CN107441954A - It is a kind of to be used to separate composite membrane of purpose leucocyte and preparation method thereof in liquid - Google Patents
It is a kind of to be used to separate composite membrane of purpose leucocyte and preparation method thereof in liquid Download PDFInfo
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- CN107441954A CN107441954A CN201710698068.5A CN201710698068A CN107441954A CN 107441954 A CN107441954 A CN 107441954A CN 201710698068 A CN201710698068 A CN 201710698068A CN 107441954 A CN107441954 A CN 107441954A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/12—Composite membranes; Ultra-thin membranes
- B01D69/125—In situ manufacturing by polymerisation, polycondensation, cross-linking or chemical reaction
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0002—Organic membrane manufacture
- B01D67/0023—Organic membrane manufacture by inducing porosity into non porous precursor membranes
- B01D67/0032—Organic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods
- B01D67/0034—Organic membrane manufacture by inducing porosity into non porous precursor membranes by elimination of segments of the precursor, e.g. nucleation-track membranes, lithography or laser methods by micromachining techniques, e.g. using masking and etching steps, photolithography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/50—Polycarbonates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2325/00—Details relating to properties of membranes
- B01D2325/02—Details relating to pores or porosity of the membranes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention provides a kind of composite membrane for being used to separate purpose leucocyte in liquid, the composite membrane include trace-etching-film and be connected in a manner of direct or indirect on the film surface of trace-etching-film and/or in duct for one or more antibody molecules for being specifically bound with purpose leucocyte, the connection of the trace-etching-film and antibody includes being connected chemically more than at one, the track etching membrane aperture is 5~40 microns, the duct of size uniform is mainly used in filtering the component containing non-purpose leucocyte on trace-etching-film, the antibody molecule connected on the trace-etching-film is used to purpose leukocytic immunity being trapped in trace-etching-film.The manageable sample size of method of the compound UF membrane purpose leucocyte of the present invention is big, it is often more important that the efficiency of this method separation leucocyte increases substantially.
Description
Technical field
The present invention relates to filter membrane field, and in particular to it is a kind of be used to separating in liquid the composite membrane of purpose leucocyte and its
Preparation method.
Background technology
Leucocyte is generally divided into the variety classeses such as lymphocyte, granulocyte and monocyte.It is existing to be separated from blood
Leucocyte or the method for separating certain specific leucocyte are usually using paramagnetic particle method sorting or selected by flow cytometry apoptosis.
Specifically, the antibody connected on magnetic bead is combined with purpose leucocyte, then enrichment is connected to magnetic bead under magnetic fields
On leucocyte.But paramagnetic particle method separation leucocyte at least has the disadvantage that：1st, need first to use erythrocyte cracked liquid by blood
In erythrocyte splitting, 2, the volume of the blood sample of paramagnetic particle method processing it is very limited, it is impossible in large quantities, on a large scale separation it is white
Cell, 3, paramagnetic particle method separation need to use magnetic frame that magnetic field can be provided.
And selected by flow cytometry apoptosis method is to mark cell by fluorescence antibody, single cell suspension is put into sample cell, carefully
Born of the same parents are by high pressure press-in flow chamber.Is full of sheath fluid in flow chamber, under the parcel of sheath fluid and promotion, cell be ordered in it is single-row, with
Certain speed sprays from flow chamber spout.Piezo-electric crystal equipped with a hyperfrequency on the spout of flow chamber, vibrates after charging,
The liquid stream of ejection is set to be fractured into uniform drop, cell to be measured is disperse among these drops.Liquid is irradiated with high energy laser
Drop, the cell with fluorescence can be detected by detector, and these drops are filled with positive and negative different electric charge, when drop flows through
When crossing with several kilovolts of deflecting plates, deflected in the presence of high voltage electric field, fall into respective collection vessel, do not charge
Drop falls into the waste fluid container of centre, so as to realize the separation of cell.Selected by flow cytometry apoptosis method has the disadvantage that：1st, sample
Need handle early stage, including splitting erythrocyte, fixation, mark fluorescent antibody etc., 2, due to being needed to each cell in sample
Analyzed and processed, therefore the volume of the blood sample handled is very limited, it is impossible to separate in large quantities, on a large scale white thin
Born of the same parents, 3, the flow cytometer device with sorting function it is sufficiently complex and expensive.
