CN103756967B - Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting - Google Patents
Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting Download PDFInfo
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Abstract
The invention discloses a kind of application of the monoclonal antibody of the anti-HLA G coupling immunomagnetic beads in tumour cell sorting.The present invention also provides a kind of method for separating of tumour cell, include the following steps：（1）The monoclonal antibody coupling immunomagnetic beads of anti-HLA G obtain HLA G immunomagnetic beads；（2）It will be added in cell liquid to be sorted after HLA G immunomagnetic beads mixings, detached with immuno magnetic cell separation method.The beneficial effects of the invention are as follows：（1）It is sorting tumour cell marker to select HLA G, and HLA G are the broad spectrum activity marker of tomour specific, and after anti-HLA G antibody coupling immunomagnetic beads, specificity is good, and sorting tumour is efficient;（2）Sensibility and repeatability are preferable after HLA G coupling immunomagnetic beads, can detect relevant tumour cell.
Description
Technical field
It is a kind of to apply anti-human leukocyte antigen G (human the present invention relates to a kind of method of sorting tumour cell
Leukocyte antigen-G, HLA-G) antibody-coupled magnetic beads sorting tumour cell method.
Background technology
Quantity of the circulating tumor cell in peripheral blood is few, usually in about 100,000,000 leucocytes and 50,000,000,000 red blood cells
Only several tumour cells, every about 105~107Just there are one circulating tumor cells in a monocyte.Therefore, in order to improve
The recall rate of circulating tumor cell, usually need to row circulating tumor cell before detection enrichment.Cell enrichment can be thin by tumour
The Specific marker (immune separation) of born of the same parents or cell morphological characteristic such as cell volume and density etc. are realized.
Common cell enrichment technology mainly has immune magnetic separation, density gradient centrifugation and cell filtration etc..
1.1 density-gradient centrifugation method：It is that one kind is settled based on physics characteristic, using under certain centrifugal force between cell
The cellifugal technology of rate difference point.This is the common technology in one, laboratory, for detaching monocyte etc..Utilize this technology
The Onclquick (Greiner Bio-One, Frickenhausen, Germany) and Ficoll-Hypaque partition methods of foundation,
Using dedicated porous barrier and density gradient separation liquid, tumour cell is isolated under certain centrifugal force.This technical equipment
Simple operations are easy, can obtain the intact form of cell, convenient for further differentiating, shown in partial clinical research certain
Clinical meaning, but sensibility and specificity is not high, tumor cell recovery is low, limits its clinical application.
1.2 membrane filter method：It is the difference using tumor cell diameter and blood cell, is filtered by the film of certain pore size
Cell and reach a point cellifugal purpose.Different with density-gradient centrifugation method, this technology is special using the morphology of cell
Point carrys out the technology of enriched tumor cell.General haemocyte diameter between 8-ll μm, and tumour cell relative diameter it is larger (such as
About 30 μm of breast cancer cell diameter), it is distinguished whereby to detach tumour cell and normal blood cell.Tumour cell and part blood
The difference of cell is not so apparent, is the aspect that the technology is denounced by people, but its advantage is equally apparent：More polymorphic type
Tumour be suitable for using size come distinguish normal blood cell without because surface marker missing or Tumor Heterogeneity caused by
Missing inspection.First use this method is ISET (Isolation by Size of Epithilial Tumor cells), the party
Method equipment is simple, easy to operate, and cell surface marker can completely retain；The tumour cell to flock together is easier to divide
From conducive to subsequent further detection.But the tumour cell aperture in cycle is not uniform, in immune system attack and
It tends to occur deform under the action of blood shearing force, therefore the size setting of membrane aperture is a problem.There is research to think 5 μm of hole
Diameter good separating effect, but sensibility is under suspicion.The ScreenCell partition methods of new development on this basis, obtained tumour
Cell has preferable sensibility except can be additionally used in cell culture in addition to conventional detection.
In view of the advantage of the separation method according to tumour cell size, at present according to the micro- of cell size separation tumour cell
Filtering technique is researched and developed successfully, and the experimental result at initial stage shows that it has higher sensitivity than antigen-antibody combination partition method, more square
Just Molecular Detection is carried out, here is relevant special description.
