A core-shell magnetic nanoparticle with high selectivity for antibody separation and a preparation method thereof

Abstract

The invention discloses a method for preparing a highly selective core-shell magnetic nanoparticle for antibody separation, comprising the following steps: preparing ferroferric oxide / silicon oxide nanoparticles with a core-shell structure, performing amine functionalization treatment on the nanoparticles, and finally coupling IgG2a on the surface thereof; dispersing the IgG2a-coupled magnetic nanoparticles in a PBS buffer, adding tetraethyl silicate to stir the reaction, adding a mixed solution of N-propyltriethoxysilane, benzyltriethoxysilane, and (3-aminopropyl)-triethoxysilane, reacting, and after the reaction is completed, dripping a mixed solution of methanol, PEG-4, and NHS to continue stirring, washing the precipitate with a PBS buffer, and finally adding the precipitate to a mixed solution of a sodium hydroxide solution and a polyethylene glycol octylphenyl ether solution for treatment, obtaining magnetic nanoparticles, and redispersing the nanoparticles in a PBS buffer for standby use. The core-shell magnetic nanoparticles provided by the invention have many surface binding sites and high selectivity, and can effectively purify monoclonal shells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a core-shell magnetic nanoparticle with high selectivity for antibody separation and a preparation method thereof. Background Art

[0002] As a highly specific and effective biotargeted drug, monoclonal antibodies have occupied half of the top ten drugs in global sales. With the application of high-expression cell line construction, large bioreactors, and new culture techniques, the antibody titer in the supernatant of Chinese hamster ovary cell fed-batch culture has increased by more than a hundredfold, reaching 3 - 13 g / L, and the PER The antibody titer in the supernatant of cell perfusion culture can be as high as 27 g / L. In sharp contrast, protein A chromatography, which is the main means of antibody capture and purification, has an average annual growth rate of only about 5.5% in its antibody binding capacity. Antibody capture and purification refer to the process of extracting, concentrating, refining, and finalizing the antibody component from tissue components or cell culture media, which together with animal cell culture constitutes an important part of monoclonal antibody drug preparation. Although protein A chromatography technology has been regarded as the "gold standard" for antibody capture and purification processes, it still suffers from problems such as low capacity, high cost, low reuse rate, and poor chemical stability. For the current monoclonal antibody production capacity of dozens or even hundreds of kilograms per batch, the inadequate ability of protein A chromatography has become the main technical "bottleneck" in monoclonal antibody drug preparation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: aiming at the deficiencies of the existing technology, to provide a core-shell magnetic nanoparticle with high selectivity for antibody separation and a preparation method thereof. The core-shell magnetic nanoparticle provided by the present invention has many surface binding sites and high selectivity, and can effectively purify monoclonal shells.

[0004] To solve the above technical problems, the technical solution of the present invention is:

[0005] A preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation, comprising the following steps:

[0006] (1) Slowly drop the iron salt solution into the heated sodium hydroxide solution, stir vigorously while dropping, stir and react for 2 - 3 h after the dropping is completed, cool to room temperature after the reaction is completed, wash the precipitate, and obtain nano-ferroferric oxide.

[0007] (2) Disperse the nano-ferroferric oxide in absolute ethanol, then drop tetraethyl orthosilicate and drop ammonia water solution, stir and react at room temperature for 10 - 15 h, filter the reaction solution after the reaction is completed, and wash the precipitate with deionized water and absolute ethanol in sequence, and dry to obtain a core-shell structured ferroferric oxide / silica nanoparticle.