Therefore, this area needs to develop a kind of new leucocyte separation method or separator.
The content of the invention
Trace-etching-film is a kind of polymeric membrane for the straight hole structure that pore distribution is uniform, aperture is consistent.Because most
A diameter of 8-11 μm of peripheral blood cells, and a diameter of 29.8-33.9 μm of circulating tumor cell (CTC).According to this characteristic,
In the prior art typically using the polycarbonate track etching-film human peripheral blood sample of certain pore size (such as aperture is 8-10 μm)
Filtered, the larger tumour cell of volume will be stayed on film, and less normal blood cell will by filter membrane, so as to
Tumour cell is enriched with polycarbonate membrane, i.e., membrane filtration crosses separation tumour cell technology (Isolation by size
of epithelial tumor cells,ISET).That is, trace-etching-film of the prior art is generally used for filtering point
From tumour cell.Inventor is had found by retrieving, in the prior art not by report of the trace-etching-film for separating leucocyte
Lead.Therefore, specific antibody is connected and forms a kind of composite membrane and be used to separate by the present inventor with trace-etching-film
Purpose leucocyte, achieve good effect.Magnetic bead in the prior art is substituted for using the compound UF membrane leucocyte
Method separates leucocyte.The red blood cell that can no longer need to use erythrocyte cracked liquid first to destroy in blood, and compound UF membrane mesh
Leucocyte the manageable sample size of method it is big, it is prior, this method separation leucocyte efficiency increase substantially.
Therefore, the present invention provides a kind of composite membrane for being used to separate purpose leucocyte in liquid, and the composite membrane includes footpath
Mark etching-film and be connected in a manner of direct or indirect on the film surface of trace-etching-film and/or in duct be used for purpose it is white
One or more antibody molecules that cell is specifically bound, the connection of the trace-etching-film and antibody include sentencing
On be connected chemically, the track etching membrane aperture be 5~40 microns, preferably 5~15 microns, size uniform on trace-etching-film
Duct be mainly used in filtering the component containing non-purpose leucocyte, the antibody molecule connected on the trace-etching-film is used for mesh
Leukocytic immunity be trapped on trace-etching-film.
Specifically, heretofore described antibody is fixedly connected on the track by biology and/or attachment chemistry and lost
On engraved film, rather than only temporarily adsorbed by physisorption on film.
In the composite membrane of the present invention, purpose leucocyte to be separated is possible to pass through the film from hole, it is also possible to by hole
Antibody retention and block the hole, certainly more purpose leucocytes are retained and stayed on film surface by the antibody on film surface.
In the present invention, trace-etching-film can be specifically aperture for 7 microns, 8 microns, 9 microns, 10 microns, 11 microns, 12
Micron and a variety of different models such as 15 microns.
In the present invention, the antibody of different specific bindings can be accordingly selected for different purpose leucocytes, can be with
It is that can specifically bind the antibody of all leucocytes or specifically bind certain leucocyte (such as T lymphocytes)
Antibody.Therefore, the purpose leukocyte antibody connected on trace-etching-film of the present invention can be one or more.
In a kind of specific embodiment, the diameter in the duct of size uniform is the μ of x ± 1 on the trace-etching-film
M, preferably x ± 0.5 μm, and x is any value in 6~14 μm.
In the present invention, the diameter in the duct of size uniform is that x ± 1 μm refers to channel diameter on the trace-etching-film
Design size, in fact because of reasons such as processing, such as a small amount of twin opening (subway) or three linkage holes can be formed and cause certain duct on film
Size substantially becomes big, and such case is allowed to and belongs to protection category of the present invention.
