CN109856388A - The catching method and capture kit of circulating tumor cell - Google Patents
The catching method and capture kit of circulating tumor cell Download PDFInfo
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- CN109856388A CN109856388A CN201811447767.3A CN201811447767A CN109856388A CN 109856388 A CN109856388 A CN 109856388A CN 201811447767 A CN201811447767 A CN 201811447767A CN 109856388 A CN109856388 A CN 109856388A
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Abstract
The invention discloses a kind of catching method of circulating tumor cell and capture kits.Wherein, which obtains tunica albuginea confluent monolayer cells the following steps are included: S1, is separated using the cell in lymphocyte separation medium human peripheral blood；S2 utilizes the circulating tumor cell in EpCAM antibody and Vimentin antibody capture tunica albuginea confluent monolayer cells.It applies the technical scheme of the present invention, by increasing interstitial phenotypic marker (Vimentin, vimentin) antibody, the capture of CTC is carried out after mixing with EpCAM antibody, conventional epitheliated type CTC can not only be captured, the bigger CTC that EMT conversion has occurred of risk can also be captured, capture rate can greatly be improved, improve the recall rate of CTC.
Description
Technical field
The present invention relates to field of biomedicine technology, a kind of catching method in particular to circulating tumor cell and
Capture kit.
Background technique
Circulating tumor cell (Circulating Tumor Cells, CTCs) is detached from and sends out from tumor focus to blood
Tumour cell in liquid is the major reason that postoperative recurrence and DISTANT METASTASES IN occurs in malignant tumor patient, and tumour is caused to be suffered from
The key factor of person's death.Many experiments have confirmed that CTCs detection facilitates the early diagnosis of tumour, judges patient's prognosis, comments
Estimate the curative effect of anti-tumor drug and formulates individualized treatment scheme.
Due to transfer be cancer related mortality the main reason for, and CTC be considered as transfer seed.It is thin based on human tumor
The conversion of the studies have shown that of born of the same parents plant and mouse model generation epithelial-mesenchymal (Epithelial-mesenchymal transition,
EMT tumour cell) plays very crucial effect in metastases.Epithelial cell-mesenchyma conversion, refers to epithelial cell
The biological process with interstitial phenotype cells is converted by specific program.By EMT, epithelial cell loses cell polar
Property, the epithelial phenotypes such as the connection with basilar memebrane are lost, higher migration and invasion, anti-apoptotic and degradation extracellular matrix are obtained
The interstitials phenotype such as ability.EMT is that the malignant cell of epithelial cell origin obtains the important biomolecule of migration and invasive ability
Process.
Immunomagnetic isolation technology (immunomagnetic separation, IMS) is the one of recent domestic research
The new immunological technique of kind, it can filter out the tumour cell with specific marker from a large amount of peripheral bloods, substantially former
Reason, which is mainly based upon cell surface antigen, to be combined with the specific monoclonal antibody for being connected with magnetic bead, and cell-antigen-antibody-is formed
Magnetic bead immune complex, which occurs mechanics movement under the action of externally-applied magnetic field, by the aim cell containing target antigen
It is separated with other cells, to achieve the purpose that specific isolation cell.It is specifically divided into positive enrichment method and negative enrichment side
Method.The former is coupled signal transduction factor (epithelial cell adhesion molecule, EpCAM) by magnetic bead
The rare cell of epithelial origin in peripheral blood is directly enriched with marks such as cytokeratins (cytokerins, CKs).The latter passes through
Magnetic bead is coupled the leucocytes mark such as CD45, removes peripheral white blood cells and the rare epithelial cell of indirect enrichment.
The technology that CTCs is enriched with or detected simultaneously currently based on immune magnetic principle mainly has CellSearch system, is immunized
Magnetic cell sorting instrument (magnetric cell sorting, MACS), AdnaTest cancer cell detection system and Lai Er system
Deng.Wherein CellSearch detection system has been approved by the fda in the United States for detection breast cancer, colorectal cancer and prostate cancer trouble
The CTCs of person's peripheral blood.The system combines the tumour cell of expression EpCAM using EpCAM antibody, is then covered with immunomagnetic beads
Lid anti-EpCAM antibody, finally under the influence of a magnetic field separates CTCs.Isolated cell is used into 4,6- after fixation
Diamidino -2-phenylindone (2- (4-amidinophenyl) -6-indole carbamidine dihydrochloride,
DAPI) fluorochrome label nucleus, CD45 fluorescence antibody and CK8, CK18, CK19 or CKmix fluorescence antibody mark cell, knot
Fruit analysis carries out under four color fluorescence microscopes.The cell of DAPI+, CD45-, CK+ and EpCAM+ are defined as CTCs.
