Immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof

A phenylboronic acid and magnetic adsorption technology, which is applied in the field of food safety testing and pretreatment, can solve the problems of low reproducibility and stability of magnetic separation, reduced effective utilization of antibodies, random and uncontrollable orientation, etc., to achieve control of batch-to-batch variation , Reduce steric hindrance, reduce the effect of antibody dosage

Active Publication Date: 2020-12-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is an obvious defect in this method, that is, because both the Fab end (antigen recognition region) and the Fc end of the antibody have a large number of amino groups, the orientation of the antibody on the surface of the magnetic carrier is random and uncontrollable, and this random orientation reduces the exposure of the Fab end. Quantity, which reduces the effective utilization of antibodies, resulting in a large amount of antibodies, high costs, low magnetic separation efficiency, low reproducibility and stability of magnetic separation, etc.

Method used

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  • Immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof
  • Immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof
  • Immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof

Examples

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Embodiment 1

[0024] In this example, the preparation of the immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibody Salmonella includes the preparation of phenylborated magnetic carrier and the coupling of antibody and magnetic carrier. The specific process steps are as follows:

[0025] 1) Preparation of phenylborated magnetic carrier,

[0026] a.Fe 3 o 4 For the synthesis of magnetic beads, add 300mL ultrapure water into a 500mL three-neck flask, 2 To remove the oxygen in the water, while preheating at 50°C for 15min. Add 3.2gFeCl 2 ·H 2 O, 5.2 g FeCl 3 After magnetic stirring and mixing, 25mL of ammonia water was added, the yellow solution quickly turned black, and the reaction was carried out at a constant temperature of 50°C for 30min. The synthesized magnetic beads were separated by magnetic suction, washed 3-5 times with ultrapure water until the pH of the solution reached neutral after magnetic suction, and then redissolved in 300mL ultrapure water. ...

Embodiment 2

[0043] In this example, the preparation of OTA immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody includes the preparation of phenylborated magnetic carrier, the coupling of antibody and magnetic carrier, the enrichment and detection of OTA, and the application of immunomagnetic adsorbent. The process steps are as follows:

[0044] 1) Preparation of phenylborated magnetic carrier

[0045] a-d steps are the same as in Example 1.

[0046] e.Fe 3 o 4 @SiO 2 Amination modification is carried out on the surface of the composite nano-microspheres, and 10 mg of Fe synthesized in the previous step is taken 3 o 4 @SiO 2 The composite nanospheres were added to 10 mL of ethanol, sonicated for 1 h to make them uniformly dispersed, and 800 μL of 3-aminopropyltriethoxysilane (APTES) reagent was added, and sonicated for 1 h to modify the surface of the microspheres, and washed with ultrapure water for 3 -5 times to remove ethanol and unreacted APTES r...

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Abstract

The invention relates to an immunomagnetic adsorbent based on a phenylboronic acid directional coupling antibody and a preparation method thereof. According to the immunomagnetic adsorbent, the antibody directional coupling on the surface of a magnetic carrier is realized by utilizing the affinity of a cis-diol structure in an antibody Fc fragment glycoprotein molecule and the specificity of a phenylboronic acid group. Compared with a traditional method, the method has the advantages that the utilization rate of the Fab end of the antibody is increased to the maximum extent, then the affinityand adsorption capacity of the immunomagnetic adsorbent to a target object are improved, the batch-to-batch difference can be effectively controlled, and the method is worthy of further application and popularization. The prepared immunomagnetic adsorbent can be used for detection pretreatment of various food samples, and can be used for enrichment and purification of harmful substances such as mycotoxin, pesticide and veterinary drug residues, heavy metals and pathogenic microorganisms in the food samples according to different coupled antibodies. The purified harmful substances can be quantitatively or qualitatively detected by high performance liquid chromatography, an immunoassay method, an enzyme-linked immunosorbent assay technology and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety detection and pretreatment, and in particular relates to an immunomagnetic adsorbent based on a phenylboronic acid directional coupling antibody and its preparation. Background technique [0002] In the detection of food hazard factors, the pretreatment steps such as sample separation and purification are indispensable, and they are the key links that affect the reliability of the analysis results. Traditional sample pretreatment methods such as liquid phase extraction, solid phase extraction, etc., are cumbersome to operate and have poor specificity, which affects the detection accuracy. Immunoaffinity chromatography is based on the specific recognition between the ligand and the analyte, and its accuracy is good, but it is costly and time-consuming. The method based on immunomagnetic adsorbent has been rapidly developed in the field of food safety testing sample pretreatment due to its adva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/31G01N30/14
CPCG01N33/5434G01N33/54346G01N21/31G01N30/14
Inventor 熊勇华郭亮冷远逵黄小林林童李响敏
Owner NANCHANG UNIV
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