Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof

A colorectal cancer and joint detection technology, applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of inaccurate detection data, poor pertinence, slow detection speed, etc., achieve high sensitivity, reduce costs, The effect of saving inspection time

Inactive Publication Date: 2015-08-26
深圳市森塔贸易有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0017] In order to overcome the deficiencies of the prior art, one aspect of the present invention is to propose a liquid-phase protein chip that jointly detects five colorectal cancer markers, and its purpose is to solve the problem that the existing colorectal cancer detection can only be aimed at the late stage detection of cancer. And the pertinence is not strong, the detection speed is slow, the detection data is inaccurate, etc.

Method used

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  • Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof
  • Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof
  • Liquid phase protein chip for combined detection of five colorectal cancer markers, and preparation method thereof

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specific Embodiment 1

[0038] Preparation of monoclonal antibodies to M2-PK, K-ras, APC, AKT and PI3K.

[0039] 1. Preparation of immune antigen

[0040] Prepare gene recombination M2-PK, K-ras, APC, AKT and PI3K protein through the engineering strain of a kind of Escherichia coli carrying M2-PK, K-ras, APC, AKT and PI3K protein gene; -PK, K-ras, APC, AKT and PI3K proteins were injected into different mice for immunization.

[0041] 2. Preparation and screening of hybridoma cells

[0042] Select the serum antibody titer to reach 1×10 6 Mice were injected with M intraperitoneally 3 days before fusion 2 - PK, K-ras, APC, AKT and PI3K antigens 100 μg. The mouse spleen was obtained in a sterile manner, and the splenocyte suspension was mixed with the mouse myeloma cell line NS-1 in the logarithmic growth phase at a ratio of 10:1, and 45% polyethylene glycol (PEG ) to facilitate integration. Specifically, polyethylene glycol (PEG) is added according to the following steps. In a water bath at 37°C,...

specific Embodiment 2

[0047] Preparation of nano-gold-coated quantum-encoded microspheres and M 2 - Conjugation of PK monoclonal antibody, K-ras monoclonal antibody, APC monoclonal antibody, AKT monoclonal antibody and PI3K monoclonal antibody.

[0048] 1. Preparation of gold nanoparticles

[0049] The glass container in the present invention is pickled and rinsed with distilled water, and then siliconized. The method of siliconization treatment is to soak for several minutes with 5% dichlorosilane in chloroform solution, rinse with distilled water and dry for later use. The preparation method and process of gold nanoparticles are as follows: the HAuC 14 First prepare a 0.01% aqueous solution, take 100mL and heat it to boiling; accurately add 10mL of 1% trisodium citrate (Na3C6H5O7 2H2O) aqueous solution under stirring, continue heating and boiling for 15min, at this time, a light yellow chloroauric acid aqueous solution can be observed After sodium citrate is added, it turns gray quickly, then ...

specific Embodiment 3

[0056] The microspheres coated with gold nanoparticles and labeled with quantum dots were coupled with monoclonal antibodies to M2-PK, K-ras, APC, AKT and PI3K.

[0057] Here, the M2-PK monoclonal antibody is taken as an example for illustration, and the coupling of other monoclonal antibodies is the same as that of the M2-PK monoclonal antibody, and will not be repeated here.

[0058] The concentration of the M2-PK monoclonal antibody in the test tube is 50nmol / L, and the phosphate buffer solution of pH=6.0 is diluted to 30 μg / mL under electromagnetic stirring, Tween-80 is added to 1% (V / V), and then Add 0.01g quantum dot coded microspheres coated with gold nanoparticle layer, continue to stir for 10 minutes, then add 5% BSA solution (1%) in the test tube to seal the unreacted site, after the sealing is completed, the The microspheres were centrifuged and washed with PBS buffer, and the washing was repeated 3 times. After calculation, the density of the probe is about 0.01 μ...

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Abstract

The invention discloses a liquid phase protein chip for combined detection of five colorectal cancer markers, and a preparation method thereof, and belongs to the technical field of liquid phase protein chips. The liquid phase protein chip comprises a carboxyl functionalized or biotinylated polystyrene microsphere, a nano-gold particle layer coated on the microsphere, and a quantum dot embedded to the nano-gold particle layer, wherein five microspheres marked with quantum dots with different emission wavelengths are selected to respectively couple with monoclonal antibodies of M2-PK, K-ras, APC, AKT and PI3K. The chip prepared through the method can simultaneously detect M2-PK, K-ras, APC, AKT and PI3K markers of colorectal cancer, can be used for detecting early stage conditions of the colorectal cancer, and has the advantages of high sensitivity, high specificity and reliable detection method.

Description

technical field [0001] The invention relates to the technical field of a liquid-phase protein chip, in particular to a liquid-phase protein chip capable of simultaneously detecting five specific markers of colorectal cancer. [0002] Another aspect involved in the present invention is the preparation method of the liquid-phase protein chip. Background technique [0003] Colorectal cancer is one of the three most common malignant tumors in the world, and its mortality rate ranks third. In recent years, with the improvement of people's living standards and changes in diet structure, its incidence has shown an obvious upward trend. However, the early diagnosis of colorectal cancer at home and abroad is still at a low level, and about half of the patients have entered the middle and late stages of colorectal cancer when they are diagnosed. Therefore, improving the early diagnosis of colorectal cancer is a key issue that urgently needs to be solved at home and abroad. [0004] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/57419G01N33/57446
Inventor 何凤屏
Owner 深圳市森塔贸易有限公司
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