The advantages of CellSearch system is that cellular prion protein can be saved preferably, i.e., sees fluorescent staining by fluorescence microscope
As a result, observing directly the form of tumour cell.
Because CellSearch system is approved by American National medical insurance Medicare, in the U.S., the system is in clinic
On be widely used.But CellSearch system itself also has insurmountable defect: be first detection sensitivity it is inadequate
It is high.Due to the EpCAM immunomagnetic ca pture CTC that the system uses, capture rate relies on the expression of cell surface antigen
It is more apparent.There is the power that research confirms and not all tumour cell can all express EpCAM and it is expressed in different cell surfaces
It differs greatly, therefore EpCAM is difficult to as general identification antigen.Meanwhile some CTC cause to lack since EMT process occurs
The expression of epiderm antigen and cannot be captured.Breast cancer cell converts once EMT occurs it is necessary to than the cancer cell that EMT does not occur
Mobility is stronger, is easy to happen the transfer such as brain, liver, lung, bone, and in addition EMT process also results in cancer cell multiplication stagnation, damage
Chemosensitivity, so the breast cancer cell that EMT is converted has occurred, survival rate is higher in blood, risk is bigger.But because
EMT conversion occurs for cell, and the marker of cell surface epitheliated type can lose, so the immune of EpCAM antibody can not be combined
Magnetic capture leads to the loss of important clinical information.In addition, part cancer metastasis phase patient is due to CTC content mistake in its blood
It is low, it can not be captured by CellSearch system, therefore there are the higher problems of false negative rate for the detection.Cellsearch system
The detection of system also needs specific instrument, reagent and consumptive material, and expensive, limit its further large-scale application in
It is clinical.
In addition, content of the CTC in peripheral blood is extremely low, it is considered that about 10 in peripheral blood5~107A monocyte
Middle just to have a CTC, the method for cellsearch is only carried out using EpCAM antibody primary positive rich in the enrichment process of CTC
Collection, will lead to a large amount of leucocyte in this way and is enriched with by nonspecific, bring very big do for the dyeing identification of subsequent CTC
It disturbs.
Summary of the invention
The present invention is intended to provide the catching method and capture kit of a kind of circulating tumor cell, to improve CTC capture
Sensibility.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of capture side of circulating tumor cell
Method.The catching method is separated the following steps are included: S1 using the cell in lymphocyte separation medium human peripheral blood, is obtained
Tunica albuginea confluent monolayer cells；S2 utilizes the circulating tumor cell in EpCAM antibody and Vimentin antibody capture tunica albuginea confluent monolayer cells.
Further, during S2 is specifically included, EpCAM antibody and Vimentin are added in the liquid of Xiang Hanyou tunica albuginea confluent monolayer cells
Antibody after incubation, abandons supernatant, antibody cell mixture is resuspended, Streptavidin MagneSphere or avidin magnetic bead incubation is added, with chain
The cell that mould affine magnetic bead or avidin magnetic bead combine is the circulating tumor cell finally captured.
Further, in S2: it further includes by magnetic bead that antibody cell mixture, which is resuspended, and is added after Streptavidin MagneSphere is incubated for
Antibody cell mixture is attached on magnetic frame, and after supernatant is limpid, supernatant is sucked out, and is added after cleaning solution mixes and is attached to magnetic again
On power frame, after supernatant is limpid, supernatant is sucked out, repetitive operation is multiple, obtains circulating tumor cell.
Further, between S1 and S2 further include: the leucocyte in removal tunica albuginea confluent monolayer cells obtains the first supernatant；
First supernatant is the liquid containing tunica albuginea confluent monolayer cells；Preferably, using white thin in CD45 magnetic bead removal tunica albuginea confluent monolayer cells
Born of the same parents.
Further, lymphocyte separation medium is Ficoll lymphocyte separation medium.
