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Immunomagnetic beads as well as preparation method and application thereof

A technology of immunomagnetic beads and cross-linking reaction, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complex preparation methods of immunomagnetic beads, harsh reaction conditions, and harsh operating conditions, so as to increase antibody loading and optimize Conditions, effects of protective activity

Inactive Publication Date: 2018-09-07
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The preparation method of traditional immunomagnetic beads is complicated and the operating conditions are harsh. It is necessary to modify special functional groups on the surface of paramagnetic substances in order to bind antibodies or antigens, and the modification process is complicated and the reaction conditions are harsh.

Method used

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  • Immunomagnetic beads as well as preparation method and application thereof
  • Immunomagnetic beads as well as preparation method and application thereof
  • Immunomagnetic beads as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] an immunomagnetic bead

[0050] Step 1: Preparation of Fe 3 o4 nano magnetic core

[0051] Weigh 20.0g of ferrous sulfate heptahydrate and dissolve it in 140.0mL of deionized water, heat in a water bath to 90°C and react for 1h under the condition of mechanical stirring at a speed of 400r / min. At the same time, another 1.6 g of potassium nitrate and 11.2 g of potassium hydroxide were dissolved in 60.0 mL of deionized water, and the prepared solution was added dropwise (1 mL / min) into the ferrous ion solution with a constant flow pump. Continue to stir at 90°C for 2 hours after the addition to obtain a black turbid solution, which is washed several times with deionized water and dried. The black solid is ferric oxide, and its SEM image is as follows figure 1 and 2 , Fe 3 o 4 The particle size of the nano-magnetic nuclei is between 100 and 300nm, the spheres of the magnetic nuclei are unstable, and some of the magnetic nuclei are irregular. Due to the relatively act...

Embodiment 2

[0092] Its difference with embodiment 1 is that AMP&ZnCl 2 The reaction conditions for hydrogel-coated magnetic beads are different, as follows.

[0093] Weigh 0.3 g of adenosine monophosphate disodium salt and dissolve it in 20 mL of HEPES buffer (10 mM, pH 7.4), then add 200 mg of ferric oxide, and stir thoroughly. Add 150mL of paraffin wax mixture containing 6.5% Span 80 dropwise under stirring, form an emulsified system after stirring at 500r / min, continue stirring for 30min and heat to 40°C in a water bath, then add 3mL of glutaraldehyde solution (C 5 h 8 o 2 , volume fraction 25%), constant temperature cross-linking reaction 30min. Lower the temperature, add 2mL 1mg / mL ZEA monoclonal antibody dropwise (1mL / min) into the reaction system with a constant flow pump at room temperature, stir well, then add 10mL 50mM ZnCl dropwise (1mL / min) with a constant flow pump 2 Solution, cross-linking reaction at room temperature for 2h.

[0094] After the reaction, wash with petro...

Embodiment 3

[0097] Its difference with embodiment 1 is that AMP&ZnCl 2 The reaction conditions for hydrogel-coated magnetic beads are different, as follows.

[0098] Weigh 0.3g of adenosine monophosphate disodium salt and dissolve in 20mL of HEPES buffer (10mM, pH 7.4), then add 200mg of ferric oxide, and stir thoroughly. Add 150mL of paraffin wax mixture containing 6.5% Span 80 dropwise under stirring, form an emulsified system after stirring at 500r / min, heat to 45°C in a water bath while stirring continuously for 30min, then add 3mL of glutaraldehyde solution (C 5 h 8 o 2 , volume fraction 25%), constant temperature cross-linking reaction 25min. Lower the temperature, add 2mL 1mg / mL ZEA monoclonal antibody dropwise (1mL / min) into the reaction system with a constant flow pump at room temperature, stir well, then add 10mL 50mM ZnCl dropwise (1mL / min) with a constant flow pump 2 Solution, cross-linking reaction at room temperature for 2h.

[0099] After the reaction, wash with petrol...

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Abstract

The invention provides immunomagnetic beads as well as a preparation method and application thereof. The preparation method of the immunomagnetic beads comprises the following steps: mixing adenosinemonophosphate or metal salt thereof, a water-phase buffer solution, ferroferric oxide, paraffin and span-80, and stirring, thus forming an emulsification system, wherein a volume ratio of the paraffinto the span-80 is 7:(93 to 100), and a mass ratio of the adenosine monophosphate or metal salt thereof to the ferroferric oxide is (0.1 to 0.3):0.2; adding glutaraldehyde in the emulsification system, and carrying out first-step cross-linking reaction at constant temperature; then adding an antibody and zinc salt, carrying out second-step cross-linking reaction, and then separating out a solid substance by using a magnet. According to the preparation method disclosed by the invention, the antibody and the ferroferric oxide are simultaneously fixed in a metal-organogel immobilizing system by utilizing properties of metal- organogel for immobilizing protein, the used reaction conditions are gentle, and special operation or complicated pretreatment procedure is not required, so that a favorable condition is broadened for wide application of the immunomagnetic beads by the preparation method.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an immunomagnetic bead and its preparation method and application. Background technique [0002] Immunomagnetic beads are conjugates of superparamagnetic substances and antibodies, which can be used to bind corresponding antigens or antibodies. Because immunomagnetic beads can move directionally under the attraction of an external magnetic field, they can be used for the separation and detection of targets, and achieve the purpose of purifying genes, proteins, cells, microorganisms, etc. Due to the rapidity and specificity of the immune response, immunomagnetic beads have better sensitivity and specificity than conventional detection or separation methods. [0003] The preparation method of traditional immunomagnetic beads is complicated and the operating conditions are harsh. It is necessary to modify special functional groups on the surface of paramagnetic substan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/543
CPCG01N33/531G01N33/54326
Inventor 丁轲孙毅蒙韩涛陈湘宁
Owner BEIJING UNIV OF AGRI
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