In a kind of specific embodiment, the antibody is lost by the connection of biotin and Avidin to be fixed on track
On engraved film.
In a kind of specific embodiment, the antibody passes sequentially through biotin, Avidin, coupling agent GMBS (N-y-
Maleimidobutyryloxysuccinimide ester, HOSu NHS acid) and silane coupler be connected to footpath
On mark etching-film.
In a kind of specific embodiment, the antibody includes the one or more in CD45, CD4, CD5 and CD19.
Those skilled in the art know, sort field in leucocyte, antibody type is very more.Such as CD45 of the present invention
Antibody is that all leucocytes are positive, and CD4 antibody can specifically cause certain class T lymphocytes (T-helper is thin
Born of the same parents) retention, CD5 antibody can be specifically so that all T lymphocytes retentions, CD19 antibody can specifically cause B
Lymphocyte retains.Certainly, it is of the present invention to be not limited in above-mentioned number with the antibody that purpose leucocyte is specifically bound
Kind.
In a kind of specific embodiment, the fenestra gross area on the film surface of the trace-etching-film is the film surface gross area
Less than 40%, preferably 1~30%, more preferably 5~20%.
In a kind of specific embodiment, the trace-etching-film is to bombard macromolecule membrane material using heavy ion avcceleration
By including the film in multiple uniform pore diameter straight hole roads obtained from chemical etching after material, preferably described trace-etching-film is polyester film
Or polycarbonate membrane.
The present invention also provides a kind of preparation method of the composite membrane, and methods described comprises the following steps：Step A, it is first right
Trace-etching-film is swung using silane coupler immersion washes the silane pretreatment for carrying out film surface；Step B, coupling agent is used to film
GMBS processing, makes GMBS be attached on film；Step C, film is handled using Avidin so that Avidin is attached on GMBS；Step
Rapid D, film is handled using biotinylated antibody so that biotinylated antibody is combined with Avidin；And in step A~D
Include cleaning after each step and remove uncombined or unreacted corresponding chemical material.
In a kind of specific embodiment, the silane coupler is 3- mercaptopropyl trimethoxysilanes.
The thickness of the trace-etching-film used in the present invention is 12~30 μm, and thickness is suitable with the size of channel diameter or omits
Greatly.The antibody used in the present invention is a kind of protein, and its size is generally below tens nanometer, and Avidin and biotin
Size it is smaller than antibody, thus the antibody in the present invention can be connected in the duct of trace-etching-film completely.
The use of GFP is that green fluorescent protein is in order that it is active somatic cell to obtain in aim cell to be separated in the present invention
In the case of Tracing detection.
The present invention at least has the advantages that：
1st, the antibody for separating purpose leucocyte is chemically crosslinked on trace-etching-film by the present invention first, provided by the invention
Composite membrane ideally combines the advantage in the immune separation of antigen-antibody and trace-etching-film uniformity straight hole road, composite membrane knot
Structure is stable, and separating effect stability and high efficiency is reliable.
2nd, sample to be separated need not be pre-processed, such as cracked red using during the compound UF membrane purpose leucocyte
Cell, fixation, mark fluorescent antibody etc., separation method is simpler.And due to need not be pre-processed to blood sample, can be with
Directly it is separated by filtration, therefore, the amount of present invention processing sample can greatly increase.This is for the rare special defects of concentration and separation
Type leucocyte has great advantage.
3rd, the manageable sample size of method of the compound UF membrane purpose leucocyte of the present invention is big, prior, this method
The efficiency of separation leucocyte increases substantially.Specifically, in the present invention, because of basic on the trace-etching-film duct of size uniform
Allow the compositions such as leucocyte (including purpose leucocyte and non-purpose leucocyte) and red blood cell in unicellular by the way that this is white to purpose
The combination of cell and antibody provides time and the space of abundance, thus the composite membrane can be comprehensively without dead angle thin in vain to purpose
Born of the same parents carry out immune sorting, are difficult mistakes and omissions purpose leucocyte in immune sorting, rather than the cell such as purpose leucocyte and red blood cell is several
All pass through from fenestra；Because blood middle leukocytes are with respect to rare numbers for red blood cell, the composite membrane can be by red blood cell and non-
Purpose leucocyte almost successfully filters completely, and the non-duct region area ratio on film surface is big, there is enough positions on film surface
Put and combined for purpose leucocyte, (i.e. enrichment combines this mode for being combined enrichment combination and separation and separation is that a step is complete
Into) operating procedure is simplified, further increase the efficiency of immune sorting purpose leucocyte.
