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A technology of immunomagnetic beads and magnetic beads, applied in the field of immunology, can solve the problems of complex operation and low purity, and achieve the effects of uniform particle size distribution, high sensitivity and good superparamagnetic properties.
Inactive Publication Date: 2017-11-24
安徽安龙基因科技有限公司
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Problems solved by technology
[0009] The technical problem to be solved by the present invention is to provide a preparation method of CD133 immunomagnetic beads in order to overcome the defects of low purity and complicated operation of tumor stem cell sorting in the prior art
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Embodiment 1
[0045] The invention discloses a preparation method of CD133 immunomagnetic beads, the specific steps are as follows:
[0046] 1. Preparation of magnetic beads:
[0047] 1.1. Under the condition of nitrogen gas, 12.1g FeSO 4 ·7H 2 O, 23.5g FeCl 3 ·6H 2 Dissolve O in 800ml of distilled water, heat to reflux, and when heated to 90°C, quickly add 30ml of ammonia water to the above solution under mechanical stirring;
[0048] 1.2. After 1 hour, the reaction is over, and the supernatant is discarded under the action of an external magnetic field;
[0049] 1.3. Add 100ml of ethanol, stir and wash the magnetic beads thoroughly, collect the magnetic beads under an external magnetic field, wash them with 100ml of distilled water, and dry them under vacuum at room temperature to obtain Fe 3 o 4 nano magnetic beads;
[0050] 2. Preparation of magnetic chitosan microspheres:
[0051] 2.1. Weigh 10 grams of chitosan and add it to 200ml 2% acetic acid solution to soak overnight to dis...
Embodiment 2
[0070] The invention discloses a preparation method of CD133 immunomagnetic beads, the specific steps are as follows:
[0071] 1. Preparation of magnetic beads:
[0072] 1.1. Under the condition of nitrogen gas, 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, 30ml of ammonia water was added to the above solution under mechanical stirring;
[0073] 1.2. After 1 hour, the reaction is over, and the supernatant is discarded under the action of an external magnetic field;
[0074] 1.3. Add 100ml of ethanol, stir and wash the magnetic beads thoroughly, collect the magnetic beads under an external magnetic field, wash them with 100ml of distilled water, and dry them under vacuum at room temperature to obtain Fe 3 o 4 nano magnetic beads;
[0075] 2. Preparation of magnetic chitosan microspheres:
[0076] 2.1. Weigh 10 grams of chitosan and add it to 200ml 2% acetic acid solution to soak overnight...
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Abstract
The invention discloses a preparation method of CD133 immunomagnetic beads. The preparation method comprises the following steps: preparing magnetic beads; preparing magnetic chitosan microspheres; weighing 10 g of chitosan, and adding the weighed chitosan into 200 ml of a 2% acetic acid solution for overnight soaking to enable dissolution; adding 1 g of Fe2O4 nano magnetic beads into the mixed solution, and performing stirring at the room temperature under the condition of 300 r/min; adding 200 ml of saxoline, and performing stirring at 40 DEG C under the condition of 300 r/min; raising the temperature to 50 DEG C, adding 40 ml of formaldehyde, and performing stirring under the condition of 300 r/min; adding 20 ml of glutaraldehyde with the volume concentration of 50% into the mixed solution, performing stirring at 60 DEG C under the condition of 170 r/min, adjusting the pH to be 10, and performing stirring for 2-6 h; adding 100 ml of petroleum ether into a reaction system after finish of reaction, fully performing stirring, performing suction filtration by use of a Buchner funnel to obtain magnetic chitosan microsphere particles, then fully performing stirring and washing by use of ethyl alcohol, washing with distilled water after removal of ethyl alcohol, and performing vacuum drying at 50 DEG C; performing carboxylation on the magnetic chitosan microspheres; and coupling immunomagnetic beads subjected to carboxylation with an anti-body. The preparation method has the advantages of good stability, high sensitivity and the like.
Description
technical field [0001] The invention relates to the field of immunology, in particular to a preparation method of CD133 immunomagnetic beads. Background technique [0002] In recent decades, the morbidity and mortality of tumors worldwide have been increasing. Although there have been many new advances in the treatment of tumors, the vast majority of tumor patients still cannot be cured, and are prone to recurrence and metastasis. The survival time and quality of life of cancer patients are always low. The first report of human tumor stem cells brings us new hope. Finding specific surface markers and sorting tumor stem cells through them is the key to further study on tumorigenesis, metastasis, recurrence and prognosis. [0003] CD133 is a unique surface marker of stem cells and tumor stem cells, with a molecular weight of 120kDa and 5 transmembrane regions. The CD133 antigen can be recognized by three CD133 antibodies: clones AC133, 293C3 and AC141, AC133 directly binds t...
Claims
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