[0008] (3) Disperse the prepared iron tetroxide / silica nanoparticles in toluene, then add 3-aminopropyltrimethoxysilane and carry out a reflux reaction. After the reaction is completed, wash the solid multiple times to obtain amine-functionalized iron tetroxide / silica nanoparticles;

[0009] (4) Mix and react mouse IgG2a and sodium periodate solution under light-shielded conditions, then add sodium sulfite solution to terminate the reaction. Then add a mixed dispersion of amine-functionalized iron tetroxide / silica nanoparticles and PBS buffer solution and react. After that, filter to obtain IgG2a-conjugated magnetic nanoparticles;

[0010] (5) Disperse the IgG2a-conjugated magnetic nanoparticles in PBS buffer solution, then add tetraethyl orthosilicate and carry out a first stirring reaction for 0.5 - 1.5 h. Then add a mixed solution of N-propyltriethoxysilane, benzyltriethoxysilane, and (3-aminopropyl)-triethoxysilane and carry out a second stirring reaction for 2 - 5 h. After the reaction is completed, add a mixed solution of methanol, PEG-4, and NHS to the reaction system and stir to react. Then wash the precipitate with PBS buffer solution. Finally, add the washed precipitate to a mixed solution of sodium hydroxide solution and polyethylene glycol octylphenyl ether solution, perform ultrasonic treatment, then filter, wash the precipitate, and obtain magnetic nanoparticles. Redisperse them in PBS buffer solution for use.

[0011] As a preference of the above technical solution, in step (1), the iron salt solution is a mixed solution of ferric chloride and ferrous chloride, and the concentrations of ferric chloride and ferrous chloride in the mixed solution are 0.015 - 0.025 mol / L and 0.005 - 0.015 mol / L respectively; the concentration of the sodium hydroxide solution is 1.4 - 1.6 mol / L, and its volume ratio to the iron salt solution is 10:1.

[0012] As a preference of the above technical solution, in step (2), the mass concentration of the ammonia water solution is 30%, and the dosage ratio of the nano iron tetroxide, tetraethyl orthosilicate, and ammonia water solution is 1 mol:(0.8 - 1) mol:10 ml.

[0013] As a preference of the above technical solution, in step (3), the dosage ratio of the iron tetroxide / silica nanoparticles and 3-aminopropyltrimethoxysilane is 1 g:(2 - 3) ml; the reaction temperature is 100 - 120 °C and the time is 10 - 15 h.

[0014] Preferably, in step (4), the concentration of the sodium periodate solution is 3.5 - 4.5 mmol / L, and the dosage ratio of the mouse IgG2a to the sodium periodate solution is 10 μg : 6 - 7 μL; the mixing reaction time is 20 - 30 min.

[0015] Preferably, in step (4), the dosage ratio of the amine-functionalized iron oxide / silica nanoparticles to the PBS buffer solution in the mixed dispersion is 1 mg : 1 ml; the reaction time is 2 - 3 h.

[0016] Preferably, in step (5), the dosage ratio of the IgG2a-conjugated magnetic nanoparticles, the PBS buffer solution, tetraethyl orthosilicate, N-propyltriethoxysilane, benzyltriethoxysilane, and (3-aminopropyl)-triethoxysilane is 2 mg : 1 ml : 0.1 ml : 5 μL : 10 μL : 10 μL.

[0017] Preferably, in step (5), the concentration of the mixed solution of methanol, PEG-4, and NHS is 0.45 - 0.55 mmol / L, and the molar ratio of methanol, PEG-4, and NHS is 1 : 1 : 1. The reaction time is 20 - 40 min.

[0018] Preferably, in step (5), the concentration of the sodium hydroxide solution is 0.05 mol / L, the concentration of the Triton X-100 solution is 0.01 - 0.02 wt%, and their volume ratio is 1 : 1; the ultrasonic treatment is 5 - 10 min, and the ultrasonic power is 100 W.

