Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip
A liquid phase chip and antibody detection technology, applied in the field of biomedical immunochemistry, can solve the problems of wasting time and labor, consuming samples and reagents, and high detection costs
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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] serial number
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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