Microchip filtering based on cell size：The concentrating platforms established based on tumour cell size gradually have
More reports, the tumour cell that this method obtains are conducive to carry out the analysis of the gene and molecule of next step.Wherein one
Kind substantially increases accumulation rate of this method to circulating tumor cell using high density micropore technology, to cultivate cell addition
Peripheral blood carries out experiment and finds that it is more than 90% to the rate of recovery of tumour cell, and the separation of 7.5mL blood preparations only needs
Time less than 3 minutes can complete.With CellSearch systematic comparisons, in 57 metastatic prostate cancers, colorectal cancer, mammary gland
In cancer and bladder cancer patients, which finds tumour cell in 51 patients, and CellSearch systems only 26 wherein
It is positive findings in blood samples of patients sample, and the circulating tumor cell quantity of chip recycling is the 5.5 of CellSearch systems
Times.There are an experiment reported _ compared according to size enrichment and CellSearch system enrichment cycles tumour cell efficiency, knot
Fruit is also similar, these all show the limitation of CellSearch systems.Microchip isolation technics based on size
Bioaccumulation efficiency depend on the density and rule of micropore, although show certain advantage, at present still without should this method
Clinical large report.
1.3 antigen-antibody combination partition methods：By antigen-antibody combine based on circulating tumor cell separation method be mesh
Preceding most common method.The different surfaces antigenic mark expressed using tumour cell and normal blood cell designs different resist
Body is in combination, and the high degree of specificity and sensitivity combined using antigen-antibody reaches difference and enrichment cycles tumour cell
Purpose (being mostly to combine antibody and magnetic bead, separation is achieved the purpose that in magnetic field).The validity of immunomagnetic beads method concentration method is wanted
It is more much higher than conventional method.It is compared with numerous beneficiation technologies, this technology is more mature, and cell recoveries are high, document report tumour
Cell recoveries are about 85% or so.It is divided into two kinds from principle based on the enrichment method of magnetic bead for immuno positive enrichment and feminine gender
It is enriched with two ways.
1.3.1 the immunomagnetic beads positive captures：
EpCAM (the withered attached factor of epithelial cell) is the target antigen of most common immunomagnetic beads positive capture.On EpCAM is
The most common surface markers of skin source tumour, EpCAM antibody combination magnetic beads, are combined with tumor cell surface antigen, in magnetic field
It is detached.But the isolation technics based on this principle needs tumor cell surface to have stable, abundant surface markers, otherwise
It is easy for generating the result of false negative.It was found that due in transfer process Epithelial and stromal conversion (EMT), tumour cell it is different
The reasons such as matter, some tumor cell surfaces EpCAM express decrease or the tumour cell of missing and non-epithelial origin not table
Up to EpCAM, such as melanoma cells.In order to improve the sensibility of the technology, it is contemplated that carried out using joint antibody magnetic bead
Capture.The biochip technology of new development can be carried using the coated chip microflutes of EpCAP with filtering blood enriched tumor cell
About 100 times of high bioaccumulation efficiency.
The characteristics of positivity is enriched with has higher bioaccumulation efficiency, since CTCs belongs to the rare cell in peripheral blood,
Can efficient recycling tumour cell be positivity enrichment sharpest edges, the multiple of enrichment is from 104To 2 × 105Differ.Cause
The enrichment of this positivity can improve the sensitivity of identification cTCs.But there are still the deficiencies of methodology for positivity enrichment, because its principle is by blood
The cell with epithelial phenotype all recycles in liquid, and the major antigen that richness is concentrated use in is EpCAM.But not there was only tumour cell
It can be also enriched with when expressing EpCAM, certain cells of blood sources or non-malignant epithelial cell expression EpCAM.Tumour is shifting
During, Partial tumors cell phenotype converts, and epithelium mesothelium converts (Epithelial to mesenchymal
Transition, EMT) phenomenon, epithelial marker antigen EpCAM will expression lower, so that the bioaccumulation efficiency of magnetic bead is reduced.In addition, swollen
One of the characteristics of oncocyte is exactly heterogeneous, some CTCs do not express EpCAM, and positive selection may result in the knot of false negative
Fruit.In addition, being still connected with magnetic bead after being detached due to tumour cell, the quality and quantity of magnetic bead identify it influence, but mesh
Preceding the volume of magnetic bead has reduced by tens times due to technological progress, such influence unobvious.