[0019] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0020] The magnetic nanoparticles provided by the present invention use silica-coated iron oxide nanoparticles as the matrix, with highly selective IgG2a conjugated on its surface, and a polymer film is modified on its surface; finally, IgG2a is removed to form multiple antibody binding sites, which have high selectivity. During the specific preparation, the present invention conjugates IgG2a on the surface of silica-coated iron oxide nanoparticles, then coats a polymer film synthesized from organosilane monomers on its surface, finally uses polyethylene glycol to block the non-specific binding sites on the surface, and finally removes IgG2a to increase the surface roughness of the magnetic nanoparticles. The prepared magnetic nanoparticles have good binding ability with antibodies and good specificity. Specific Embodiments

[0021] The present invention will be further described below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0022] Example 1

[0023] A preparation method of core-shell magnetic nanoparticles with high selectivity for antibody separation, comprising the following steps:

[0024] (1) Slowly drop 1 L of a mixed solution containing 0.015 mol of ferric chloride and 0.005 mol of ferrous chloride into 100 ml of a sodium hydroxide solution with a concentration of 1.4 mol / L and a temperature of 60 °C, while vigorously stirring during the dropping process. After the dropping is completed, stir and react for 2 h. After the reaction is completed, cool to room temperature, wash the precipitate to obtain nano-ferroferric oxide;

[0025] (2) Disperse 1 mol of nano-ferroferric oxide in 500 ml of absolute ethanol, then drop 0.8 mol of tetraethyl orthosilicate, and drop 10 ml of an ammonia water solution with a mass concentration of 30%. Stir and react at room temperature for 10 h. After the reaction is completed, filter the reaction solution, and wash the precipitate with deionized water and absolute ethanol in sequence, and dry to obtain core-shell structured ferroferric oxide / silica nanoparticles;

[0026] (3) Disperse 10 g of ferroferric oxide / silica nanoparticles in 500 ml of toluene, then add 20 3-aminopropyltrimethoxysilane, and carry out a reflux reaction at 100 °C for 10 h. After the reaction is completed, wash the solid multiple times to obtain amine-functionalized ferroferric oxide / silica nanoparticles;

[0027] (4) Mix 10 μg of mouse IgG2a and 6 μL of a sodium periodate solution with a concentration of 3.5 mmol / L and react under light-shielded conditions for 20 min. Then drop a sodium sulfite solution to terminate the reaction, and then add a mixed dispersion of 1 mg of amine-functionalized ferroferric oxide / silica nanoparticles and 1 ml of PBS buffer solution, react for 2 h, and then filter to obtain IgG2a-conjugated magnetic nanoparticles;

[0028] (5) Disperse 2 mg of IgG2a-conjugated magnetic nanoparticles in 1 ml of PBS buffer, then add 0.1 ml of tetraethyl orthosilicate, and carry out the first stirring reaction for 1.5 h. Then add a mixed solution of 5 μL of N-propyltriethoxysilane, 10 μL of benzyltriethoxysilane, and 10 μL of (3-aminopropyl)-triethoxysilane, and carry out the second stirring reaction for 2 h. After the reaction is completed, add a mixed solution of methanol, PEG-4, and NHS dropwise to the reaction system and stir for 20 min. Then wash the precipitate with PBS buffer. Finally, add the washed precipitate to a mixture of 5 ml of a sodium hydroxide solution with a concentration of 0.05 mol / L and 5 ml of a Triton X-100 solution with a concentration of 0.01 wt%, and ultrasonically treat it at a power of 100 W for 5 min. Then filter, wash the precipitate, and redisperse it in PBS buffer to obtain a dispersion of magnetic nanoparticles.

[0029] Example 2

[0030] A preparation method of core-shell magnetic nanoparticles with high selectivity for antibody separation, comprising the following steps:

[0031] (1) Slowly drop 1 L of a mixed solution containing 0.025 mol of ferric chloride and 0.015 mol of ferrous chloride into 100 ml of a sodium hydroxide solution with a concentration of 1.6 mol / L and a temperature of 70 °C while vigorously stirring. After the dropping is completed, stir and react for 3 h. After the reaction is completed, cool to room temperature, wash the precipitate, and obtain nano-sized magnetite.