4th, magnetic frame or flow cytometer are no longer needed to use in the present invention, further simplifies separator.
Brief description of the drawings
Fig. 1 is that composite filtering film is detected under the white field light of microscope to leucocyte separating effect comparison diagram, and wherein Figure 1A is to make
With the track etching filter membrane of uncrosslinked antibody, Figure 1B be crosslinked CD45 antibody filtering membrane filtration equivalent people blood extraction it is white
Cell, PBS is washed after fixing, and observes cell distribution situation on two filter membranes.Sample behaviour whole blood is separated through erythrocyte splitting
The leukocyte suspension extracted afterwards.
Fig. 2 detects composite filtering film to leucocyte separating effect figure for inverted fluorescence microscope after Acridine orange.Wherein A
It is the trace-etching-film of uncrosslinked antibody with B, C and D are the track etching filter membranes for being crosslinked with CD45 antibody.Sample is separated to behave
The leukocyte suspension that whole blood extracts after erythrocyte splitting.
Fig. 3 is that fluorescent staining and normal dyeing microscope detect combination design sketch of the composite filtering film to leucocyte.Wherein
A, B and C is the trace-etching-film of uncrosslinked antibody, and D, E and F are the track etching filter membranes for being crosslinked with CD45 antibody.Specifically,
Fig. 3 A be normal filtration film Acridine orange after, the cell distribution situation under a length of 488nm of excitation light wave fluorescence channel, Fig. 3 B
For normal filtration film situation under white field light；Fig. 3 C are normal filtration film situation under white field light after haematoxylin dyeing；Fig. 3 D are friendship
After the filter membrane Acridine orange for having joined CD45 antibody, the cell distribution situation under a length of 488nm of excitation light wave fluorescence channel,
Fig. 3 E are the filter membrane situation that CD45 antibody has been crosslinked under white field light；Fig. 3 F are the haematoxylin dyeing post-crosslinking mistake of CD45 antibody
Filter membrane cell situation under white field light.Sample behaviour whole blood is separated, it is not preprocessed directly to filter.
Embodiment
The present invention is illustrated by following examples, but protection scope of the present invention is not limited in following embodiments.
Embodiment 1
Contrast is not connected to the trace-etching-film of antibody and connects the footpath of purposeful leucocyte sorting antibody in the present embodiment
The separating resulting of mark etching-film.The antibody used in the present embodiment is CD45, and used trace-etching-film is polycarbonate membrane
(8 microns of aperture), i.e. PC filtration membranes.Blood used pre-processes by splitting erythrocyte.