1.3.2 immunomagnetic beads feminine gender sorts：Similar with positive capture, what feminine gender sorting also utilized is magnetic bead sorting principle.
But the antibody that negative sorting is combined with magnetic bead is the antigen for combining normal blood cell surface, tumor cell surface does not express this
Class antigen, such as CD45.The principle of negativity enrichment is all to remove other cells in blood, and then remaining cell is to include
CTCs whole rare cells, then row identification inside.Red blood cell in blood is cleaved, in magnetic after leucocyte binding antibody and magnetic bead
It is detained in, remaining is tumour cell and a small amount of red, leucocyte, then recycles immunofluorescence dyeing identification and counts swollen
Oncocyte.The shortcomings that feminine gender enrichment is the rate of recovery to be enriched with not as good as positive, and advantage is the CTCs of identification not by marked by magnetic bead, in sample
The present treatment and process of enrichment is relatively fewer is affected.Compared with the positive captures, feminine gender sorting is not anti-by tumor cell surface
The influence of original variation, therefore in circulating tumor cell surface antigen expresses inconsistent and specific, the lower tumour of sensibility
It can also apply, such as melanoma etc., and sensibility is relatively high, but interference cell residual is more.This technology is at present not
Automatic system is developed, operation is complex, and human factor influence is relatively large, but is still the following possible development side
To.
1.3.3 CellSearch detecting systems
CellSearch systems are the only approved circulating tumor cell beneficiation technologies of current FDA, and the system is through excessive rule
Mould clinical verification is approved for the prognostic evaluation and examination of curative effect of metastatic hole gland cancer, colorectal cancer and prostate cancer.It is multinomial to grind
Studying carefully confirms that the life cycle of metastatic breast cancer, colorectal cancer and patients with prostate cancer can be effectively predicted in the system, it is often more important that energy
The early prediction curative effect after a period conventional therapy, therefore obtain FDA approvals and be applied to above several late tumor patients.
CellSearch systems are the cycle splenoma cell detecting systems of an automation, and detection stability obtains at multiple centers
Verification.The principle that the system is captured using the immunomagnetic beads positive.
Its workflow is：Tumour cell is combined in magnetic field using immunomagnetic beads combination EpCAM antibody, then uses angle again
Albumen (CK) antibody detects circulating tumor cell, while can also measure CD45, using CrCD45_ as tumour cell standard, most
It is counted afterwards to circulating tumor cell.The system can also provide the data such as karyon endochylema ratio of part cytomorphology, and can
Artificial identification is carried out to collect obtained tumour cell.CellSearch systems complete aforesaid operations and generally require 7.5mL
Peripheral blood, if necessary can be with the expression of additional detected HER2, but ability is general.
The characteristics of immunomagnetic beads are with the sensitivity, specificity and quick separating of its height, in circulating tumor cell detection
More application is obtained.But should and prognosis correlation research technology it is maximum the disadvantage is that, due to many types reality
Widely there is Epithelial and stromal conversion (EMT) phenomenon in body tumor, for tumour cell during entering the circulatory system, some epitheliums are special
Anisotropic antigen presentation weakens or disappears；Simultaneously into the tumour cell of the circulatory system in human autoimmune power and hemorheology
Under the action of mechanics, itself occur adjusting to die weakening with antigen table Da, these all may cause positive capture technique to circulating tumor
The detectability of cell weakens.Therefore, for certain tumours, which has some limitations.
1.3.4 the microchip technology based on antigen-antibody combines：There are several microchip technologies to have been reported for cycle swollen
Oncocyte detects, and the microchip reported recently uses surface array, and coated EpCAM antibody research claims its recycling in a row
Efficiency is more than 60%.The chip can be applied to the detection of kinds of tumors circulating tumor cell, including lung cancer, prostate simultaneously
Cancer, cancer of pancreas, breast cancer and colorectal cancer etc., and be verified in prognostic experiment to lung cancer the effect of, which obtains swollen
Oncocyte can carry out the detection of the EGFR of next step.Microchip technology needs blood flow to pass through so that anti-with extremely a little slower speed
Original antibody fully combines, and 7.5mL blood takes around l0h.