[0032] (2) Disperse 1 mol of nano-sized magnetite in 500 ml of absolute ethanol, then dropwise add 1 mol of tetraethyl orthosilicate, and dropwise add 10 ml of an ammonia water solution with a mass concentration of 30%. Stir and react at room temperature for 15 h. After the reaction is completed, filter the reaction solution, and wash the precipitate with deionized water and absolute ethanol in sequence, and dry to obtain core-shell structured magnetite / silica nanoparticles.

[0033] (3) Disperse 10 g of magnetite / silica nanoparticles in 500 ml of toluene, then add 30 ml of 3-aminopropyltrimethoxysilane, and carry out a reflux reaction at 120 °C for 15 h. After the reaction is completed, wash the solid multiple times to obtain amine-functionalized magnetite / silica nanoparticles.

[0034] (4) Mix 10 μg of mouse IgG2a and 7 μL of a sodium periodate solution with a concentration of 4.5 mmol / L and react in the dark for 30 min. Then dropwise add a sodium sulfite solution to terminate the reaction, and then add a mixed dispersion of 1 mg of amine-functionalized magnetite / silica nanoparticles and 1 ml of PBS buffer, and react for 3 h. Then filter to obtain IgG2a-conjugated magnetic nanoparticles.

[0035] (5) Disperse 2 mg of IgG2a-conjugated magnetic nanoparticles in 1 ml of PBS buffer, then add 0.1 ml of tetraethyl orthosilicate, and conduct the first stirring reaction for 1.5 h. Then add a mixed solution of 5 μL of N-propyltriethoxysilane, 10 μL of benzyltriethoxysilane, and 10 μL of (3-aminopropyl)-triethoxysilane, and conduct the second stirring reaction for 5 h. After the reaction is completed, dropwise add a mixed solution of methanol, PEG-4, and NHS to the reaction system and stir for 40 min. Then wash the precipitate with PBS buffer. Finally, add the washed precipitate to a mixed solution of 5 ml of a sodium hydroxide solution with a concentration of 0.05 mol / L and 5 ml of a Triton X-100 solution with a concentration of 0.02 wt%, and ultrasonically treat it at a power of 100 W for 10 min. Then filter, wash the precipitate, and redisperse it in PBS buffer to obtain a dispersion of magnetic nanoparticles.

[0036] Example 3

[0037] A preparation method of core-shell magnetic nanoparticles with high selectivity for antibody separation, comprising the following steps:

[0038] (1) Slowly drop 1 L of a mixed solution containing 0.015 mol of ferric chloride and 0.015 mol of ferrous chloride into 100 ml of a sodium hydroxide solution with a concentration of 1.4 mol / L and a temperature of 70 °C, while vigorously stirring during the dropping process. After the dropping is completed, stir and react for 2 h. After the reaction is completed, cool to room temperature, wash the precipitate, and obtain nano-ferroferric oxide.

[0039] (2) Disperse 1 mol of nano-ferroferric oxide in 500 ml of absolute ethanol, then dropwise add 1 mol of tetraethyl orthosilicate, and dropwise add 10 ml of an ammonia water solution with a mass concentration of 30%. Stir and react at room temperature for 11 h. After the reaction is completed, filter the reaction solution, and wash the precipitate with deionized water and absolute ethanol in sequence, and dry to obtain core-shell structured ferroferric oxide / silica nanoparticles.

[0040] (3) Disperse 10 g of ferroferric oxide / silica nanoparticles in 500 ml of toluene, then add 20 ml of 3-aminopropyltrimethoxysilane, and conduct a reflux reaction at 110 °C for 11 h. After the reaction is completed, wash the solid multiple times to obtain amine-functionalized ferroferric oxide / silica nanoparticles.