Further, S1 is specifically included: being diluted peripheral blood with PBS, the peripheral blood after being diluted；Ficoll leaching
Bar cell separating liquid is contained in centrifuge tube, and the peripheral blood after dilution is added to the upper layer of Ficoll lymphocyte separation medium, room
Temperature centrifugation, takes intermediate tunica albuginea layer to be washed with PBS, obtains tunica albuginea confluent monolayer cells.
Further, catching method further include: S4, the cell quantity of immunofluorescence dyeing detection capture.
Further, S4 is specifically included: S41, uses DAPI fluorescent dye after isolated circulating tumor cell is fixed
Mark nucleus；S42, with CD45 green fluorescence antibody labeled cells；S43, with CKmix, EpCAM, vimentin red fluorescence
Antibody labeled cells；S44 carries out interpretation of result under three fluorescence microscope, by DAPI+, CD45-, CKmix+ or EpCAM+
Or the cell of vimentin+ is defined as CTCs.
According to another aspect of the present invention, a kind of capture kit of circulating tumor cell is provided.The kit includes:
Lymphocyte separation medium, CD45 magnetic bead and EpCAM antibody.
Further, capture kit further includes Vimentin antibody；Preferably, capture kit further includes that strepto- is affine
Magnetic bead or avidin magnetic bead.
It applies the technical scheme of the present invention, by increasing interstitial phenotypic marker (Vimentin, vimentin) antibody, with
The capture that CTC is carried out after the mixing of EpCAM antibody, can not only capture conventional epitheliated type CTC, moreover it is possible to it is bigger to capture risk
The CTC that EMT conversion has occurred, can greatly improve capture rate, improve the recall rate of CTC.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
The technical issues of for mentioning in background technique, the invention proposes following technical proposals.
A kind of typical embodiment according to the present invention, provides a kind of catching method of circulating tumor cell.The capture side
Method is separated the following steps are included: S1 using the cell in lymphocyte separation medium human peripheral blood, and tunica albuginea confluent monolayer cells are obtained；
S2 utilizes the circulating tumor cell in EpCAM antibody and Vimentin antibody capture tunica albuginea confluent monolayer cells.Preferably, lymphocyte
Separating liquid is Ficoll lymphocyte separation medium.
It applies the technical scheme of the present invention, by increasing interstitial phenotypic marker (Vimentin, vimentin) antibody, with
The capture that CTC is carried out after the mixing of EpCAM antibody, can not only capture conventional epitheliated type CTC, moreover it is possible to it is bigger to capture risk
The CTC that EMT conversion has occurred, can greatly improve capture rate, improve the recall rate of CTC.
Preferably, EpCAM antibody is added during S2 is specifically included, in the liquid of Xiang Hanyou tunica albuginea confluent monolayer cells and Vimentin is anti-
Body after incubation, abandons supernatant, antibody cell mixture is resuspended, Streptavidin MagneSphere or avidin magnetic bead incubation is added, with strepto-
The cell that affine magnetic bead or avidin magnetic bead combine is the circulating tumor cell finally captured.
It is first in conjunction with cell incubation using the antibody with biotin labeling, it is then added with marked by streptavidin
Magnetic bead is captured, all smaller without the antibody volume and weight of magnetic bead, can be uniformly distributed in the liquid phase, sufficiently with cell phase
In conjunction with.After antibody incubation, with the antibody cell conjugate of the magnetic capture and biotin labeling marked with streptomysin,
Middle Streptavidin can be with high degree of specificity in conjunction with four molecular biosciences elements, and affinity between the two is extremely strong, so
It being capable of quick special capture CTC.
It is furthermore preferred that in S2: it further includes by magnetic bead that antibody cell mixture, which is resuspended, and is added after Streptavidin MagneSphere is incubated for
Antibody cell mixture is attached on magnetic frame, and after supernatant is limpid, supernatant is sucked out, and is added after cleaning solution mixes and is attached to magnetic again
On power frame, after supernatant is limpid, supernatant is sucked out, repetitive operation is multiple, obtains circulating tumor cell.Further improve CTC
The efficiency of capture improves the recall rate of CTC.