First, experimentation：
1. the antibody linked PC filtrations film preparations of biotinylation CD45
(1) the PC filter membranes that hole density is evenly distributed are taken, are completely soaked respectively to containing 3ml 4% (v/v) silane-alcohol
In the small glass container of solution, closing, it is put on shaking table and shakes, room temperature 45 minutes, silane is fully pre-processed film surface；
(2) film is gone in the small culture dish of the absolute ethyl alcohol containing 2ml, reclaims 4% (v/v) silane-alcoholic solution, swing washout
Unreacted silane is removed, is washed 3 times, each 7min；
(3) after the completion of washing, alcohol is exhausted, is added toward container in 1ml 1mM GMBS solution, swings washed filter membrane reaction 15
Minute, GMBS is attached on film；
(4) 1mM GMBS solution is reclaimed, 2ml absolute ethyl alcohols is separately added into and swings and wash filtration membrane, washout is gone unreacted
GMBS, wash 3 times, each 7min；
(5) after the completion of washing, add in 1ml 10ug/ml neutrality avidin solution to container, be completely soaked filter membrane,
It is placed in 4 DEG C of refrigerators overnight, Avidin is attached to GMBS；
(6) after reclaiming 10ug/ml neutrality avidin solutions, add PBS and swing washed filter membrane 3 times, each 7min, remove not anti-
The Avidin answered；
(7) after the completion of washing, add in 1ml 10ug/ml biotinylation CD45 antibody liquids to container, be completely soaked filtering
Film, swing wash reaction 30 minutes at room temperature, biotinylated antibody is combined with Avidin；
(8) after reclaiming CD45 antibody liquids, PBS swings washed filter membrane 3 times, each 7min, removes uncombined antibody；
(9) PBS solution is suctioned out, antibody membrane is in the moist environment containing a small amount of PBS solution；Seal the small culture of membrane closure
Ware, the interior storage of 4 DEG C of refrigerators (normal temperature maintains up to 3 weeks).
2. extract human peripheral leucocytes
(1) 15ml centrifuge tubes are taken, add about 13ml 1 × erythrocyte cracked liquid (being free of tris)；
(2) 2ml people's anticoagulated whole blood is added in above-mentioned pipe, gently overturns and mix, cracking 15 minutes is limpid to solution；
(3) 2000 leave the heart 5 minutes, topple over and remove upper strata red blood cell liquid, stay sedimentation cell (leucocyte)；
(4) plus 5ml erythrocyte cracked liquids blow even resuspension sedimentation cell, mix；
(5) 2000 leave the heart 5 minutes, abandon supernatant, obtain sedimentation cell (leucocyte)；
(6) wash：Cell is resuspended in 5ml PBS solutions, and 2000 leave the heart 5 minutes, abandon supernatant, obtain sedimentation cell；
(7) cell is resuspended in 2ml PBS, dispenses and is transferred in 1.5ml EP centrifuge tubes after mixing, often pipe 1ml.This is to carry
The human blood leukocyte taken.
3. variable is set：
Experimental group：The PC filtration membranes of crosslinked bio elementization CD45 antibody；
Control group：The PC filtration membranes of uncrosslinked antibody.
4. membrane filtration is tested
(1) respectively using the filter membrane of uncrosslinked antibody, be crosslinked CD45 antibody filter membrane assemble film filter, row
Triple valve is closed after gas, stays 2ml standby after adding the filtering of 15ml brines.
(2) the 1ml leucocyte solution extracted in above-mentioned people's blood is taken respectively, after fully mixing, is added with pipette tips to Filter column
It is interior, mouth triple valve under Filter column is opened after mixing, solution is slowly flowed out from lower mouth, treats that leucocyte solution enters Filter column substantially
When middle, triple valve is closed.
(3) Filter column is positioned on 50ml centrifuge tubes respectively, it is small that standing 1 in 37 DEG C of insulating boxs is put it into after putting surely
When, allow leucocyte in Filter column to be settled down on filter membrane, it is fully contacted.
(4) Filter column is taken out after 1h and is placed on iron stand fixed, addition 15ml physiological saline, opening the triple valve of lower mouth makes
Liquid flows out, and physiological saline 15ml is added when liquid has flowed soon, abundant washing and filtering device simultaneously removes unlocked on filter membrane
Cell；It is repeated 4 times.
(5) filtering of 1-2ml4% paraformaldehydes will gradually be added when will filter to fix, and be added about 10ml paraformaldehydes altogether and is allowed to
Fully fixed 15min.
(6) after the fully fixed 15min of room temperature, add 15ml physiological saline and clean 2 times.
(7) filtration membrane is taken out respectively, is put in six orifice plates, PBST washes 5min × 3 time on shaking table.
(8) cell distribution situation on film is observed under inverted microscope.
