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Protein chip of directional immobilizing antibody, its preparing method and use thereof

A protein chip and directional immobilization technology, applied in biological testing, material inspection products, etc., can solve the problems of losing the ability to bind antigen molecules, unfavorable antibody-antigen binding, and increased steric hindrance, so as to reduce non-specific adsorption , Improve analytical sensitivity, reduce the effect of steric hindrance

Inactive Publication Date: 2003-03-12
INST OF MECHANICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biological activity of antibody molecules immobilized directly on the solid phase surface is usually lower than that of antibody molecules in solution. The main reason for the decrease in biological activity is that the steric hindrance of antibody molecules immobilized directly on the solid phase surface increases, which is not conducive to Antibody-antigen binding, as described in the document "Effectiveness of protein A for antibody immobilization for a fiber optic biosensor" (Biosensors & Bioelectronics, Vol.12 No.4, 329-336, 1997); another reason is that it is directly fixed on When the functional domain of the antibody molecule on the solid phase surface tends downward or towards the side on the solid phase surface, it is not conducive to the binding of antibody-antigen; and if the functional domain of the antibody molecule that binds to the antigen is adsorbed on the solid phase surface, Antibody molecules cannot bind to antigens, such as figure 1 As shown, antibody molecule 5 contains two antigen-binding domains, one of which binds to solid surface 1, thus losing the ability to bind to antigen molecule 6

Method used

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  • Protein chip of directional immobilizing antibody, its preparing method and use thereof
  • Protein chip of directional immobilizing antibody, its preparing method and use thereof
  • Protein chip of directional immobilizing antibody, its preparing method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Prepare a protein chip on the surface of a silicon chip for directional immobilization of antibody human immunoglobulin G (antiIgG), its structure comprising: a silicon chip 1 for integrated circuits with a thickness of 0.5mm, and a monomolecular layer of dichlorodichloromethylene formed on the silicon chip 1 The methyl silane hydrophobic polarized surface layer 2, the monomolecular layer protein A film layer 3 formed on the hydrophobic polarized surface layer 2, and the monomolecular layer antiIgG sensing film layer 4 formed on the protein A film layer 3.

[0047] The preparation of the protein chip is carried out as follows:

[0048] 1. Selection of solid substrate and surface cleaning treatment

[0049] A commercially available silicon wafer for integrated circuits with a thickness of 0.5 mm is selected as a solid substrate, and its surface is cleaned by a conventional cleaning treatment method.

[0050] 2. Formation of the second hydrophobic polarized surface layer...

Embodiment 2

[0060] Preparation of Protein Chip with Oriented Immobilization of AntiBSA Antibody on Composite Substrate

[0061] 1. Selection of solid substrate and surface cleaning treatment

[0062] A 2 μm thick gold plate is plated on a 0.5 mm thick silicon wafer, which is used as a composite solid substrate, and its surface is cleaned by a conventional cleaning treatment method.

[0063] 2. Preparation of Protein A Film Layer

[0064] Immerse the solid substrate that has been cleaned in step 1 into a protein A solution with a concentration of 1 mg / ml, soak for 30 minutes, take it out and wash it with deionized water, and a saturated adsorption film layer of protein A is formed on the surface of the gold sheet .

[0065] 3. Preparation of induction film layer

[0066] Immerse 2 / 3 of the protein A saturated adsorption film layer formed on the gold sheet in the above step 2 into the antiBSA solution with a concentration of 1 mg / ml, soak for 30 minutes, take it out and wash it with deio...

Embodiment 3

[0071] Preparation of a protein chip for directional immobilization of human serum prothrombin antibody (antiFIB) on the surface of a silicon wafer:

[0072] 1. Selection of solid substrate and surface cleaning treatment

[0073] A commercially available silicon wafer with a thickness of 0.5 mm for integrated circuits is selected as a solid substrate, and its surface is cleaned by a conventional cleaning treatment method.

[0074] 2. Formation of the second hydrophobic polarized surface layer

[0075] (1) surface hydrophilization treatment: put the clean silicon chip of the above-mentioned step 1 into 30wt% H with a volume ratio of 1:3 2 o 2 and 98 wt% H 2 SO 4 After soaking in the mixed solution for 20 minutes, take it out and wash it with deionized water for 3 times until it becomes neutral, and then obtain a silicon wafer with surface hydrophilization treatment.

[0076] (2) Surface hydrophobization treatment: put the above-mentioned hydrophilized silicon wafer into a ...

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Abstract

A protein chip with directional fixed antibody for immunodetection medium screening, etc is composed of solid substrate, protein A film, antibody induction film,and hydrophobic polarized layer. Its preparing process includes choosing and cleaning solid substrate, preparing hydrophobic polarized layer, preparing protein A film, and preparing antibody induction film.

Description

technical field [0001] The invention relates to a chip for immunological detection, in particular to a protein chip capable of directional immobilizing antibodies on a solid surface, a preparation method and application thereof. Background technique [0002] Antibody molecules have extremely high specific selection natural properties for target antigens, and they have been widely used in various immunodiagnostic methods and various immunosensors, such as T.M.Phillips et al. in the literature "Protein A coated glass beads universal support medium for high -performance immunoaffinity chromatography" (Journal of Chromatography, 327 (1985) 213-219). Many of these are solid-phase immunoassays, in which antibody molecules are immobilized on a solid surface to detect antigens. The biological activity of antibody molecules immobilized directly on the solid phase surface is usually lower than that of antibody molecules in solution. The main reason for the decre...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 靳刚王战会
Owner INST OF MECHANICS - CHINESE ACAD OF SCI
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