A kind of typical embodiment according to the present invention, between S1 and S2 further include: white in removal tunica albuginea confluent monolayer cells
Cell obtains the first supernatant；First supernatant is the liquid containing tunica albuginea confluent monolayer cells；Preferably, it is gone using CD45 magnetic bead
Except the leucocyte in tunica albuginea confluent monolayer cells, naturally it is also possible to it is a large amount of white thin to reach removal using other methods such as Leukapheresis
Born of the same parents, to separate the purpose of tumour cell.Wherein, first remove peripheral blood in a large amount of leucocytes, can further increase EpCAM and
Specificity and sensibility of the Vimentin antibody in conjunction with CTC are gone using the leucocyte in CD45 magnetic bead removal tunica albuginea confluent monolayer cells
Except rate can achieve 99.99%.
In an embodiment of the present invention, S1 is specifically included: peripheral blood is diluted with PBS, it is outer after being diluted
All blood；Ficoll lymphocyte separation medium is contained in centrifuge tube, and Ficoll lymphocyte point is added in the peripheral blood after dilution
The upper layer of chaotropic, room temperature centrifugation, takes intermediate tunica albuginea layer to be washed with PBS, obtains tunica albuginea confluent monolayer cells.Wherein, peripheral blood allusion quotation
Type is the peripheral blood of patient with breast cancer.
A kind of typical embodiment according to the present invention, catching method further include: S4, immunofluorescence dyeing detection capture
Cell quantity；Preferably, S4 is specifically included: S41, uses DAPI fluorescent dye mark after isolated circulating tumor cell is fixed
Remember nucleus；S42, with CD45 green fluorescence antibody labeled cells；S43, it is anti-with CKmix, EpCAM, vimentin red fluorescence
Body marks cell；S44 carries out interpretation of result under three fluorescence microscope, by DAPI+, CD45-, CKmix+ or EpCAM+ or
The cell of vimentin+ is defined as CTCs.
A kind of typical embodiment, catching method include: according to the present invention
1. the separation of monocyte
1) Peripheral Blood In Patients With Breast Cancer 8mL is acquired with EDTA pipe.
2) PBS is by 1 times of 8mL hemodilution.
3) 15mL Ficoll lymphocyte separation medium is added in 50mL centrifuge tube, and the blood after dilution is careful
Separating liquid upper layer is added.
4) 400g room temperature is centrifuged 30min.
5) it is layered after being centrifuged, takes intermediate tunica albuginea layer, as cellular layer, in the 15mL centrifuge tube new to one.
6) PBS is washed 2 times.
2. leucocyte removal
1) the CD45 magnetic bead of 250 μ L is added, on rotation blending instrument, 4 DEG C of incubation 30min.
2) cell magnetic bead mixed liquor is attached on magnetic frame.
3) limpid to supernatant, magnetic bead is bonded with magnet, and supernatant is sucked out, and is transferred in new pipe.
3.CTC capture
1) 1 μ gEpCAM antibody and 1 μ g Vimentin antibody are separately added into.
2) on rotation blending instrument, it is incubated at room temperature 30min.
3) 300g room temperature is centrifuged 10min, sucks supernatant.
4) antibody cell mixture is resuspended with 1mL PBS.
5) 25 μ L Streptavidin MagneSpheres are added, on rotation blending instrument, are incubated at room temperature 5min.
6) magnetic bead antibody cell mixture is attached on magnetic frame.
7) limpid to supernatant, after magnetic bead is bonded with magnet, supernatant is sucked out.
8) 800 μ L cleaning solutions are added, after mixing, are attached on magnetic frame again.
9) it is bonded to magnetic bead with magnet, supernatant is sucked out, 800 μ L cleaning solutions are added.
10) (5) (6) are repeated step 2 times.
11) cell in conjunction with magnetic bead is the CTC finally captured.
4. the cell quantity of immunofluorescence dyeing detection capture
1) isolated cell is used to DAPI fluorochrome label nucleus after fixation.
2) CD45 green fluorescence antibody labeled cells.
3) CKmix, EpCAM, vimentin red fluorescence antibody labeled cells are used.
4) interpretation of result carries out under three fluorescence microscope.By DAPI+, CD45-, CKmix+ or EpCAM+ or
The cell of vimentin+ is defined as CTCs.
In order to facilitate the implementation of above-mentioned technical proposal of the present invention, a kind of typical embodiment more of the invention provides one
The capture kit of kind circulating tumor cell.The kit includes: lymphocyte separation medium, CD45 magnetic bead and EpCAM antibody.It is excellent
Choosing, capture kit can also include Vimentin antibody, strepto- is affine magnetic bead or avidin magnetic bead etc..It is understood that
It is that required reagent and instrument may include in this kit in the catching method for implementing circulating tumor cell of the present invention
It is interior.
Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.
Embodiment 1
Independent EpCAM capture peripheral blood CTC and EpCAM+Vimentin capture peripheral blood CTC capture rate verifying.
Specific step is as follows:
1) healthy human peripheral blood 8mL is acquired with EDTA pipe, is mixed into the thin of equal number of MCF-7 cell and MDA-MB-231
Born of the same parents.
2) one times is diluted with PBS.
3) 15mL Ficoll lymphocyte separation medium is added in 50mL centrifuge tube, and the blood after dilution is careful
Separating liquid upper layer is added.
4) 400g room temperature is centrifuged 30min.
5) it is layered after being centrifuged, takes intermediate tunica albuginea layer, as cellular layer, in the 15mL centrifuge tube new to one.
6) PBS is washed 2 times.
7) the CD45 magnetic bead of 250 μ L is added, on rotation blending instrument, 4 DEG C of incubation 30min.
8) cell magnetic bead mixed liquor is attached on magnetic frame.
9) limpid to supernatant, magnetic bead is bonded with magnet, and supernatant is sucked out, and is transferred in new pipe.
10) 1 μ gEpCAM antibody is only added in one group, is separately added into 1 μ gEpCAM antibody and 1 μ g in another set
Vimentin antibody.
11) on rotation blending instrument, it is incubated at room temperature 30min.
12) 300g room temperature is centrifuged 10min, sucks supernatant.
13) antibody cell mixture is resuspended with 1mL PBS.
14) 25 μ L Streptavidin MagneSpheres are added, on rotation blending instrument, are incubated at room temperature 5min.
15) magnetic bead antibody cell mixture is attached on magnetic frame.
16) limpid to supernatant, after magnetic bead is bonded with magnet, supernatant is sucked out.
17) 800 μ L cleaning solutions are added, after mixing, are attached on magnetic frame again.
18) it is bonded to magnetic bead with magnet, supernatant is sucked out, 800 μ L cleaning solutions are added.
19) (5) (6) are repeated step 2 times.
20) cell in conjunction with magnetic bead is the CTC finally captured.
21) isolated cell is used to DAPI fluorochrome label nucleus after fixation.
22) CD45 green fluorescence antibody labeled cells.
23) CKmix, EpCAM, vimentin red fluorescence antibody labeled cells are used.
24) interpretation of result carries out under three fluorescence microscope.By DAPI+, CD45-, CKmix+ or EpCAM+ or
The cell of vimentin+ is defined as CTCs.
It has done altogether three times, every time three repetitions, the cell number result such as the following table 1 captured:
Table 1
Embodiment 2
Leucocyte is not removed using CD45 magnetic bead, directly individually EpCAM captures peripheral blood CTC and EpCAM+Vimentin
Capture the verifying of peripheral blood CTC capture rate.
Specific step is as follows:
1) healthy human peripheral blood 8mL is acquired with EDTA pipe, is mixed into the thin of equal number of MCF-7 cell and MDA-MB-231
Born of the same parents.
2) one times is diluted with PBS.
3) 15mL Ficoll lymphocyte separation medium is added in 50mL centrifuge tube, and the blood after dilution is careful
Separating liquid upper layer is added.
4) 400g room temperature is centrifuged 30min.
5) it is layered after being centrifuged, takes intermediate tunica albuginea layer, as cellular layer, in the 15mL centrifuge tube new to one.
6) PBS is washed 2 times.
7) 1 μ gEpCAM antibody is only added in one group, is separately added into 1 μ gEpCAM antibody and 1 μ g in another set
Vimentin antibody.
8) on rotation blending instrument, it is incubated at room temperature 30min.
9) 300g room temperature is centrifuged 10min, sucks supernatant.
10) antibody cell mixture is resuspended with 1mL PBS.
11) 25 μ L Streptavidin MagneSpheres are added, on rotation blending instrument, are incubated at room temperature 5min.
12) magnetic bead antibody cell mixture is attached on magnetic frame.
13) limpid to supernatant, after magnetic bead is bonded with magnet, supernatant is sucked out.