5. Acridine orange is tested
(1) after the completion of washing, filter membrane detergent PBST liquid is sucked, 1.8ml PBS liquid is separately added into and fully soaks filtering
Film.
(2) respectively toward 0.1% acridine orange solution 200ul is added in two films, it is 0.01% to make Acridine orange concentration；Concussion
It is placed in after mixing on shaking table and dyes 5min.
(3) acridine orange dye liquor is sucked, 3ml PBST liquid is added and shakes washed filter membrane, each 5min, until solution is clarified
Without dye liquor.
(4) cell distribution situation, Fluirescence observation cell fluorescence situation etc. can be observed under inverted microscope after the completion of washing.
(5) 40% glycerine mountant mountings, observation experiment result and photograph to record under fluorescence microscope.
2nd, experimental result：
1. filter bores distribution situation is basically identical on two films before filtering.
2. cell distribution situation is observed under the white field light of microscope：
Fig. 1 is that membrane filtration crosses cell distribution comparison diagram after leucocyte is fixed, and wherein Figure 1A is the track using uncrosslinked antibody
Filter membrane is etched, Figure 1B is the leucocyte for the filtering membrane filtration equivalent people blood extraction for being crosslinked CD45 antibody, and PBS is washed after fixing
Wash, observe cell distribution situation on two filter membranes.Cell on the filter membrane of CD45 antibody has been crosslinked by Figure 1A and Figure 1B are visible
Quantity is obvious more compared with normal trace-etching-film.
3. inverted fluorescence microscope observation cell distribution situation after Acridine orange washing：
Fig. 2 is inverted microscope observation cell distribution situation after Acridine orange washing.Wherein A and B is uncrosslinked antibody
Trace-etching-film, C and D are the track etching filter membranes for being crosslinked with CD45 antibody.
Respectively using 0.01% acridine orange solution at room temperature the normal filtration film to uncrosslinked antibody, be crosslinked CD45 antibody
Filter membrane dyeing 5min, PBS washing after cell distribution situation on two filter membranes is observed under inverted microscope.It is normal to scheme A
Filter membrane situation under a length of 488nm of excitation light wave fluorescence channel, figure B are normal filtration film situation under white field light；C is schemed to hand over
The filter membrane of the CD45 antibody situation under green fluorescence channel is joined, figure D is the filter membrane that CD45 antibody has been crosslinked under white field light
Situation.As it is clear from fig. 2 that fluorescence channel of the normal filtration film in a length of 488nm of excitation light wave only has weaker green fluorescence, with reference to
Caused by finding considers the predominantly acridine orange fluorescein stain of filter bores residual under the light of white field, quantity of leucocyte is few；It is crosslinked
Cell quantity is obvious more compared with normal film on the filter membrane of CD45 antibody.
3rd, experiment conclusion
The track etching filter membrane for being crosslinked CD45 antibody can specific adsorption leucocyte, hence it is evident that enhancing track etching filtering
Interception capacity of the film to leucocyte.
Embodiment 2
Contrast is not connected to the trace-etching-film of antibody and connects the footpath of purposeful leucocyte sorting antibody in the present embodiment
The separating resulting that mark etching-film directly filters to people's whole blood.The antibody used in the present embodiment is CD45, used track erosion
Engraved film is polycarbonate membrane (8 microns of aperture), i.e. PC filtration membranes.The not advance splitting erythrocyte processing of blood, is directly filtered.
First, experimentation：
1. the antibody linked PC filtrations film preparations of biotinylation CD45
The step is identical with the 1st step in embodiment 1.
2. variable is set：
Experimental group：The PC filtration membranes of crosslinked bio elementization CD45 antibody；
Control group：The PC filtration membranes of uncrosslinked antibody.
3. membrane filtration is tested
(1) respectively using the filter membrane of uncrosslinked antibody, be crosslinked CD45 antibody filter membrane assemble film filter, row
Triple valve is closed after gas, stays 2ml standby after adding the filtering of 15ml brines.
