind of method for preparing 12 kinds of allergen probes and total IgE probes used in the liquid phase chip of the present invention

[0083] (1) In the present embodiment, the formulations of various solutions are as follows:

[0084] 1. 50mM MES buffer, pH6.0

[0085]

[0086] 2. Phosphate buffer saline, namely PBS, pH7.4

[0087]

[0088] 3. Storage buffer, that is, PBS containing 0.5% BSA, 0.05% Tween-20, 0.1% NaN3, pH7.4.

[0089]

[0090] After the solution is prepared, it is filtered through a 0.22 μM filter, and stored in the dark at 2-8 degrees Celsius.

[0091] (2) In this example, the sources of main reagents and consumables are as follows,

[0092] 12 kinds of allergen freeze-dried powder: mite, cockroach, ragweed, mugwort, dog hair, cat hair, soybean, peanut, milk, egg, shrimp, crab allergen extract freeze-dried powder was purchased from Greer Laboratory in the United States;

[0093] Carboxylated microspheres with different flu...

Embodiment 3

[0124] Embodiment 3: Using the liquid phase chip of the present invention to detect 12 kinds of allergen-specific IgE antibodies in serum

[0125] (1) The steps are as follows,

[0126] 1. Take out all reagents before use, and place them to equilibrate to room temperature (20-25 degrees Celsius).

[0127] 2. Dilution of the standard: the concentration of the stock solution of the IgE standard is 100ng / ml; using the analysis buffer, the standard is diluted in a ratio of 1:2 to obtain the required final concentration. The dilution scheme of each tube, the expected concentration and the content of IgE in the reaction system are as follows,

[0128] Numbering

Standard series

diluent volume

Expected concentration of IgE

Amount of IgE / 100μL

Std1

stock solution

none

100ng / mL

10ng

Std2

200μL stock solution

400 μL assay buffer

33.3ng / mL

3.33ng

Std3

200 μL Std2

400 μL assay buffer

11.1ng / mL

...

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PUM

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Abstract

The invention discloses a liquid phase chip for detecting an allergen specific antibody. The liquid phase chip mainly comprises (1) a plurality of common allergen probes, comprising 12 fluorescence coded microspheres respectively coated with mite, cockroach, ragweed, felon herb, dog hairs, cat hairs, soybean, peanut, milk, egg, shrimp and crab allergen extracts; (2) a biotin labeling detection antibody; (3) a streptavidin phycoerythrin; (4) a total IgE (immunoglobulin E) probe used when semiquantitative or quantitative detection is carried out on the allergen specific IgE antibody, namely, a fluorescence coded microsphere coated with a rat anti-human IgE monoclonal antibody. The invention also discloses a preparation method of the liquid phase chip probe. The liquid phase chip disclosed by the invention is mainly used for detecting the allergen specific IgE antibody in a serum sample, also for detecting an allergen specific IgG4 (Intravenous Gamma Globulin 4) antibody and a total IgE antibody, and has the advantages of few used samples, parallel detection of a plurality of indexes, high flux, high sensitivity and the like.

Description

technical field [0001] The invention belongs to the field of biomedical immunochemistry, and relates to a liquid phase chip for detecting allergen-specific antibodies and a preparation method thereof. Background technique [0002] Allergy is a common disease. According to the World Health Organization, 20-40% of people have suffered from allergic diseases. It is induced by allergens (or allergens) and mediated by IgE antibodies bound to mast cells and basophils, the so-called type I allergy. Allergens are exogenous proteins that can bind to IgE antibodies on the surface of mast cells and basophils after entering the human body and cause allergic reactions. [0003] Allergy diagnosis includes three parts: clinical history, skin test and allergen-specific IgE detection. Only when the three are consistent can the diagnosis be confirmed. Allergen-specific IgE antibody detection, also known as in vitro allergen detection, aims to understand the IgE antibody content of a specifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N21/64
Inventor 程爱阳
Owner NANJING BOMINDA BIO TECH
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