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Preparation method of protein chip

A protein chip and substrate technology, applied in the field of protein chips or immunoassays, can solve the problems of signal interaction, high signal background, shedding, etc.

Inactive Publication Date: 2004-10-06
上海裕隆神光医学检验实验室有限公司
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Problems solved by technology

[0012] However, the biggest disadvantage of using a membrane as a support is that the immobilization of the membrane to the protein is only due to the adsorption of the membrane to the protein, the immobilization force is weak, and it will cause shedding after repeated washing.
Using the solid phase carrier of the protein chip for the membrane, the signal background is high and uneven
Moreover, the protein lattice is easy to diffuse during the reaction process, especially when the density of the array is high, it is easy to cause the signals of different points to affect each other due to the close distance.

Method used

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  • Preparation method of protein chip

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preparation example Construction

[0044] The invention also provides a preparation method of the protein chip solid phase carrier. Taking NC film as an example, its processing method includes the following steps:

[0045] (1) Pre-closed

[0046] The protein chip solid-phase carrier membrane (substrate) is pre-blocked with a biotin-labeled sealer, thereby forming a sealer layer on the solid-phase carrier.

[0047] A preferred method is to block the solid-phase carrier membrane used as a protein chip with a weight concentration of 2%-10% biotin-labeled blocker, at room temperature for 1-2 hours or overnight at 4°C.

[0048] As for soaking temperature and time, there is no particular limitation. Typically, the soaking time in the avidin-biotin complex solution is 90-150 minutes or longer at room temperature, or overnight at 4°C.

[0049] The concentration of the biotin-labeled blocker in the pre-blocking solution is usually 5-1000ug / ml, preferably 100ug / ml-500ug / ml, calculated according to the content of bioti...

Embodiment 1

[0064] Hereinafter, the detection of hepatitis C anti-HCV antibody is taken as an example to further illustrate the present invention, but it is not limited to the scope of the present invention.

[0065] (a) Preparation of protein chip solid phase carrier

[0066] Choose a hydrophilic NC membrane with a pore size of 0.4mm produced by Whatman, block with long-arm biotinylated (BCNHS) 2-10%, pH 7.8 PBS deesterified milk powder buffer, and block for 1-2 hours at room temperature or 4°C over liquid. Wash with pH 7.8 PBS buffer three times, dry at room temperature; soak in 0.5% streptavidin solution for 30 minutes, and dry in air.

[0067] (b) Preparation of protein chip

[0068] Use a high-speed spotting instrument to spot the genetically engineered fusion antigen (including Core\NS3\NS4\NS5) of hepatitis C long-arm biotinylated (BCNHS) on the above-mentioned treated NC membrane, each array 16 (4 × 4 ) samples, each with a diameter of 0.4mm, and dry at room temperature.

[00...

Embodiment 2

[0073] The sequencing of Example 1 was repeated, the only difference being that different concentrations of avidin and biotin, different ratios of avidin:biotin and different solid phase carriers were used (see Table 2 for details).

[0074] Solid phase carrier

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Abstract

A solid carrier of protein chip is composed of a substrate, a biotin marked enclosing substance layer on said substrate, and an avidin layer on said enclosing substance layer. Its advantages are less diffusion of sample applied point and high sensitivity. A process for preparing the solid carrier of protein chip is also disclosed.

Description

technical field [0001] The invention belongs to the field of protein chip or immunoassay, and more specifically relates to a new protein chip carrier and a processing method thereof. Background technique [0002] In order to improve the detection throughput and detection efficiency, a microarray enzyme-linked immunoassay technology, also known as protein chip technology, has been developed in recent years. It uses the specificity of antibody-antigen binding, that is, the immune response to detect samples. [0003] The simplified model of protein chip construction is: select a solid phase carrier that can firmly bind protein molecules (antigens or antibodies), thus forming a protein microarray, that is, a protein chip. The protein microarray is constructed on the substrate, and each point in the array represents an independent biochemical reaction, so that many reactions are integrated on a small carrier. [0004] In the process of protein chip manufacturing, the preparation...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/50G01N33/53G01N33/543G01N33/68
Inventor 穆海东
Owner 上海裕隆神光医学检验实验室有限公司
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