CN102527353A - Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells - Google Patents
Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells Download PDFInfo
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- CN102527353A CN102527353A CN201110433615XA CN201110433615A CN102527353A CN 102527353 A CN102527353 A CN 102527353A CN 201110433615X A CN201110433615X A CN 201110433615XA CN 201110433615 A CN201110433615 A CN 201110433615A CN 102527353 A CN102527353 A CN 102527353A
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Abstract
The invention belongs to the technical field of biomedicine and discloses a nanometer magnetic immunomicrosphere for enriching circulating cancer cells and a use method thereof. A nanometer carbon coated pure ferromagnetic microsphere (Fe@C) is taken as an inner core and is connected with a cancer cell antibody, so as to prepare the nanometer magnetic immunomicrosphere for enriching the circulating cancer cells. By utilizing the magnetic immunomicrosphere to enrich the circulating cancer cells, the sensitivity of the detection for the circulating cancer cells is efficiently increased. The immunomicrosphere has the characteristics of strong magnetic reactivity, easiness in operation, and the like, and is applied to the quick enriching of the circulating cancer cells.
Description
Technical field
The invention belongs to biomedical sector, be specifically related to a kind of preparation and method for using thereof of nona-magnetic immuno-microsphere of enrichment circulating tumor cell.
Technical background
Approach according to the tumour cell micrometastasis; The tumour cell that in blood or lymphatic vessel, circulates is called circulating tumor cell (circulating tumor cell; CTC); By some circulating tumor cells assemble the cell mass that forms be called as the little bolt of circulating tumor (circulating tumor microemboli, CTM).CTM is tumour cell " collective migration " behavior, because of it can be resisted Apoptosis, keep ability of cell proliferation to have higher metastatic potential.Because circulating tumor cell content in blood or lymph liquid is considerably less and lack effective specificity marker thing, the progress of relevant CTC is slower.Existing separation, detection technique method cut both ways, and a unified experimental program and standard are not arranged so far as yet.
The content of CTC in peripheral blood is few, possibly only contain a few CTC or CTM in every 10ml blood, and 10ml blood then contains about 100,000,000 leucocytes and 50,000,000,000 red blood cells.Therefore, have only at first the CTC in the clinical blood sample is carried out enrichment, just can detect and biological property and genetic analysis then.
The method of existing enrichment CTC has three kinds usually: 1) the gradient density centrifugal method is promptly separated according to cell settlement coefficient difference.2) polycarbonate membrane in filtration method 8 μ m apertures can make less lymphocyte and neutrophil leucocyte pass through, and bigger tumour cell then is arrested on the film.This method is not only simple to operate, and relatively more responsive, can avoid that loaded down with trivial details multistep is rapid separates the destruction of the rare cells that causes and lose.3) the immunomagnetic isolation method this be CTC enrichment method at present commonly used.Antibody to epithelial antigen EpCAM, BerEP4, CK or organ specificity mark (as: mammoglobul in, PSA, CEA and HER-2) is used to separation of C TC usually, and bioaccumulation efficiency can reach 1 * 10
4～2 * 10
5Doubly.In order further to reduce haemocyte content, improve bioaccumulation efficiency, can negative sorting be combined with positive sorting.The mark that negative sorting is adopted is the CD61 that expresses of the CD45 that expresses of leucocyte or macrophage, blood platelet normally.In addition, in order to improve the specificity of CTC enrichment, nearest research is tended to physical separation method and immunomagnetic isolation method are combined.As: earlier, then carry out the immune magnetic positive, negative sorting, the last prostate tumor cells of on the basis of fluorescent staining, collecting efficiently concentrating with micromanipulative technique with the Ficoll-Hypaque centrifugation.Immunomagnetic isolation method efficient is high, and in whole mistake, can avoid cytolysis, makes the counting of purpose cell become possibility.
Detect the powerful measure that existence can become early diagnosis of cancer or whether help discriminating tumour soaks in tumor biopsy of tumour cell in the blood at present.The detection method commonly used of tumour cell micrometastasis comprise RT-PCR technology, Flow Cytometry (flow cytometry, FCM) and immunocytochemical method (ICC) or the like.Though RT-PCR method sensitiveness is higher, has higher false positive rate.The FCM detection method costs an arm and a leg and operates to waste time and energy, and the information of relevant tumour cell form aspect can not be provided, and also needs to carry out ICC dyeing with the tumour cell specific antibody, is difficult to be applied to clinical as the conventional sense method.The ICC method has higher specificity, but the rate that picks is very low.At present, the birth of immune nano magnetic microballoon provides advanced separation means for the tumour cell biological diagnosis, and it has high specific, highly enriched property, high separation rate, and does not influence characteristics such as cytoactive, has report can improve separation rate 10 abroad
3-10
4Doubly (Martin et al.1998).