14) 800 μ L cleaning solutions are added, after mixing, are attached on magnetic frame again.
15) it is bonded to magnetic bead with magnet, supernatant is sucked out, 800 μ L cleaning solutions are added.
16) (5) (6) are repeated step 2 times.
17) cell in conjunction with magnetic bead is the CTC finally captured.
18) isolated cell is used to DAPI fluorochrome label nucleus after fixation.
19) CD45 green fluorescence antibody labeled cells.
20) CKmix, EpCAM, vimentin red fluorescence antibody labeled cells are used.
21) interpretation of result carries out under three fluorescence microscope.By DAPI+, CD45-, CKmix+ or EpCAM+ or
The cell of vimentin+ is defined as CTCs.
22) it has done three times altogether, three repetitions every time, the cell number result such as the following table 2 captured:
Table 2
Embodiment 3
Antibody first with cell combination, antibody elder generation in conjunction with avidin magnetic bead after in conjunction with cell, with common EPCAM antibody
Coated magnetic capture peripheral blood CTC capture rate verifying.
1) healthy human peripheral blood 8mL is acquired with EDTA pipe, is mixed into the thin of equal number of MCF-7 cell and MDA-MB-231
Born of the same parents.
2) one times is diluted with PBS.
3) 15mL Ficoll lymphocyte separation medium is added in 50mL centrifuge tube, and the blood after dilution is careful
Separating liquid upper layer is added.
4) 400g room temperature is centrifuged 30min.
5) it is layered after being centrifuged, takes intermediate tunica albuginea layer, as cellular layer, in the 15mL centrifuge tube new to one.
6) PBS is washed 2 times.
7) the CD45 magnetic bead of 250 μ L is added, on rotation blending instrument, 4 DEG C of incubation 30min.
8) cell magnetic bead mixed liquor is attached on magnetic frame.
9) limpid to supernatant, magnetic bead is bonded with magnet, and supernatant is sucked out, and is transferred in new pipe.
10) 1 μ g EpCAM antibody is only added in one group.
11) on rotation blending instrument, it is incubated at room temperature 30min.
12) 300g room temperature is centrifuged 10min, sucks supernatant.
13) antibody cell mixture is resuspended with 1mL PBS.
14) 25 μ L Streptavidin MagneSpheres are added, on rotation blending instrument, are incubated at room temperature 5min.
15) 1 μ g EpCAM antibody is first combined with 25 μ L Streptavidin MagneSpheres in another set, is incubated at room temperature 20min, then
Cell suspension is added, on rotation blending instrument, is incubated at room temperature 30min.
16) the coated magnetic bead 20ul of common EPCAM antibody is directly added into third group, on rotation blending instrument, room temperature is incubated
Educate 30min.
17) three groups of magnetic bead antibody cell mixtures are attached on magnetic frame.
18) limpid to supernatant, after magnetic bead is bonded with magnet, supernatant is sucked out.
19) 800 μ L cleaning solutions are added, after mixing, are attached on magnetic frame again.
20) it is bonded to magnetic bead with magnet, supernatant is sucked out, 800 μ L cleaning solutions are added.
21) (5) (6) are repeated step 2 times.
22) cell in conjunction with magnetic bead is the CTC finally captured.
23) isolated cell is used to DAPI fluorochrome label nucleus after fixation.
24) CD45 green fluorescence antibody labeled cells.
25) CKmix, EpCAM, vimentin red fluorescence antibody labeled cells are used.
26) interpretation of result carries out under three fluorescence microscope.By DAPI+, CD45-, CKmix+ or EpCAM+ or
The cell of vimentin+ is defined as CTCs.
It has done altogether three times, every time three repetitions, the cell number result such as the following table 3 captured:
Table 3
It can be seen from the above description that the above embodiments of the present invention realized the following chievements:
1) most of leucocyte is removed by CD45, CTC capture can be improved in the case where reducing antibody dosage in this way
Sensibility.
2) other than common EpCAM antibody combination CTC, Vimentin antibody is increased, EMT conversion occurs for capturing
Loss epithelium characteristic CTC, can be improved capture sensibility and increase the detection of CTC stronger to harmfulness.
3) antibody elder generation and cell combination can be contacted more fully with cell, increase joint efficiency.