[0041] (4) Mix 10 μg of mouse IgG2a and 6 μL of sodium periodate solution with a concentration of 3.5 mmol / L under light protection and react for 30 min. Then, add sodium sulfite solution to terminate the reaction. Next, add a mixed dispersion of 1 mg of amine-functionalized iron oxide / silica nanoparticles and 1 ml of PBS buffer solution, and react for 2 h. After that, filter to obtain IgG2a-conjugated magnetic nanoparticles;

[0042] (5) Disperse 2 mg of IgG2a-conjugated magnetic nanoparticles in 1 ml of PBS buffer solution. Then, add 0.1 ml of tetraethyl orthosilicate and conduct the first stirring reaction for 1 h. Then, add a mixed solution of 5 μL of N-propyltriethoxysilane, 10 μL of benzyltriethoxysilane, and 10 μL of (3-aminopropyl)-triethoxysilane, and conduct the second stirring reaction for 3 h. After the reaction ends, add a mixed solution of methanol, PEG-4, and NHS to the reaction system and stir for 30 min. Then, wash the precipitate with PBS buffer solution. Finally, add the washed precipitate to a mixed solution of 5 ml of sodium hydroxide solution with a concentration of 0.05 mol / L and 5 ml of polyethyleneglycol octylphenyl ether solution with a concentration of 0.01 wt%, and ultrasonically treat at a power of 100 W for 10 min. After that, filter, wash the precipitate, and redisperse it in PBS buffer solution to prepare a dispersion of magnetic nanoparticles.

[0043] Example 4

[0044] A preparation method of core-shell magnetic nanoparticles with high selectivity for antibody separation, comprising the following steps:

[0045] (1) Slowly drop 1 L of a mixed solution containing 0.02 mol of ferric chloride and 0.015 mol of ferrous chloride into 100 ml of sodium hydroxide solution with a concentration of 1.5 mol / L and a temperature of 60 °C while vigorously stirring. After the dropping is completed, stir and react for 2 h. After the reaction ends, cool to room temperature, wash the precipitate, and prepare nano-ferric oxide;

[0046] (2) Disperse 1 mol of nano-ferric oxide in 500 ml of absolute ethanol. Then, drop 1 mol of tetraethyl orthosilicate and 10 ml of ammonia water with a mass concentration of 30%, and stir and react at room temperature for 12 h. After the reaction ends, filter the reaction solution, and wash the precipitate with deionized water and absolute ethanol in sequence, and dry to prepare iron oxide / silica nanoparticles with a core-shell structure;

[0047] (3) Disperse 10 g of iron oxide / silica nanoparticles in 500 ml of toluene. Then, add 20 ml of 3-aminopropyltrimethoxysilane and conduct a reflux reaction at 110 °C for 13 h. After the reaction ends, wash the solid multiple times to prepare amine-functionalized iron oxide / silica nanoparticles;

[0048] (4) Mix 10 μg of mouse IgG2a and 7 μL of sodium periodate solution with a concentration of 4 mmol / L under light protection and react for 30 min. Then, add sodium sulfite solution to terminate the reaction. Next, add a mixed dispersion of 1 mg of amine-functionalized iron oxide / silica nanoparticles and 1 ml of PBS buffer solution and react for 3 h. After that, filter to obtain IgG2a-conjugated magnetic nanoparticles.

[0049] (5) Disperse 2 mg of IgG2a-conjugated magnetic nanoparticles in 1 ml of PBS buffer solution. Then, add 0.1 ml of tetraethyl orthosilicate and conduct the first stirring reaction for 1 h. Then, add a mixed solution of 5 μL of N-propyltriethoxysilane, 10 μL of benzyltriethoxysilane, and 10 μL of (3-aminopropyl)-triethoxysilane and conduct the second stirring reaction for 4 h. After the reaction, add a mixed solution of methanol, PEG-4, and NHS to the reaction system and stir for 40 min. Then, wash the precipitate with PBS buffer solution. Finally, add the washed precipitate to a mixture of 5 ml of sodium hydroxide solution with a concentration of 0.05 mol / L and 5 ml of Triton X-100 solution with a concentration of 0.015 wt%, ultrasonically treat at a power of 100 W for 10 min, then filter, wash the precipitate, and redisperse it in PBS buffer solution to prepare a dispersion of magnetic nanoparticles.