(2) 2 milliliters of people's anticoagulated whole blood is taken respectively, is added with pipette tips to Filter column, and mouth three under Filter column is opened after mixing
Port valve, solution is slowly flowed out from lower mouth, when blood enters in Filter column substantially, close triple valve.
(3) Filter column is positioned on 50ml centrifuge tubes respectively, it is small that standing 1 in 37 DEG C of insulating boxs is put it into after putting surely
When, allow leucocyte in Filter column to be settled down on filter membrane, it is fully contacted.
(4) Filter column is taken out after 1h and is placed on iron stand fixed, addition 15ml physiological saline, opening the triple valve of lower mouth makes
Liquid flows out, and physiological saline 15ml is added when liquid has flowed soon, abundant washing and filtering device simultaneously removes unlocked on filter membrane
Cell；It is repeated 4 times.
(5) filtering of 1-2ml4% paraformaldehydes will gradually be added when will filter to fix, and be added about 10ml paraformaldehydes altogether and is allowed to
Fully fixed 15min.
(6) after the fully fixed 15min of room temperature, add 15ml physiological saline and clean 2 times.
(7) filtration membrane is taken out respectively, is put in six orifice plates, PBST washes 5min × 3 time on shaking table.
(8) cell distribution situation on film can be observed under inverted microscope.
4. Acridine orange is tested
The step is identical with the 5th step in embodiment 1.
5. haematoxylin dyeing
(1) after the completion of washing, filter membrane detergent PBST liquid is sucked, 1.8ml PBS liquid is separately added into and fully soaks filtering
Film.
(2) 1min is dyed toward addition haematoxylin dyeing liquid 1ml in two films respectively.
(3) suck haematoxylin dyeing liquid, add 3ml PBS liquid and shake washed filter membrane, each 5min, totally 3 times.
(4) 40% glycerine mountant mountings, observation experiment result and photograph to record under microscope.
2nd, experimental result：
1. filter bores distribution situation is basically identical on two films before filtering.
Cell distribution situation after 2. filtering is fixed：
Fig. 3 is knot of the trace-etching-film to leucocyte that fluorescent staining and normal dyeing microscope inspection test cross join CD45 antibody
Close design sketch.Wherein A, B and C are the trace-etching-films of uncrosslinked antibody, and D, E and F are the track etchings for being crosslinked with CD45 antibody
Filter membrane.Specifically, it is thin under a length of 488nm of excitation light wave fluorescence channel after Fig. 3 A are normal filtration film Acridine orange
Born of the same parents' distribution situation, Fig. 3 B are normal filtration film situation under white field light；Fig. 3 C are normal filtration film after haematoxylin dyeing in white field light
Lower situation；Fig. 3 D are after being crosslinked the filter membrane Acridine orange of CD45 antibody, in a length of 488nm of excitation light wave fluorescence channel
Lower cell distribution situation, Fig. 3 E are the filter membrane situation that CD45 antibody has been crosslinked under white field light；Fig. 3 F be haematoxylin dyeing after hand over
The filter membrane of the CD45 antibody cell distribution situation under white field light is joined.Wherein white arrow is designated as leucocyte, dark arrow
It is designated as a diameter of 8 microns on film of filter bores.
Normal filtration film only has weaker green fluorescence under a length of 488nm of excitation light wave fluorescence channel as seen from Figure 3,
Caused by the acridine orange fluorescein stain that predominantly filter bores remain is considered with reference to finding under white field light, quantity of leucocyte is few；Crosslinking
Cell quantity is obvious compared with normal film more on the filter membrane of CD45 antibody.
3rd, experiment conclusion
The track etching filter membrane for being crosslinked CD45 antibody can specific adsorption leucocyte, hence it is evident that enhancing track etching filtering
Interception capacity of the film to leucocyte.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
Claims (9)
- Be used to separating the composite membrane of purpose leucocyte in liquid 1. a kind of, the composite membrane include trace-etching-film and with directly or Indirect mode, which is connected on the film surface of trace-etching-film and/or in duct, to be used to be specifically bound with purpose leucocyte One or more antibody molecules, the connection of the trace-etching-film and antibody includes being connected chemically more than at one, described Track etching membrane aperture is 5~40 microns, and preferably 5~15 microns, the duct of size uniform is mainly used in filtering on trace-etching-film The component containing non-purpose leucocyte is crossed, the antibody molecule connected on the trace-etching-film is used to retain purpose leukocytic immunity On trace-etching-film.