4) it introduces Streptavidin MagneSphere and biotin labelled antibodies, the binding constant of the two is at least common primary antibody secondary antibody
100 times of binding constant, more stable in conjunction with magnetic bead using this set system acquisition CTC, CTC, speed is faster.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of catching method of circulating tumor cell, which comprises the following steps:
S1 is separated using the cell in lymphocyte separation medium human peripheral blood, obtains tunica albuginea confluent monolayer cells；
S2 utilizes the circulating tumor cell in tunica albuginea confluent monolayer cells described in EpCAM antibody and Vimentin antibody capture.
2. catching method according to claim 1, which is characterized in that during the S2 is specifically included, to contain the tunica albuginea
EpCAM antibody and Vimentin antibody are added in the liquid of confluent monolayer cells, after incubation, abandons supernatant, antibody cell mixture is resuspended and adds
Enter Streptavidin MagneSphere or avidin magnetic bead is incubated for, the cell in conjunction with the affine magnetic bead of strepto- or avidin magnetic bead is finally to catch
The circulating tumor cell received.
3. catching method according to claim 2, which is characterized in that in the S2: antibody cell mixture is resuspended and is added
Streptavidin MagneSphere further includes that magnetic bead antibody cell mixture is attached on magnetic frame after being incubated for, will be upper after supernatant is limpid
It is clear to be sucked out, it is added after cleaning solution mixes and is attached on magnetic frame again, after supernatant is limpid, supernatant is sucked out, repetitive operation is multiple,
Obtain the circulating tumor cell.
4. catching method according to claim 2, which is characterized in that between the S1 and S2 further include: described in removal
Leucocyte in tunica albuginea confluent monolayer cells obtains the first supernatant；First supernatant is the liquid for containing the tunica albuginea confluent monolayer cells
Body；
Preferably, the leucocyte in the tunica albuginea confluent monolayer cells is removed using CD45 magnetic bead.
5. catching method according to claim 1, which is characterized in that the lymphocyte separation medium is that Ficoll lymph is thin
Born of the same parents' separating liquid.
6. catching method according to claim 5, which is characterized in that the S1 is specifically included:
The peripheral blood is diluted with PBS, the peripheral blood after being diluted；
The Ficoll lymphocyte separation medium is contained in centrifuge tube, and the Ficoll is added in the peripheral blood after the dilution
The upper layer of lymphocyte separation medium, room temperature centrifugation, takes intermediate tunica albuginea layer to be washed with PBS, obtains the tunica albuginea confluent monolayer cells.
7. catching method according to claim 1, which is characterized in that the catching method further include: S4, immunofluorescence dye
The cell quantity of color detection capture.
8. catching method according to claim 7, which is characterized in that the S4 is specifically included:
S41 uses DAPI fluorochrome label nucleus after fixing the isolated circulating tumor cell；
S42, with CD45 green fluorescence antibody labeled cells；
S43, with CKmix, EpCAM, vimentin red fluorescence antibody labeled cells；
S44 carries out interpretation of result under three fluorescence microscope, by DAPI+, CD45-, CKmix+ or EpCAM+ or vimentin
+ cell be defined as CTCs.
9. a kind of capture kit of circulating tumor cell characterized by comprising lymphocyte separation medium, CD45 magnetic bead and
EpCAM antibody.
10. capture kit according to claim 9, which is characterized in that the capture kit further includes Vimentin
Antibody；
Preferably, the capture kit further includes the affine magnetic bead of strepto- or avidin magnetic bead.
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| CN110251164A (en) * | 2019-06-14 | 2019-09-20 | 上海交通大学医学院附属仁济医院 | A kind of dyeing interpretation method of cell capture device, cystoscope and sample cell |
| CN111812324A (en) * | 2020-06-22 | 2020-10-23 | 东南大学 | Method for detecting lung cancer circulating tumor cells |
| CN113789303A (en) * | 2021-09-24 | 2021-12-14 | 安徽贝铭生物科技有限公司 | Method for improving blood circulation tumor cell capture |
| CN114729055A (en) * | 2022-02-24 | 2022-07-08 | 青岛华赛伯曼医学细胞生物有限公司 | Methods for detecting and isolating cell populations co-expressing CD45 and EpCAM and uses thereof |
| CN115896026A (en) * | 2022-11-29 | 2023-04-04 | 四川大学华西医院 | Separation and purification method of circulating tumor cells |
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