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An immunomagnetic bead and antibody technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of poor stability and poor specificity, and achieve the effects of good stability, good specificity and excellent dispersion.
Inactive Publication Date: 2017-12-19
安徽安龙基因科技有限公司
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[0005] The technical problem to be solved by the present invention is to provide a preparation method of EpCAM antibody immunomagnetic beads in order to overcome the defects of poor stability and poor specificity in the prior art
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preparation example Construction
[0046] The invention discloses a preparation method of EpCAM antibody immune magnetic beads, the specific steps are as follows:
[0047] 1. Synthesis of oleic acid-Fe 3 o 4 :
[0048] (1.1), under the condition of passing nitrogen, FeSO 4 ·7H 2 O, FeCl 3 ·6H 2 O was dissolved in 800ml of distilled water, heated to reflux, and when heated to 90°C, while stirring, 30ml of ammonia water was added to the above solution;
[0049] (1.2), add 20ml oleic acid, stop the reaction after stirring continuously for 1 hour at 90°C;
[0050] (1.3) After the solution is cooled to room temperature, apply an external magnetic field to absorb the precipitate, and then wash it with distilled water at least once, and the product is oleic acid-Fe3O4;
[0051] 2. Fe 3 o 4 / PVAc magnetic microsphere preparation:
[0052] (2.1), add 100ml of water to the four-necked flask equipped with agitator, condenser, thermometer and air guide tube, heat and reflux in a water bath, and feed nitrogen for ...
Embodiment 1
[0079] The invention discloses a preparation method of EpCAM antibody immune magnetic beads, the specific steps are as follows:
[0080] 1. Synthesis of oleic acid-Fe 3 o 4 :
[0081] (1.1), under the condition of nitrogen, 12.1g FeSO 4 ·7H 2 O, 23.5g FeCl 3 ·6H 2 O was dissolved in 800ml of distilled water, heated to reflux, and when heated to 90°C, while stirring, 30ml of ammonia water was added to the above solution;
[0082] (1.2), add 20ml oleic acid, stop the reaction after stirring continuously for 1 hour at 90°C;
[0083] (1.3) After the solution is cooled to room temperature, apply an external magnetic field to absorb the precipitate, then wash it twice with distilled water, and the product is oleic acid-Fe3O4;
[0084] 2. Fe 3 o 4 / PVAc magnetic microsphere preparation:
[0085] (2.1), add 100ml of water to the four-necked flask equipped with agitator, condenser, thermometer and air guide tube, heat and reflux in a water bath, and feed nitrogen for degassin...
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Abstract
The invention discloses a preparation method of EpCAM (Epithelial Cell Adhesion Molecule) antibody immunomagnetic beads. The preparation method comprises the following specific steps: synthesizing oleic acid-Fe3O4; preparing Fe3O4 / PVAc (Polyvinyl Acetate) magnetic microspheres; preparing (Fe3O4 / PVA) / SiO2 magnetic microspheres; adding aminated magnetic nanoparticles into a PBS (Polybutylene Succinate) solution of glutaraldehyde and stirring for 3h; modifying an aldehyde-group functional group on the surfaces of the magnetic particles through a Schiff base formed between an amino group and an aldehyde group; after finishing reaction, separating out the particles from a reaction medium by utilizing an external magnetic field and dispersing the particles into the PBS solution; carrying out hydrazino modification on an antibody: adding a dimethylformamide solution containing p-hydrazone pyridine formic acid N-hydroxysuccinyl imide into an EpCAM antibody solution; after reacting for 2h, purifying by utilizing an ultra-filtration column to obtain a hydrazino-modified EpCAM antibody; preparing the immunomagnetic beads: mixing the aldehyde-group-modified magnetic microspheres and the hydrazino modified EpCAM antibody and mixing to obtain the EpCAM antibody immunomagnetic beads of EpCAM antibody coupled magnetic microspheres. The EpCAM antibody immunomagnetic beads disclosed by the invention have the advantages of good stability, strong specificity and the like.
Description
technical field [0001] The invention relates to the field of preparation of immunomagnetic beads, in particular to a preparation method of EpCAM antibody immunomagnetic beads. Background technique [0002] Immunomagnetic bead separation (IMB) is a new immunological technology that has been studied more at home and abroad in recent years. At present, this technology has made great progress in cell separation, protein purification, immunology and microbiology detection. The separation method has the advantages of high sensitivity, fast detection speed and high specificity. However, the magnetic beads used in the Chinese market are generally imported from abroad and are expensive, which greatly limits the application of immune magnetic beads in medicine and scientific research in my country. [0003] There are many methods for preparing magnetic nanoparticles, including physical methods, chemical methods and other special preparation methods. Chemical precipitation is the mo...
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