1.4 other beneficiation technologies：
New technology based on micro-fluidic technologies and cell size partition method is also developing, and allows blood sample to pass through first certain
The duct in space, then forms the certain space of size spacing by microtrabeculae aligned transfer again, and the cell more than a certain size obtains
It is other to retain, it is detached.Using the aperture of different spacing, the separation of different cells and blood plasma may be implemented.
The introducing growth-promoting of microelectrode array technology bidimensional electrophoretic separation technology carrys out enrichment cycles tumour cell.Different characteristic
Cell can find the position of oneself in the different distance of microelectrode and assemble the cell of different characteristic because hydrodynamics and leading
Electrical difference is detached, while the feature of cell itself is able to complete preservation.The skill is examined with the tumour cell of culture
The accumulation rate of art finds that its rate of recovery is more than 90%.The shortcoming of these technologies is that its blood using amount is very little and it is necessary to use phase
The equivalent working solution answered dilutes, if tested according to clinically used blood volume, the working solution scale of construction up to 70mL, and
Only have 0.5mL/h, the dilution of liquid that can equally cause the loss of circulating tumor cell by speed.
1.5 HLA-G:
The half of fetus gene comes from father, and fetus is one and half allosomes for mother, but how fetus is resistant to
The immune system of mother is repelledIt is verified by studying during the last ten years：It is mainly distributed on the white of placental villi cell surface
Cellular antigens G (HLA-G) plays main function, and HLA-G has suppression of natural killer cell and cytotoxic T lymphocyte
Function, play an important role in the mechanism of the immune tolerance of fetus.
For tumour in occurrence and development change procedure, immune system has the work(for repelling and eliminating to the cell of these generation cancerations
Can, the referred to as immunosurveillance of host.But suffering from cancer in patient body, cancer cell has significantly escaped from exempting from for host
Epidemic disease monitors that this phenomenon is known as the immunologic escape of tumour, and its mechanism is：Tumour cell occurs have anti-ancestral's phenomenon in early days,
It can secrete, express HLA-G, it is made to escape from the immunosurveillance of host and killing.
From (1996) in last century the nineties, having a large amount of result of study proves：Most of common pernicious swollen
Tumor, such as breast cancer, lung cancer, cancer of the esophagus, gastric cancer, straight-colon cancer, oophoroma, carcinoma of endometrium, the part of cervix cancer or big portion
Tumour cell is divided the unconventionality expression of HLA-G occur, this is a universal phenomenon in oncobiology.
Only there is unconventionality expression in malignant tumour in HLA-G, except extraplacental normal structure is then without expression, is being a kind of new
, tumor markers with high malignancy tumour-specific.In the Second HLA-G scientific seminars that Paris in 2003 is held
On, Forum Summary is explicitly pointed out from the HLA-G progress of research after the meeting of first academic discuss in 1999：Tumour cell occurs
HLA-G unconventionality expressions have been the facts out of question.
Invention content
In view of the above-mentioned deficiencies in the prior art, the technical problems to be solved by the invention provide a kind of sorting mammary gland
The method of cancerous swelling oncocyte.
To achieve the goals above, the following technical solutions are proposed by the present invention：
Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting.
The tumour cell includes breast cancer tumor cells, cancer of the esophagus, non-small cell lung cancer, uterine cancer, gastric cancer, colon cancer
Deng.
A kind of method for separating of tumour cell, includes the following steps：
(1) the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G must resist HLA-G immunomagnetic beads；
(2) anti-HLA-G immunomagnetic beads are added in cell liquid to be sorted, are divided with immuno magnetic cell separation method
From.
Preferably, the monoclonal antibody of the anti-HLA-G of the coupled bead is MEM-G/2, MEM-G/9 or 4H84.
Preferably, between the monoclonal antibody and immunomagnetic beads of the anti-HLA-G it is the full conjunction coupling of antibody, preferably materials matter
Amount is than being 200 μ g antibody:1000 μ g activated magnetic beads.
Preferably, the preparation of the immunomagnetic beads：It learns from else's experience the magnetic bead of activation, is added the full anti-HLA-G's for closing coupling dosage
Monoclonal antibody and PBS, at room temperature circumference mixing 3h, PBS cleanings three times, are added glycine mixing, close remaining aldehyde radical；
The PBS solution containing BSA is added and mixes 30min, closes nonspecific adsorption site, PBS is cleaned 3 times, and PBS buffer solution is added
Whirlpool concussion is to get to the immunomagnetic beads for being uniformly modified with primary antibody.
