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Method for preparing enzyme labeled antibody by utilizing metal organic framework material

A metal-organic framework, enzyme-labeled antibody technology, applied in the field of immunoassays, can solve the problems of low protein loading, insufficient enzyme action, protein variability, etc. Lost effect

Inactive Publication Date: 2019-03-19
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these supports have some disadvantages, such as low protein loading capacity in non-porous systems; disordered sols are easily destroyed when used as carriers, so the immobilized proteins are volatile; silica mesoporous materials have a large pore size. , high porosity and orderly structure, etc., and it is regarded as a highly anticipated enzyme support. However, because it is a one-dimensional channel structure, the surface of the inner channel and the insufficient force of the enzyme often lead to enzyme leakage during recycling. , and enzymatic polymerization also occurs in the inner channel

Method used

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  • Method for preparing enzyme labeled antibody by utilizing metal organic framework material
  • Method for preparing enzyme labeled antibody by utilizing metal organic framework material
  • Method for preparing enzyme labeled antibody by utilizing metal organic framework material

Examples

Experimental program
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Embodiment 1

[0030] according to figure 1 The synthetic route shown prepares the enzyme-labeled antibody complex, and the specific method is as follows:

[0031] 1. Synthesis of PCN-333(Al)

[0032] Take 25mg 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine and 50mg AlCl 3 ·6H 2O. Put 20mg of PVP (average molecular weight 40,000) in a 50mL round bottom flask, add 20mL of N,N-diethylformamide, ultrasonically disperse for 10min, ensure that the added drug is completely dissolved, then add 0.25mL of trifluoroacetic acid, at 120°C Heat and stir in an oil bath for 20 hours, centrifuge, and wash the solid with N,N-dimethylformamide and ethanol to obtain PCN-333(Al). Depend on figure 2 and image 3 It can be seen that 300nm PCN-333(Al) has been successfully synthesized, and the synthesized PCN-333(Al) has uniform size and uniform distribution.

[0033] 2. Functionalized PCN-333(Al)

[0034] Add 1mg PCN-333(Al) to 100μL ultrapure water, then add 100μL 25mmol / L N-(3-dimethylaminopropyl)-N'-ethylc...

Embodiment 2

[0039] In Step 1 of this example, 12.5 mg of 2,4,6-tri(4-carboxyphenyl)-1,3,5-triazine)) and 25 mg of AlCl 3· 6H 2 O. Put 50mg of PVP (average molecular weight 10000) in a 25mL round bottom flask, add 10mL of N,N-diethylformamide, ultrasonically disperse for 10min to ensure that the added drug is completely dissolved, then add 0.125mL of trifluoroacetic acid, and incubate at 120°C Heat and stir in an oil bath for 20 hours, centrifuge, and wash the solid with N,N-dimethylformamide and ethanol to obtain PCN-333(Al). The other steps were the same as in Example 1 to obtain the enzyme-labeled antibody complex, which was stored at 4°C.

Embodiment 3

[0041] In step 1 of this embodiment, take 25mg 2,4,6-tri(4-carboxyphenyl)-1,3,5-triazine)) and 30mgFeCl 3 , 50mg PVP (average molecular weight 10000) in a 25mL round bottom flask, add 10mL N,N-diethylformamide, ultrasonically disperse for 10min to ensure that the added drug is completely dissolved, then add 0.25mL trifluoroacetic acid, oil at 120°C Heated and stirred in the bath for 20 hours, centrifuged, and the solid was washed with N,N-dimethylformamide and ethanol to obtain PCN-333(Fe). The other steps were the same as in Example 1 to obtain the enzyme-labeled antibody complex, which was stored at 4°C.

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Abstract

The invention discloses a method for preparing an enzyme labeled antibody by utilizing a metal organic framework material. The method comprises the following steps: reacting carboxyl on a metal organic framework material PCN-333 with EDC and sulfo-NHS, functionalizing the PCN-333, adding an antibody, carrying out an acylation reaction on amino on the antibody and the functionalized PCN-333 so as to produce a stable amido bond, namely the antibody is immobilized onto the surface of the PCN-333; and finally, encapsulating the enzyme into the PCN-333, and forming an enzyme labeled antibody complex. According to the method disclosed by the invention, by virtue of a coupled reaction between the antibody and MOFs (Metal Organic Frameworks), tedious and high-efficiency enzyme and protein (antibody) chemical crosslinking is avoided, the prepared complex can be separated from excessive antibodies or enzymes by virtue of simple centrifugation, tedious separation and purification steps are avoided, such as gel filtration, long-term dialysis and the like, the activity of the enzymes and antibodies is further prevented from being lost, the coupling process between the antibody and the enzyme inthe traditional meaning is changed, the enzyme labeled antibody process is simplified, the enzyme labeled quantity is improved, and the cost of preparing the enzyme labeled antibody is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of immune analysis, and in particular relates to a preparation method of an enzyme-labeled antibody. Background technique [0002] The development of diagnostic techniques for the efficient detection of disease biomarkers has become an important part of biomedical research and clinical care, and has important implications for modern analytical systems. Over the past decades, researchers have actively explored various techniques based on signal sensing principles, notable examples include fluorescence, chemiluminescence, colorimetric, electrochemical, Raman, and plasmonic methods. Detection of disease biomarkers with simple and affordable technologies is essential to improve the living standards of people, especially in resource-poor regions and countries. Enzyme-based colorimetric assays (enzyme and bioreceptor binding in advance, transfer catalysis of chromogenic substrate to generate color signal), such a...

Claims

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Application Information

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IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 张耀东李瑶夏梦凡赵梦园
Owner SHAANXI NORMAL UNIV
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