[0050] Example 5

[0051] A preparation method of core-shell magnetic nanoparticles with high selectivity for antibody separation, comprising the following steps:

[0052] (1) Slowly drop 1 L of a mixed solution containing 0.025 mol of ferric chloride and 0.015 mol of ferrous chloride into 100 ml of sodium hydroxide solution with a concentration of 1.5 mol / L and a temperature of 65 °C, stirring vigorously while dropping. After dropping, stir and react for 3 h. After the reaction, cool to room temperature, wash the precipitate, and prepare nano-sized iron oxide.

[0053] (2) Disperse 1 mol of nano-sized iron oxide in 500 ml of absolute ethanol, then drop 1 mol of tetraethyl orthosilicate, and drop 10 ml of ammonia water with a mass concentration of 30%. Stir and react at room temperature for 14 h, and dry to prepare iron oxide / silica nanoparticles with a core-shell structure.

[0054] (3) Disperse 10 g of iron oxide / silica nanoparticles in 500 ml of toluene, then add 20 ml of 3-aminopropyltrimethoxysilane, and conduct a reflux reaction at 110 °C for 14 h. After the reaction, wash the solid multiple times to prepare amine-functionalized iron oxide / silica nanoparticles.

[0055] (4) Mix 10 μg of mouse IgG2a with 7 μL of sodium periodate solution at a concentration of 4 mmol / L and react under light avoidance conditions for 20 min. Then, add sodium sulfite solution to terminate the reaction. Next, add a mixed dispersion of 1 mg of amine-functionalized iron oxide / silica nanoparticles and 1 ml of PBS buffer solution and react for 3 h. After that, filter to obtain IgG2a-conjugated magnetic nanoparticles.

[0056] (5) Disperse 2 mg of IgG2a-conjugated magnetic nanoparticles in 1 ml of PBS buffer solution. Then, add 0.1 ml of tetraethyl orthosilicate and carry out the first stirring reaction for 1 h. Then, add a mixed solution of 5 μL of N-propyltriethoxysilane, 10 μL of benzyltriethoxysilane, and 10 μL of (3-aminopropyl)-triethoxysilane and carry out the second stirring reaction for 4 h. After the reaction is completed, add a mixed solution of methanol, PEG-4, and NHS to the reaction system and stir for 30 min. Then, wash the precipitate with PBS buffer solution. Finally, add the washed precipitate to a mixed solution of 5 ml of sodium hydroxide solution at a concentration of 0.05 mol / L and 5 ml of Triton X-100 solution at a concentration of 0.015 wt% and perform ultrasonic treatment at a power of 100 W for 10 min. After that, filter and wash the precipitate to prepare magnetic nanoparticles.

[0057] Perform antibody separation tests on fetal bovine serum solution containing human IgG using 1 mg of the magnetic nanoparticles prepared in the above example. The test results are shown in Table 1.

[0058] Table 1

[0059]

[0060] It can be seen from the above test results that the magnetic nanoparticles provided by the present invention have excellent effects when used for antibody purification and separation.