Preferably, the dosage volume ratio of the cell liquid to be sorted and HLA-G immunomagnetic beads is 100:(20-100).
Preferably, the specific method of the step (2) is：100 μ L (cell number 10/mL) of cell liquid to be sorted are taken, are added
Enter after being incubated 30 minutes in HLA-G immunomagnetic beads L, 2mg/mL, 28 DEG C of incubators of 40 μ, immunomagnetic beads magnetic point is utilized on magnetic frame
From method, suspension is abandoned it, is washed repeatedly 3-4 times with suitable 0.01M PBS (PH=7.4), suspension is abandoned it, finally
After magnetic bead is resuspended per effective 100 μ L PBS, tumor cell number is counted using cell counting board.
The present invention also provides a kind of anti-HLA-G immunomagnetic beads for detaching tumour cell.And it is immune containing anti-HLA-G
The sorting kit of magnetic bead.
The beneficial effects of the invention are as follows：
(1) it is sorting tumour cell marker to select HLA-G, and HLA-G is the broad spectrum activity marker of tomour specific, and coupling is exempted from
After epidemic disease magnetic bead, specificity is good, and sorting tumour is efficient；
(2) sensibility and repeatability are preferable after HLA-G couplings immunomagnetic beads, can detect relevant tumour cell.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obviously, or practice through the invention is recognized.
Specific implementation mode
Technical scheme of the present invention is described further below by embodiment, but should not be understood as the limit to the present invention
System.
1, anti-HLA-G monoclonal antibodies
Monoclonal antibody MEM-G/2, MEM-G/9 and 4H84 of HLA-G, source company in project：Santa Cruz biology skill
Art company.
2, prepared by nano immune magnetic bead
The preparation of nano immune magnetic bead includes the coupling committed step of activated magnetic beads and monoclonal antibody.By the grain for optimizing nano-particle
The joint efficiency of diameter and antibody improves the adsorption efficiency of nano immune magnetic bead confrontation archaeocyte.
The selection and modification of 2.1 nanometer magnetic beads
Test method：
1, the purchase of activated magnetic beads：The present embodiment uses the magnetic bead that Mei Tian Ni companies, Kisker companies produce.
2, magnetic bead selects 50nm activated magnetic beads.
2.2 immunomagnetic beads are prepared with monoclonal antibody coupling
Test method：
1, antibody coupling：1) before antibody coupling, anti-HLA-G antibody (does reality using MEM-G/2, MEM-G/9 and 4H84 respectively
Test) to PBS at 4 DEG C, by dialysing three times, overnight, be stored in PBS, concentration be adjusted to 5mg/mL.
2) preparation of the HLA-G immunomagnetic beads：
Activated magnetic beads 500 μ L, 2mg/mL (in terms of the quality of active magnetic bead) are taken, is added the anti-HLA-G's of 40 μ L 5mg/mL
Monoclonal antibody, in 1.5ML micro-centrifuge tubes, three times, 500 μ L are added in circumference mixing 3h, 1ML PBS cleanings at room temperature
0.2mol/L glycine mixing 30min, close remaining aldehyde radical；1MLPBS is cleaned three times；It is added and contains mass percent 2.5%
The PBS solution of BSA mixes 30min, closes nonspecific adsorption site, and PBS is cleaned 3 times, and 500 μ L PBS buffer solution whirlpools are added
Whirlpool concussion is to get to the uniform immunomagnetic beads for being modified with primary antibody of a concentration of 2.0mg/mL.
2, the reaction condition of monoclonal antibody marking nano magnetic bead is optimized, selects best reaction condition method as follows：
(1) selection of reaction medium：500 μ L nanometer magnetic beads are taken respectively, are placed in 7 centrifuge tubes, are numbered, in externally-applied magnetic field
Under the action of, change medium in 1,2 pipes into phosphate buffer, concentration is as 0.1M PB (pH=7.4) and 0.01M PB
(pH=7.4), medium is respectively 0.1M PBS (pH=6.0), 0.01M PBS (pH=6.0), 0.1M PBS (pH in 3-6 pipes
=7.4) and 0.01M PBS (pH=7.4), medium is deionized water (pH=6.0) in the 7th pipe, and 40 μ L are added into pipe respectively
(5mg/mL) antibody is put on constant temperature oscillator after reacting 6h (150r/min), is placed in magnetic field and detaches supernatant, at ultraviolet point
OD280nm is surveyed under light photometer, determines best reaction medium.