Summary of the invention
During the object of the invention is intended to detect to circulating tumor cell in the sample tumour cell concentration extremely low, be unfavorable for the problem that detects a kind of nona-magnetic immuno-microsphere and method for using thereof that is used for the circulating tumor cell enrichment being provided.
To achieve these goals; The present invention has announced a kind of new nona-magnetic immuno-microsphere, and it is a kernel with nano-sized carbon bag pure iron magnetic microsphere, connects the epithelial cell specific antibody; Combine through antigen-antibody reaction with circulating tumor cell; Tumour cell to existing in the peripheral blood carries out fast enriching, and (ICC) combines with immunocytochemistry, and patient's peripheral blood tumour cell is detected.Detection sensitivity is brought up to 5 * 10
7Can detect a tumour cell in the individual cell.Be convenient to find the recessive micrometastasis of early stage patient, monitoring postoperative patient tumor recurrence and transfer, assess prognosis and formulate therapeutic scheme.
Aforesaid nano-magnetic microsphere, carbon bag pure iron (FeC) nano-magnetic microsphere that it is characterized in that having superparamagnetism;
Aforesaid nano-magnetic microsphere is characterized in that inside that carbon bag pure iron can be positioned at magnetic microsphere Anywhere;
Aforesaid nano-magnetic microsphere is characterized in that can there be one or more carbon bag pure iron particle magnetic microsphere inside.
Aforesaid nano-magnetic microsphere is characterized in that the matrix of magnetic microsphere can be used to do the material of magnetic microsphere matrix for shitosan, polystyrene, silica, shitosan or any other.
Aforesaid nano-magnetic microsphere, the magnetic-particle of its characteristic diameter between 10nm-50nm, it can be an Any shape, and is preferred spherical;
Aforesaid magnetic immuno-microsphere is characterized in that this magnetic microsphere can pass through covalent coupling, hydrophobic effect power or intermolecular force and antibody coupling together.
Aforesaid nona-magnetic immuno-microsphere is characterized in that not being only applicable to the separation of tumour cell, also is applicable to separate other cell or molecule.
Aforesaid specific antibody is characterized in that the antibody that can combine with circulating tumor cell surface protein (antigen) specificity, and it can be a monoclonal antibody, also can be polyclonal antibody.
To achieve these goals; The present invention has also announced a kind of method with nona-magnetic immuno-microsphere enrichment circulating tumor cell; It is characterized in that nona-magnetic immuno-microsphere catches tumour cell; Under the active force of magnetic field, will combine sample separation, realize enrichment all kinds tumour cell in peripheral blood or the body fluid with magnetic immuno-microsphere.Again magnetic immuno-microsphere-tumour cell compound is washed, remove residual cell and thin other impurity, obtain the tumour cell-magnetic microsphere compound of high concentration at last.
The method of aforesaid enrichment circulating tumor cell; It is characterized in that immune magnetic microsphere can be through magnetic microsphere surface fixing antibody and the reaction of tumor cell surface corresponding antigens; Form magnetic microsphere-antibody-antigenic compound, thereby the realization immune magnetic microsphere is caught tumour cell.Can also carry out through following mode; Biotinylated antibody and the corresponding antigens reaction of circulating tumor cell surface; Form biotin-antibody-tumour cell compound, add the magnetic microsphere that the surface is fixed with Avidin again, the Avidin and the biotin reaction on magnetic microsphere surface; Form magnetic microsphere-Avidin-biotin-antibody-circulating tumor cell compound, thereby realize immune magnetic microsphere catching circulating tumor cell.
It is strong that the disclosed nona-magnetic immuno-microsphere of the present invention has a magnetic responsiveness, and particle diameter is little, characteristics such as easy operating.
The specific embodiment
Although content of the present invention is to combine this instance to describe, can not think restriction to patent of the present invention, scope of the present invention is limited appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms belong to protection scope of the present invention equally.
