Immunomagnetic bead and preparation method thereof

A technology of immunomagnetic beads and magnetic microspheres, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of inactivation of active substances such as proteins, unfavorable protein coupling, and changes in isoelectric point, etc., to achieve affinity. High, not easy to deteriorate, and the effect of increasing the amount of coating

Active Publication Date: 2017-02-15
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each peptide chain of streptavidin contains 159 amino acid residues, of which the content of basic amino acids such as lysine and arginine is less than that of general avidin, and it contains more acidic amino acids, and its isoelectric point is 6.0 , so too much reaction of the amino group on the streptavidin will easily lead to a change or inactivation of the isoelectric point; and this method also needs to add a strong reducing agent (such as sodium borohydride or sodium cyanoborohydride) when reducing, It is easy to cause the inactivation of active substances such as proteins, and glutaraldehyde will cause cross-linking of the magnetic particles themselves and the protein itself, which is not conducive to the coupling of proteins

Method used

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  • Immunomagnetic bead and preparation method thereof
  • Immunomagnetic bead and preparation method thereof
  • Immunomagnetic bead and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] (1) Preparation of magnetic nanoclusters: Preparation of magnetic nanoclusters Fe by co-precipitation method 3 o 4 1;

[0031] (2) Preparation of amino-modified magnetic microspheres: add 10 mg magnetic nanocluster Fe 3 o 4 Add 125 mg of ammonia water, 30 mg of tetraethyl orthosilicate and 30 mg of (3-aminopropyl) triethoxysilane to the solution of 1, conduct magnetic separation after 1 day of reaction, and alternately wash twice with ethanol and water to obtain amino Modified magnetic microsphere 1;

[0032] (3) Preparation of biotinylated magnetic microspheres: Disperse 20 mg of amino-modified magnetic microspheres 1 in dimethyl sulfoxide, then add 10 mg of biotin active ester for amidation reaction, perform magnetic separation after overnight reaction, and use Biotinylated magnetic microspheres 1 were obtained after washing with PBS solution with a pH of 7.2;

[0033](4) Combining biotinylated magnetic microspheres with streptavidin: react 1 mg of biotinylated...

Embodiment 2

[0038] (1) Preparation of magnetic nanoclusters: Preparation of magnetic nanoclusters Fe by co-precipitation method 3 o 4 2;

[0039] (2) Preparation of amino-modified magnetic microspheres: add 10 mg magnetic nanocluster Fe 3 o 4 Add 250 mg of ammonia water, 20 mg of tetraethyl orthosilicate and 10 mg of (3-aminopropyl) triethoxysilane to the solution of 2, react for 2 days, carry out magnetic separation, and alternately wash twice with ethanol and water to obtain amino Modified magnetic microsphere 2;

[0040] (3) Preparation of biotinylated magnetic microspheres: Disperse 20 mg of amino-modified magnetic microspheres 2 in N,N-dimethylformamide, then add 5 mg of biotin active ester for amidation reaction, and react overnight Magnetic separation was carried out, and biotinylated magnetic microspheres 2 were obtained after washing with a PBS solution with a pH of 7.2;

[0041] (4) Combining biotinylated magnetic microspheres with streptavidin: 1 mg biotinylated magnetic...

Embodiment 3

[0045] (1) Preparation of magnetic nanoclusters: Preparation of magnetic nanoclusters Fe by co-precipitation method 3 o 4 3;

[0046] (2) Preparation of amino-modified magnetic microspheres: add 10 mg magnetic nanocluster Fe 3 o 4 Add 125 mg of ammonia water, 40 mg of tetraethyl orthosilicate and 5 mg of (3-aminopropyl) triethoxysilane to the solution of 3, conduct magnetic separation after 3 days of reaction, and alternately wash twice with ethanol and water to obtain amino Modified magnetic microsphere 3;

[0047] (3) Preparation of biotinylated magnetic microspheres: Disperse 20 mg of amino-modified magnetic microspheres part 1 in dimethyl sulfoxide or N,N-dimethylformamide, then add 20 mg of biotin active ester for amide Biotinylated magnetic microspheres 3 were obtained after being washed with a PBS solution with a pH of 7.2;

[0048] (4) Binding biotinylated magnetic microspheres to streptavidin: 1 mg of biotinylated magnetic microspheres 3 and 200 μL streptavidin...

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Abstract

The invention provides an immunomagnetic bead and a preparation method thereof. The immunomagnetic bead comprises a magnetic microsphere and a biological ligand, wherein the structure of the magnetic microsphere is magnetic microsphere-biotin-streptavidin-biotin-biological ligand from inside to outside. The immunomagnetic bead provided by the invention is stable in property, good in magnetism and small in particle size. A connecting mode of biotin-streptavidin-biotin is adopted, and the biotinylated magnetic microsphere is firstly obtained through amidation reaction and then is combined with the streptavidin. As the combination of biotin-streptavidin has the characteristics of high affinity and specificity, the magnetic microsphere and the biological ligand can be directionally combined, cross-linking is not easy, and a change in the structure of the streptavidin is not easily caused. Meanwhile, the preparation method provided by the invention is simple and mild in reaction conditions, and the biological ligand can retain the biological activity.

Description

technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to an immunomagnetic bead and a preparation method thereof. Background technique [0002] Immonumagnetic beads (IMB for short) are composed of magnetic microspheres and biological ligands. The combination of magnetic microspheres and biological ligands generally has physical combination and chemical coupling methods. The chemical coupling method has a large binding capacity and good stability, and the prepared immunomagnetic beads can usually be stored for several months. However, in order to prevent antibody inactivation, the chemical coupling method can only use relatively mild conditions, which will lead to poor coating efficiency of the antibody; and chemical coupling will use too many antibody active groups, and the controllability is low. lead to inactivation of antibodies. The physical adsorption method is the first method adopted by JohnUgelstad et al. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/531
CPCG01N33/531G01N33/54326
Inventor 刘关蔡红东陈昌岳张培培张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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