(2) determination of optimum temperature：The antibody of optimum concentration is added in by magnetic bead after surface modification, room is respectively adopted
4h is reacted under warm (25 DEG C) and 37 DEG C of two kinds of temperature conditions, is then washed for several times with 0.01M PBS (pH=7.4), its original is restored
Volume simultaneously surveys OD280nm values in its supernatant.
(3) determination of Best Times：Take 6 1.5mL centrifuge tubes, number consecutively 1-6 that 500 μ L activation are added into every pipe
The nanometer magnetic bead of processing is washed 2-3 times with 0.01M PBS (pH=7.4), 40 μ L (5mg/mL) antibody is added, after addition immediately
Supernatant OD280nm values are surveyed, then survey an OD280nm value every 1h, until 6h.
(4) saturated concentration of antibody-coupled magnetic beads：500 μ L 2mg/mL nanometer magnetic beads are respectively with different amounts of antibody 5mg/
ML, 10 μ L, 30 μ L, 40 μ L, 60 μ L, 100 μ L, it is 600 μ L to supply total volume, is coupled to room temperature 30 minutes, is surveyed immediately after addition
Supernatant OD280nm values after the completion of coupling, then survey an OD280nm value, determine saturation coupled antibody magnetic bead ratio.
3, the detection of antibody coupling amount：
Microplate reader (the U.S. is combined using BCA kits (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd)
PerKinEImer companies) after detection coupling reaction in supernatant antibody content.The monoclonal antibody solution of anti-HLA-G is used
PH7.4,0.01moL phosphate tween (0.05%Tween-20) solution is diluted to the standard solution of 0.5mg/mL, takes 0 respectively,
For 1,2,4,8,12,16,20 μ L titers in enzyme reaction item, each standard solution does 1 parallel repetition, adds pH7.4 per hole,
It is 20 μ L that 0.01mol/L phosphate tween (O.05%Tween-20) solution, which supplies total volume,；By supernatant 12000r/ to be detected
Min takes 20 μ L in enzyme reaction item after centrifuging 10min, and each supernatant to be detected is cooked 1 parallel repetition；Mark is added to all
200 μ L BCA reaction solutions, 60 DEG C of reaction 30min are added in the enzyme reaction hole of quasi- liquid or supernatant；Each hole is detected with microplate reader
Absorbance (A) value.Standard curve is made according to the concentration of titer and light absorption value, further according to the absorbance value of supernatant to be detected
The amount of antibody in supernatant is obtained, to obtain the antibody content of unit mass magnetic bead surfaces coupling.
Test result：
1, the determination of reaction medium：The reaction medium of suitable ion concentration and pH, it is efficient with protein for magnetic bead
The non-specific adsorption that coupling is reduced between them plays an important roll.Experimental result shows that three kinds of antibody are applicable in, and work as medium
For 0.01M PBS (pH=7.4) when, non-specific adsorption unobvious, OD280 values are higher, can as coating magnetic bead reaction
Medium.
2, the determination of optimum temperature：In 37 DEG C of temperature drifts, part solution is caused to volatilize, and measured compared with room temperature condition
Supernatant in OD280 numerical value it is extremely unstable, therefore the experimental selection room temperature of three kinds of antibody coating.
3, most preferably it is coated with the time：Satisfy from table 1 it follows that three kinds of antibody have substantially achieved after magnetic bead adsorbs 3h
With, OD280 values tend towards stability, it is thus determined that coating the time be 3h.
1 antibody of table is coated with magnetic bead Best Times (by taking 4H84 as an example)
4, antibody magnetic bead conjugation ratios：As a result the 4H84 monoclonal antibodies for finally showing anti-HLA-G, antibody concentration is set to
5mg/mL, a concentration of 2mg/mL of magnetic bead, antibody and magnetic bead mass ratio are 200 μ g:1000 μ g carry out magnetic bead coupling, antibody at this time
Coupling can reach saturation substantially.