The preparation of embodiment 1 nano-magnetic microsphere
The 100mg sodium alginate solution is dissolved in the 4ml pure water, adds 2ml 5%FeC magnetic fluid solution, 42 ℃ make it abundant mixing dissolving after, ultrasonic 15min uses 10%Na
2CO
3The pH that regulates mixed liquor is about 10, is warming up to 60 ℃.Under the condition of ultrasonic and high-speed stirred, it is in 60 ℃ the 90ml AOT/ normal heptane oil phase that mixed liquor is dropwise joined temperature, forms transparent grey black reverse microemulsion system.Under ultrasonic and stirring condition with 30% 4.5ml CaCl
2Solution joins in the microemulsion system, carries out washing with acetone, ethanol and distilled water respectively after magnetic separates, and centrifugal classification is then preserved after the vacuum freeze drying.
The preparation of embodiment 2 magnetic microspheres-antibody coupling matter
Get the magnetic microsphere that 1mg prepares, with phosphate buffer washing 3 times, with the constant volume of 0.01mol/L PBS PH7.0 to 4ml; Add water-soluble EDC 5mg and 7.5mg suflo-NHS, abundant mixing, room temperature reaction 15min; The 6-aminocaprolc acid that adds 50mg then after 3h is stirred in the room temperature rotation, adds 500 μ l (40 μ g/ml) EpCAM monoclonal antibody (BD Pharmingen; The U.S.), stir 6h gently, seal with 0.2M glycine solution (containing 0.2%BSA) 1ml; The magnetic separating, washing adds 4 ℃ of preservations of storage liquid.
The transmission electron microscope observing microballoon is spherical in shape, and good dispersiveness is arranged, and particle diameter is distributed between the 45-89nm, and average grain diameter is 56nm.Utilize the Bradford detection method to measure protein content in the nano immune magnetic microballoon, every milligram of immune magnetic microsphere can connect 108.6 μ g antibody.Utilize immunofluorescence dyeing, flow cytometer detects and shows that 97.9% nano immune magnetic microsphere surface is connected with EpCAM antibody.
Embodiment 3 breast cancer tumour cell enrichment and detections
It is an amount of to add nano immune magnetic microballoon in every duplicate samples, and 30min is stirred in the room temperature rotation behind the mixing, and magnetic separator separates; With PBS or physiological saline washing magnetic microsphere several times; Remove impurity, at last magnetic microsphere is resuspended in the suitable liquid that obtain concentrating, pure circulating tumor cell.
The preparation of embodiment 3 Streptavidin immune magnetic microspheres
Get the magnetic microsphere that 1mg prepares,, to 4ml, add water-soluble EDC 5mg and 7.5mg suflo-NHS with the constant volume of 0.01mol/L PBS PH7.0 with phosphate buffer washing 3 times; Abundant mixing, room temperature reaction 15min adds the 6-aminocaprolc acid of 50mg, after 3h is stirred in the room temperature rotation then; Add 500 μ l (40 μ g/ml) Streptavidin, stir 6h gently, seal with 0.2M glycine solution (containing 0.2%BSA) 1ml; Magnetic separates, and washing adds 4 ℃ of preservations of storage liquid.
The enrichment of embodiment 4 tumour cells
Add the antibody of biotinylated antitumor cell surface antigen in the sample, mixing leaves standstill 30min; Add an amount of Streptavidin magnetic microsphere, vortex mixing, incubated at room 15min; The centre is mixing repeatedly, and magnetic separator separates, with PBS or physiological saline washing magnetic microsphere several times; Remove impurity, at last magnetic microsphere is resuspended in the suitable liquid that obtain concentrating, pure circulating tumor cell.
Claims (11)
1. the present invention has announced a kind of new nona-magnetic immuno-microsphere; It is kernel with the nano-magnetic microsphere; Connect the epithelial cell specific antibody, combine through antigen-antibody reaction, the tumour cell that exists in the peripheral blood is carried out fast enriching with circulating tumor cell.
2. like right 1 described nano-magnetic microsphere, it is characterized in that having carbon bag pure iron (FeC) nano-magnetic microsphere of superparamagnetism.
3. like right 1 described nano-magnetic microsphere, it is characterized in that activated carbon parcel nanometer pure iron.
4. like right 1 described nano immune magnetic microballoon, it is characterized in that can there be one or more nano-sized carbon bag pure iron microballoon immune microsphere inside.
5. like right 1 described nano-magnetic microsphere, it is characterized in that the matrix of magnetic microsphere can be used to do the material of magnetic microsphere matrix for shitosan, polystyrene, silica, shitosan or any other.