[0061] In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A preparation method of core-shell magnetic nanoparticles with high selectivity for antibody separation, characterized in that, It includes the following steps: (1) Slowly drip the iron salt solution into the heated sodium hydroxide solution, control the volume ratio of the sodium hydroxide solution to the iron salt solution to be 10:1, stir vigorously while dripping, stir and react for 2 - 3 h after the dripping is completed, cool to room temperature after the reaction is completed, wash the precipitate, and prepare nano-ferroferric oxide; (2) Disperse the nano-ferroferric oxide in absolute ethanol, then drip tetraethyl orthosilicate and ammonia water solution, stir and react at room temperature for 10 - 15 h, filter the reaction solution after the reaction is completed, and wash the precipitate with deionized water and absolute ethanol in sequence, and dry to prepare ferroferric oxide / silica nanoparticles with a core-shell structure; the dosage ratio of the nano-ferroferric oxide, tetraethyl orthosilicate, and ammonia water solution is 1 mol:(0.8 - 1) mol:10 ml; (3) Disperse the prepared ferroferric oxide / silica nanoparticles in toluene, then add 3-aminopropyltrimethoxysilane, carry out a reflux reaction, wash the solid multiple times after the reaction is completed, and prepare amine-functionalized ferroferric oxide / silica nanoparticles; the dosage ratio of the ferroferric oxide / silica nanoparticles to 3-aminopropyltrimethoxysilane is 1 g:(2 - 3) ml; (4) Mix and react mouse IgG2a and sodium periodate solution under light-shielding conditions, then drip sodium sulfite solution to terminate the reaction, then add a mixed dispersion of amine-functionalized ferroferric oxide / silica nanoparticles and PBS buffer solution, react, and then filter to obtain IgG2a-conjugated magnetic nanoparticles; the dosage ratio of the mouse IgG2a to the sodium periodate solution is 10 μg:6 - 7 μL; the dosage ratio of the amine-functionalized ferroferric oxide / silica nanoparticles to the PBS buffer solution in the mixed dispersion is 1 mg:1 ml; (5) Disperse the IgG2a-conjugated magnetic nanoparticles in PBS buffer solution, then add tetraethyl orthosilicate, carry out the first stirring reaction for 0.5 - 1.5 h, then add a mixed solution of N-propyltriethoxysilane, benzyltriethoxysilane, and (3-aminopropyl)-triethoxysilane, carry out the second stirring reaction for 2 - 5 h, after the reaction is completed, drip a mixed solution of methanol, PEG-4, and NHS into the reaction system and stir to react, then wash the precipitate with PBS buffer solution, finally add the washed precipitate into a mixed solution of sodium hydroxide solution and polyethylene glycol octylphenyl ether solution, carry out ultrasonic treatment, then filter, wash the precipitate, prepare magnetic nanoparticles, and redisperse them in PBS buffer solution for use; the dosage ratio of the IgG2a-conjugated magnetic nanoparticles, PBS buffer solution, tetraethyl orthosilicate, N-propyltriethoxysilane, benzyltriethoxysilane, and (3-aminopropyl)-triethoxysilane is 2 mg:1 ml:0.1 ml:5 μL:10 μL:10 μL; the molar ratio of methanol, PEG-4, and NHS is 1:1:1; the volume ratio of the polyethylene glycol octylphenyl ether solution to the sodium hydroxide solution is 1:

1.

2. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (1), the iron salt solution is a mixed solution of ferric chloride and ferrous chloride, and the concentrations of ferric chloride and ferrous chloride in the mixed solution are 0.015 - 0.025 mol / L and 0.005 - 0.015 mol / L respectively; the concentration of the sodium hydroxide solution is 1.4 - 1.6 mol / L.

3. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (2), the mass concentration of the ammonia water solution is 30%.

4. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (3), the temperature of the reaction is 100 - 120 °C, and the time is 10 - 15 h.

5. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (4), the concentration of the sodium periodate solution is 3.5 - 4.5 mmol / L; the time of the mixed reaction is 20 - 30 min.

6. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (4), the time of the reaction is 2 - 3 h.

7. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (5), the concentration of the mixed solution of methanol, PEG-4, and NHS is 0.45 - 0.55 mmol / L; the time of the reaction is 20 - 40 min.

8. The preparation method of a core-shell magnetic nanoparticle with high selectivity for antibody separation according to claim 1, characterized in that, In step (5), the concentration of the sodium hydroxide solution is 0.05 mol / L, and the concentration of the Triton X-100 solution is 0.01 - 0.02 wt%; the ultrasonic treatment is 5 - 10 min, and the ultrasonic power is 100 W.

9. A core-shell magnetic nanoparticle with high selectivity for antibody separation, characterized in that, Prepared by the method according to any one of claims 1 to 8.

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