5, magnetic bead surfaces antibody quantitatively uses the anti-HLA-G antibody of BCA protein quantification kits detection magnetic bead surfaces connection
Amount.Obtain the absorbance value corresponding to protein standard solution known to concentration.According to protein content contained by various criterion solution with
Its absorbance value, does standard curve.By taking 4H84 as an example, supernatant samples absorbance value is substituted into formula, obtains supernatant samples
Middle protein content is respectively 71.2, is 200 μ g because reagin is added, i.e., the amount of antibody of every 1000 μ g magnetic bead surfaces connection is
128.8；In summary it can be calculated, the anti-128.8 μ g/mg of HLA-G amount of antibody average out to of magnetic bead surfaces connection.
3, anti-HLA-G immunomagnetic beads enriched tumor cell experiment
The tumor cell line that the three kinds of anti-HLA-G immunomagnetic beads prepared using the present embodiment step 2.2 are used to manually cultivate
Cell carries out tumour cell collection；The tumor cell line cell manually cultivated and healthy human blood are mixed with Artificial sample,
Using anti-HLA-G immunomagnetic beads, tumour cell in Artificial sample is enriched with, cell count is carried out to enriched sample, is calculated
Bioaccumulation efficiency.
Test method：
1, cell sample preparation and anti-HLA-G immunomagnetic beads enriched tumor cell:
1) 6 kind tumor cell line cells of the laboratory cultures from ATCC:Breast cancers (MCF-7), cancer of the esophagus
(EC109), non-small cell lung cancer (A549), cervix cancer (Hela), gastric cancer (NCI-N87), colon cancer (HT29) prepare 10/
The various tumour cell samples of mL (10/ μ L) cultures；Various tumour cells, 100 μ L are taken to be added in 1.5mL centrifuge tubes, each is thin
Born of the same parents are three pipes；
2) it takes the above-mentioned various tumour cells prepared of 5 μ L to be mixed with 5mL healthy human bloods, forms tumour cell in periphery
Artificial sample in blood, tumor cell number:10/mL blood.Each cell prepares three pipes；
3) the anti-HLA-G immunomagnetic beads for preparing 40 μ L, are added mixing in 100 μ L tumor cells specimens, are incubated, then make
It with the LS columns and high-intensity magnetic field magnetic separtor of German Mei Tian mud company, is detached, PBS cleanings, then by magnetic bead adherent cell
100 μ L PBS are resuspended in, cell count is carried out, the cell of enrichment is detected using cell counting board, count tumour
Cell accounts for the ratio of total number of cells before enrichment, calculates anti-HLA-G enrichment with magnetic bead tumour cell efficiency.
4) the anti-HLA-G immunomagnetic beads for preparing 40 μ L 2mg/mL, are added in the artificial tumour cell blood samples of 1mL,
It is detached in magnetic field, then magnetic bead adherent cell is resuspended in 1mL PBS by PBS cleanings；5 μ L samples are taken, are added dropwise in load
On slide, the IF such as paraformaldehyde is fixed prepare, and using the anti-CK8/18/19-FICT of BD companies, are incubated, aobvious using fluorescence
Micro mirror is counted, and statistics recycling tumour cell accounts for the ratio of total number of cells before enrichment, calculates anti-HLA-G enrichment with magnetic bead blood sample
Tumour cell efficiency in product.
2, nano immune magnetic bead optimum amount determines：5 parts of artificial tumor samples of MCF-7, every part of 100 μ L are taken to be separately added into
10,20,40,60,100 μ L of nano immune magnetic bead after being incubated 1h in 28 DEG C of incubators, utilize immunomagnetic beads Magneto separate on magnetic frame
Suspension is abandoned it by method, is washed repeatedly 2-3 times with suitable 0.01M PBS (PH=7.4), suspension is abandoned it, last every
After magnetic bead is resuspended in effective 100 μ L PBS, tumor cell number is counted using cell counting board.
3, the determination of nano immune magnetic bead and tumour cell optimum reacting time：It is 1mL's by four parts of volumes of MCF-7 cells
Artificial tumor-blood sample is separately added into 40 μ L of magnetic bead, after being incubated 10min, 30min, 60min respectively in 28 DEG C of incubators, in magnetic
Immunomagnetic beads magnetism separate method is utilized on power frame, suspension will be abandoned it, repeats to wash with suitable 0.01M PBS (pH=7.4)
It washs 2-3 times, suspension is abandoned it, it is dense to tumour cell using cell counting board after magnetic bead is resuspended in last every effective 100 μ LPBS
Degree is counted.
