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Preparation method of immune-magnetic bead

A technology of immunomagnetic beads and magnetic beads, applied in the field of immunological detection, can solve the problems of easy agglomeration of magnetic nanoparticles, unsuitable for large-scale samples, and difficult to control the differences between batches, so as to reduce the interaction and achieve rapid and thorough solid-liquid separation. , good water solubility

Active Publication Date: 2014-07-16
山东辛丁技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are problems with the currently used solid-phase carriers: batch-to-batch differences are not easy to control, the coating operation is complicated, the degree of automation is not high, and it is not suitable for the operation of large batches of samples.
However, due to the nano-Fe 3 o 4 Due to the effects of small size effect, magnetic dipole attraction, etc., magnetic nanoparticles are easy to agglomerate, and the chemical stability is not high, easy to be oxidized, and the surface hydroxyl group is insufficient, which makes it difficult to be directly applied to the biological field.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] S1. Preparation of silica-based magnetic beads

[0040] Weigh 300g of anhydrous ferric chloride and dissolve in 1L of ultrapure water, weigh 392g of ferrous ammonium sulfate hexahydrate and dissolve in 1L of ultrapure water, transfer the two solutions into a 5L reactor and stir at 200r / min for 30min , weigh 45g of polyethylene glycol-8000, dissolve it in 400ml of ultrapure water, and transfer it to the reaction kettle; pass argon to remove oxygen for 30 minutes, adjust the reaction temperature to 60°C, adjust the speed to 500r / min, and drop 25% Ammonia water until the pH value of the system is 11, heat up to 80°C, adjust the rotation speed to 100r / min and react for 3h; The concentration was 1% ammonia water, 5% NaCl and deionized water, and the precipitate was repeatedly washed 3 times until the washing liquid was neutral, and freeze-dried at -10°C for 48 hours to obtain superparamagnetic Fe 3 o 4 nanoparticles;

[0041] Weighing superparamagnetic Fe 3 o 4 Disperse...

Embodiment 2

[0050] S1. Preparation of silica-based magnetic beads

[0051] Same as step S1 in embodiment one;

[0052] S2. Preparation of carboxyl magnetic beads

[0053] Take 10 g of freeze-dried silicon-based magnetic beads and disperse them into 700ml of N,N-dimethylformamide solution for 60 minutes of ultrasonic treatment, then add 20ml of N,N-dimethylformamide with a concentration of 50g / L trisodium citrate Formamide solution, after stirring at 500r / min for 30min, add dropwise 100ml of maleic acid solution dissolved in N,N-dimethylformamide, the concentration of which is 10g / L, and the rate of addition is controlled to be 0.5ml / min. The temperature is 60°C, and react for 10 hours after the dropwise addition; after the reaction, wash the magnetic separation with absolute ethanol and deionized water several times in sequence until the pH of the washing liquid is 6, ultrasonically treat for 30 minutes, and disperse in 2 / 10,000 NaN 3 In water, adjust its concentration to 10mg / ml to obt...

Embodiment 3

[0060] S1. Preparation of silica-based magnetic beads

[0061] Same as step S1 in embodiment one;

[0062] S2. Preparation of carboxyl magnetic beads

[0063] Take 100 g of freeze-dried silicon-based magnetic beads and disperse them into 3L N,N-dimethylformamide solution for 60 minutes of ultrasonic treatment, then add 100ml of N,N-dimethylformamide with a concentration of 50g / L trisodium citrate Formamide solution, after stirring at 500r / min for 30min, add dropwise 500ml of maleic acid solution dissolved in N,N-dimethylformamide, the concentration of which is 5g / L, and the rate of addition is controlled to be 0.5ml / min. The temperature is 60°C, and react for 18 hours after the dropwise addition; after the reaction, wash the magnetic separation with absolute ethanol and deionized water several times in sequence until the pH of the washing liquid is 6, ultrasonically treat for 30 minutes, and disperse in 2 / 10,000 NaN 3 In water, adjust its concentration to 10mg / ml to obtain c...

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Abstract

The invention discloses a preparation method of an immune-magnetic bead. The preparation method comprises the following steps: firstly, dispersing freeze-dried silicon-based magnetic beads in a polar organic solvent, treating with ultrasonic wave, subsequently adding a dispersant dissolved in the polar organic solvent, stirring, dripping organic small molecules which are dissolved in the polar organic solvent and contain double-end carboxyl, and then reacting for 6-18 hours after the dripping is ended; magnetically separating out carboxyl magnetic beads after the reaction ends, sequentially and repeatedly washing precipitates for a plurality of times by use of absolute ethyl alcohol and deionized water until the pH value of a washing liquid is 6-7, performing ultrasonic treatment for 15-30 minutes, and dispersing into 0.02% of NaN3 water to obtain the carboxyl magnetic beads; washing the carboxyl magnetic beads by use of an MES (Methyl Ester Sulfonate) buffer solution at least once, re-suspending the carboxyl magnetic beads in the MES buffer solution, activating the magnetic beads by using an activating agent carbodiimide, and then coupling an antibody in the environment of the MES buffer solution. The preparation method disclosed by the invention is simple in technological process, simple and convenient to operate and suitable for batch production.

Description

technical field [0001] The invention belongs to the technical field of immunological detection, in particular to a preparation method of immunomagnetic beads. Background technique [0002] At present, the method used in immunoassay and immunodiagnosis is to bind antigen or antibody to the surface of some kind of solid phase carrier and maintain its immunological activity. During the determination, the test specimen (to determine the antigen or antibody in it) and the enzyme-labeled antigen or antibody react with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by washing, and the amount of enzyme bound to the solid-phase carrier is directly related to the amount of the substance to be tested in the sample, so it can be qualitatively published according to the depth of the color reaction. or quantitative analysis. Due to the high frequency o...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531B82Y15/00B82Y40/00B82Y30/00
CPCB82Y15/00B82Y30/00B82Y40/00G01N33/54326G01N33/54346G01N2446/00
Inventor 杜德光高祥辉姜夏马睿
Owner 山东辛丁技术有限公司
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