- 2. composite membrane according to claim 1, it is characterised in that the duct of size uniform is straight on the trace-etching-film Footpath is x ± 1 μm, preferably x ± 0.5 μm, and x is any value in 6~14 μm.
- 3. composite membrane according to claim 1, it is characterised in that the antibody by the connection of biotin and Avidin and It is fixed on trace-etching-film.
- 4. composite membrane according to claim 3, it is characterised in that the antibody passes sequentially through biotin, Avidin, coupling Agent GMBS and silane coupler are connected on trace-etching-film.
- 5. composite membrane according to claim 1, it is characterised in that the antibody is included in CD45, CD4, CD5 and CD19 It is one or more.
- 6. composite membrane according to claim 1, it is characterised in that the fenestra gross area on the film surface of the trace-etching-film For less than the 40% of the film surface gross area, preferably 1~30%, more preferably 5~20%.
- 7. composite membrane according to claim 1, it is characterised in that the trace-etching-film is to be banged using heavy ion avcceleration Hit by including the film in multiple uniform pore diameter straight hole roads obtained from chemical etching after macromolecule member material, preferably described track erosion Engraved film is polyester film or polycarbonate membrane.
- A kind of 8. preparation method of the composite membrane as described in any one in claim 1~7, it is characterised in that methods described bag Include following steps：Step A, first trace-etching-film is swung using silane coupler immersion and washes the silane pretreatment for carrying out film surface；Step B, coupling agent GMBS processing is used to film, GMBS is attached on film；Step C, film is handled using Avidin so that Avidin is attached on GMBS；Step D, film is handled using biotinylated antibody so that biotinylated antibody is combined with Avidin；And step A~ Include cleaning after each step in D and remove uncombined or unreacted corresponding chemical material.
- 9. preparation method according to claim 8, it is characterised in that the silane coupler is 3- mercapto propyl trimethoxies Silane.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201819826U (en) * | 2010-09-15 | 2011-05-04 | 肖乐义 | Cell separation and identification device |
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN104694381A (en) * | 2015-04-01 | 2015-06-10 | 刘韬 | Filter and integration device for separating tumor cells from blood fluid |
| CN105169963A (en) * | 2015-09-15 | 2015-12-23 | 中国原子能科学研究院 | Making method of nuclear track etching membrane |
| CN105233700A (en) * | 2015-09-15 | 2016-01-13 | 中国原子能科学研究院 | Manufacturing method for nuclear track etched membrane with single taper micropore |
| CN105483082A (en) * | 2015-12-24 | 2016-04-13 | 苏州市中心血站 | Method for obtaining leukocytes by adopting LRS and application of obtained leukocytes in basic experiment |
-
2017
- 2017-08-15 CN CN201710698068.5A patent/CN107441954A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN201819826U (en) * | 2010-09-15 | 2011-05-04 | 肖乐义 | Cell separation and identification device |
| CN104694381A (en) * | 2015-04-01 | 2015-06-10 | 刘韬 | Filter and integration device for separating tumor cells from blood fluid |
| CN105169963A (en) * | 2015-09-15 | 2015-12-23 | 中国原子能科学研究院 | Making method of nuclear track etching membrane |
| CN105233700A (en) * | 2015-09-15 | 2016-01-13 | 中国原子能科学研究院 | Manufacturing method for nuclear track etched membrane with single taper micropore |
| CN105483082A (en) * | 2015-12-24 | 2016-04-13 | 苏州市中心血站 | Method for obtaining leukocytes by adopting LRS and application of obtained leukocytes in basic experiment |
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