Method for extracting and purifying horse radish peroxidase from horse radish by using monoclonal antibody affinity adsorption technology

A horseradish peroxidase and monoclonal antibody technology, which is applied in the field of horseradish peroxidase extraction and purification, can solve the problems of low recovery rate, purity and enzyme activity, and achieve the improvement of enzyme activity and strong market competitiveness , cost-saving effect

Active Publication Date: 2009-09-16
HUZHOU YINGCHENG BIOLOGICAL TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] In order to solve the technical defects of low recovery rate, purity and enzyme activity in the existing HRP production method, the object of the invention is to provide a method for extracting horseradish peroxidase using monoclonal antibody affin...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Screening of high-specificity and high-affinity monoclonal antibody

[0017] Purchase specific monoclonal antibodies against HRP from the market, respectively numbered: 01-13, and screen according to the following steps:

[0018] ①High affinity monoclonal antibody screening

[0019] The ability of monoclonal antibody to capture HRP was tested in a 96-well microtiter plate: first, the specific monoclonal antibody was coated in 96 wells at different concentrations with coating buffer (50mM carbonate, pH 9.6), 100L / well In the microtiter plate, overnight at 4°C. After washing the plate with PBS / Tween-20 (0.05%), add the HRP solution to the wells coated with specific monoclonal antibodies and blank wells, 100 μL / well, and incubate at 37°C for 30 minutes; after washing again, add ready-to-use type 3, 3',5,5'-Tetramethylbenzidine (TMB) Chromogenic Solution II (Bi opanda), react at room temperature for 10 minutes, add 100 μL stop solution (2M H 2 SO 4 ) to ...

Embodiment 2

[0022] Example 2 Screening of Carrier Packing for Monoclonal Antibody Coupling

[0023] The screening scope of the present invention mainly includes agarose gel 4B, agarose and immunomagnetic beads, and the screening mainly considers the following factors: the coupling efficiency of the carrier, stability, recyclable times and carrier filler cost, and the screening procedure is as follows:

[0024] ①Coupling of monoclonal antibody and carrier: operate in accordance with the instructions of each product. After coupling of monoclonal antibody and carrier, it is referred to as monoclonal antibody-coupled product.

[0025] ②Coupling substance adsorption efficiency test: The sample selected for this test is commercially available HRP, and the main test is the ability of monoclonal antibody conjugates to adsorb HRP.

[0026] Use 3 times the volume of the coupling product to equilibrate the coupling material, take a sufficient amount of HRP (dissolved in PBS solution, 10mg / ml) and ...

Embodiment 3

[0030] Example 3 Optimization of purification conditions

[0031] By changing the different ion concentrations of the eluent (NaCl concentrations are 0, 50mM, 100mM, 200mM, 500mM), pH value (2.7, 3.0, 4.0, 5.0) and experimental temperature (4°C, 20°C, 37°C), Orthogonal experiments were carried out on the above conditions, and the experimental plan was carried out according to the test method for the adsorption efficiency of coupling substances. And test and compare the change of enzyme activity before and after purification, the enzyme activity test is carried out according to the following experimental scheme:

[0032] The specific monoclonal antibody was coated in a 96-well ELISA plate at different concentrations with coating buffer (50 mM carbonate, pH 9.6) at 100 μL / well, overnight at 4°C. Dilute the HRP purified under the above conditions to the same concentration, add 5 μl of HRP solution to the wells coated with specific monoclonal antibodies and blank enzyme labels,...

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PUM

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Abstract

The invention relates to a method for extracting and purifying horse radish peroxidase, in particular to a method for extracting and purifying horse radish peroxidase from horse radish by using monoclonal antibody affinity adsorption technology. A method for extracting horse radish peroxidase by using monoclonal antibody affinity adsorption technology uses monoclonal antibodies having high specificity and high affinity aiming at horse radish peroxidase and conjugation of carrier padding to extract and purify horse radish peroxidase from horse radish by using a method of affinity chromatography. The invention uses monoclonal antibody affinity adsorption to purify HRP, thereby not only saving time and cost but also not producing much water material; moreover, compared with the traditional method, the activity of enzyme is greatly improved so as to accord with the requirement for environment-friendly and social-saving social construction. The HRP product extracted and purified by the technology has good quality and low cost, has stronger market competition, and also accords with better and better quality requirement of technical development for HRP; therefore, the invention will replace the traditional deposition purification method.

Description

technical field [0001] The invention relates to a method for extracting and purifying horseradish peroxidase, in particular to a method for extracting and purifying horseradish peroxidase from horseradish by applying monoclonal antibody affinity adsorption technology. technical background [0002] Peroxidase (Horseradish Peroxidase, HRP, EC 1.11.1.7), usually derived from horseradish (Armoracia rusticana) (a perennial herb growing in parts of Europe and Asia), so it is called horseradish peroxidase, the enzyme It is a commonly used enzyme in clinical test reagents. It is not only used in multiple biochemical test items, but also widely used in immune test tests. Impact matters. [0003] Horseradish peroxidase HRP has high specific activity, stable properties and small molecular weight, so it is widely used in various fields of life sciences. HRP is widely distributed in the plant kingdom, and its content is high in horseradish. It is a glycoprotein composed of colorless en...

Claims

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Application Information

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IPC IPC(8): C12N9/08
Inventor 阎可廷费小战关义海
Owner HUZHOU YINGCHENG BIOLOGICAL TECH
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