6. like right 1 described nano-magnetic microsphere, the nano magnetic particle of its characteristic diameter between 10nm-50nm, it can be an Any shape, and is preferred spherical.
7. like right 1 described nano-magnetic microsphere, it is characterized in that this magnetic microsphere can pass through covalent coupling, hydrophobic effect power or intermolecular force and antibody coupling together.
8. like right 1 described nona-magnetic immuno-microsphere, it is characterized in that not being only applicable to the separation of circulating tumor cell, also be applicable to the separation of other cell or molecule.
9. like right 1 described specific antibody, it is characterized in that the antibody that can combine with circulating tumor cell surface protein (antigen) specificity, it can be a monoclonal antibody, also can be polyclonal antibody.
10. the present invention has also announced a kind of method with nona-magnetic immuno-microsphere enrichment circulating tumor cell; It is characterized in that immune magnetic microsphere catches tumour cell; Under the active force of magnetic field, will combine sample separation, realize enrichment all kinds tumour cell in peripheral blood or the body fluid with magnetic immuno-microsphere.Again magnetic immuno-microsphere-tumour cell compound is washed, remove residual cell and thin other impurity, obtain the tumour cell-magnetic microsphere compound of high concentration at last.
11. method like right 9 described enrichment circulating tumor cells; It is characterized in that magnetic immuno-microsphere can be through magnetic immuno-microsphere surface fixing antibody and the reaction of tumor cell surface corresponding antigens; Form magnetic microsphere-antibody-antigenic compound, thereby the realization magnetic immuno-microsphere is caught tumour cell.Can also carry out through following mode; Biotinylated antibody and the corresponding antigens reaction of circulating tumor cell surface; Form biotin-antibody-tumour cell compound, add the magnetic microsphere that the surface is fixed with Avidin again, the Avidin and the biotin reaction on magnetic microsphere surface; Form magnetic microsphere-Avidin-biotin-antibody-circulating tumor cell compound, thereby realize magnetic immuno-microsphere catching circulating tumor cell.
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| CN102980793A (en) * | 2012-11-20 | 2013-03-20 | 武汉友芝友生物制药有限公司 | Circulating tumor cell dyeing kit and use thereof |
| CN103278521A (en) * | 2012-11-30 | 2013-09-04 | 中国检验检疫科学研究院 | Magnetic resonance immune sensing method for detecting biomacromolecule |
| CN103275934A (en) * | 2013-06-05 | 2013-09-04 | 南昌大学 | Separation method of micro circulating tumor cells |
| CN103439515A (en) * | 2013-08-14 | 2013-12-11 | 南方医科大学 | Method for detecting valence of antibody |
| CN103630440A (en) * | 2013-11-28 | 2014-03-12 | 武汉大学 | Enriching method of circulating tumor cells |
| CN103923192A (en) * | 2013-01-15 | 2014-07-16 | 国家纳米科学中心 | Polypeptide for enrichment, separation and detection of circulating tumor cells and application thereof |
| CN103940997A (en) * | 2014-03-21 | 2014-07-23 | 上海柏慧康生物科技有限公司 | Breast cancer circulating tumor cells detection system and kit |
| CN104568923A (en) * | 2014-12-01 | 2015-04-29 | 浙江省肿瘤医院 | Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection |
| CN105779393A (en) * | 2016-04-25 | 2016-07-20 | 武汉大学 | Lymph node metastasis carcinoma diagnostic method based on immunomagnetic beads and kit |
| CN105891492A (en) * | 2016-05-17 | 2016-08-24 | 浙江天科高新技术发展有限公司 | Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit |
| CN106367393A (en) * | 2016-08-26 | 2017-02-01 | 中国人民解放军第四军医大学 | Mouse prostate cancer circulating tumor cell line and prostate cancer circulating tumor cell isolating and culturing method |
| CN108546676A (en) * | 2018-04-21 | 2018-09-18 | 深圳市红莓生物科技有限公司 | The method that tunnel magnetic field technology detaches and is enriched with rare cell in peripheral blood |
| CN108982839A (en) * | 2018-06-12 | 2018-12-11 | 广州四叶草健康科技有限公司 | Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer |
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| CN117607441B (en) * | 2024-01-23 | 2024-04-12 | 杭州华得森生物技术有限公司 | Circulating tumor cell TROP-2 immune chromogenic detection reagent |
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Application publication date: 20120704 |