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Preparation method of enzyme-labeled antibody

An enzyme-labeled antibody and antibody technology, which is applied to measuring devices, instruments, scientific instruments, etc., to achieve the effects of enhanced stability, low price, and high efficiency

Pending Publication Date: 2018-12-07
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current studies mainly focus on encapsulating natural enzymes to enhance their catalytic activity, and there are few reports on encapsulating other biomolecules with MOFs.

Method used

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  • Preparation method of enzyme-labeled antibody
  • Preparation method of enzyme-labeled antibody
  • Preparation method of enzyme-labeled antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Follow the steps below to prepare RIgG@Cu-MOF:

[0028] (1) 0.7mg copper chloride dihydrate is dissolved in 1mL water to obtain an aqueous solution of copper chloride dihydrate;

[0029] (2) Mix ethanol and water according to v / v=1:1 to form 1mL ethanol-water mixed solution, and then add 2.4mg 4,4'-bipyridine ligand to obtain 4,4'-bipyridine ligand Ethanol-water mixed solution;

[0030] (3) Mix 1mL copper chloride dihydrate aqueous solution with 1mL ligand ethanol-water mixed solution, add 0.1mg RIgG, stir at 4°C for 12h, centrifuge (13000rpm, 5min), and wash 3 times with water to obtain RIgG@ Cu-MOFs.

Embodiment 2

[0032] Combined with the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation experiment, the absorbance corresponding to its maximum absorption peak was recorded by a UV spectrophotometer to prove the simulated enzyme activity of Cu-MOF. Its method steps are:

[0033] (1) Sodium acetate (NaAc) with pH = 4.0 was used as a buffer solution with a total volume of 100 μL and a reaction temperature of 37° C.;

[0034] (2) Use three 0.5mL centrifuge tubes, numbered a, b, and c respectively. The preparation method of the solution in centrifuge tube a is as follows: Take 3 μL of 2 mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) in 97 μL of sodium acetate (NaAc) buffer solution with pH=4.0, and place in a 37°C water bath React for 30 minutes;

[0035] (3) The preparation method of the solution in the centrifuge tube b is: take 3 μL 2mg / mL TMB, 2 μL 100mM hydrogen peroxide H 2 o 2 In 95 μL of sodium acetate (NaAc) buffer solution with pH=4.0, react in a water bath at 37°C for 30 minutes;

[0...

Embodiment 3

[0039] To prove that RIgG@Cu-MOF can recognize mIgG, the solution was prepared in a 96-well plate, and then the absorbance corresponding to the maximum absorption peak was recorded by a UV spectrophotometer. The specific method is:

[0040] (1) Using a 96-well plate, number the reaction wells as a, b, and c respectively, and the reaction wells a, b, and c are respectively coated with buffer solution (carbonate buffer solution with pH=9.6 and a concentration of 0.05 mol / L) Dilute RIgG to a final concentration of 10 μg / mL, react at 4°C for 12 h, wash the reaction wells 4 times with 400 μL of washing buffer (pH=7.4, 0.15 mol / L carbonate buffer containing 0.05% Tween-20), and Remove unfixed RIgG and pour off the washing solution;

[0041] (2) Add 400 μL of blocking agent (washing buffer containing 1% BSA) to reaction wells a, b and c respectively, react at 37°C for 40 min, and wash 3 times;

[0042] (3) Add mIgG with a final concentration of 100ng / mL to reaction wells a, b and c...

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Abstract

The invention discloses a novel method for preparing an enzyme-labeled antibody. Some unavoidable problems, particularly the tedious preparation of the enzyme-labeled antibody based on covalent binding usually exist in an enzyme-linked immunosorbent assay method. Therefore, the antibody is bound with a metal organic framework (MOF) with catalytic activity to peroxidase so as to form a bidirectional MOF / antibody composite material, and the bidirectional MOF / antibody composite material can be applied to a colorimetric immunoassay method. The MOF does not influence the capture capacity of the antibody to the antigen, and the antibody is not influenced by high temperature and biological degradation, so that the stability of the antibody is improved. More importantly, the signal amplification of the MOF with catalytic activity to peroxidase can be realized in a colorimetric immunoassay experiment, so that the detection sensitivity is improved. The method has the advantages of simplicity andconvenience in operation, low cost, high efficiency and the like.

Description

technical field [0001] The invention relates to the preparation of enzyme-labeled antibody by combining catalytic MOF with antibody for colorimetric immunoassay, especially the method of preparing enzyme-labeled antibody by combining MOF with antibody, which belongs to the field of preparation and application of MOF as a biosensor material. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) is a common analytical method, which is widely used in disease diagnosis, environmental analysis, food monitoring and so on. This is because it has the advantages of simple operation, high sensitivity and good specificity. However, there are still some problems in practical application, among which the most worthy of attention is the preparation and purification of enzyme-labeled antibodies. Traditional enzyme-labeled antibodies are usually prepared by covalent bonding of enzymes and antibodies. Due to the cumbersome covalent bonding process, some enzymes will inevit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 谭宏亮王彩红
Owner JIANGXI NORMAL UNIV
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