Test result：
1, after tested, in the various tumour cell samples of preparation and tumour cell in blood-Artificial sample, anti-HLA-
G immunomagnetic beads can reach rate of recovery 85%-93% to corresponding tumor cell enrichment rate.To tumor cell enrichment rate in blood
77%-84% is can reach, this explanation, the accumulation rate of the anti-HLA-G immunomagnetic beads of preparation is higher, and repeatability preferably, can return
Receive relevant tumour cell.
2 anti-HLA-G immunomagnetic beads of table compare different tumor cell enrichment sensibility
2, nano immune magnetic pearl dosage：It is shown in Table 3 after tested, tumor sample is 100 μ L (10 with nanometer magnetic bead ratio7/mL):40
When μ L (2mg/mL), concentration effect is preferable and magnetic bead will not be caused to waste.
The relationship of table 3 nanometer magnetic bead dosage and tumor cell enrichment rate
3, nano immune magnetic bead and tumour cell optimum reacting time：4 are shown in Table, it is observed that magnetic bead is reacted with tumor sample
When 30min, tumour cell quantity is more, and the normal cell being mixed with is few.And magnetic bead is with tumor sample when reacting 60min, normal cell
It is more.Magnetic bead is long or too short with sample binding time, can all increase nonspecific reaction.Therefore nano immune magnetic bead and sample
The Best Times of reaction are 30min.
4 nano immune magnetic bead of table and tumor sample optimum reacting time (by taking 4H84 as an example)
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of being detached from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The range of invention is limited by claim and its equivalent.
Claims (3)
1. a kind of method for separating of tumour cell, which is characterized in that include the following steps：
（1）The monoclonal antibody coupling immunomagnetic beads of anti-HLA-G obtain HLA-G immunomagnetic beads；The monoclonal antibody of the anti-HLA-G
For MEM-G/2, MEM-G/9 or 4H84；The preparation of the immunomagnetic beads：
Activated magnetic beads 500 μ L, 2mg/mL are taken, in terms of the quality of active magnetic bead, the Dan Ke of the anti-HLA-G of 40 μ L 5mg/mL is added
Grand antibody, in 1.5 mL micro-centrifuge tubes, three times, 500 μ L are added in circumference mixing 3h, 1 mL PBS cleanings at room temperature
0.2mol/L glycine mixing 30min, close remaining aldehyde radical；1 mL PBS cleanings are three times;Addition contains mass percent
The PBS solution of 2.5%BSA mixes 30min, closes nonspecific adsorption site, and PBS is cleaned 3 times, and 500 μ L PBS bufferings are added
The concussion of liquid whirlpool is to get to the uniform immunomagnetic beads for being modified with primary antibody of a concentration of 2.0mg/mL；
（2）It will be added in cell liquid to be sorted after HLA-G immunomagnetic beads mixings, divided with immuno magnetic cell separation method
From；Specific method is：100 μ L of cell liquid to be sorted are taken, HLA-G immunomagnetic beads 40 the μ L, 2mg/mL, 28 of mixing is added
After being incubated 30 minutes in DEG C incubator, immunomagnetic beads magnetism separate method is utilized on magnetic frame, suspension is abandoned it, uses 0.01M
PBS, pH=7.4 are washed repeatedly 2-3 times, and suspension is abandoned it, after magnetic bead is resuspended in last every effective 100 μ L PBS, using thin
Born of the same parents' tally counts tumor cell number.
2. method for separating according to claim 1, which is characterized in that the monoclonal antibody of the anti-HLA-G and immune magnetic
Being that antibody is full between pearl closes coupling, and materials mass ratio is 200 μ g：1000µg.
3. method for separating according to claim 1, which is characterized in that magnetic is immunized with HLA-G in the cell liquid to be sorted
The dosage volume ratio of pearl is 100：(20-100).
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| PCT/CN2014/093816 WO2015101163A1 (en) | 2013-12-31 | 2014-12-15 | Application in tumor cell sorting of coupling anti-hla-g monoclonal antibody to immunomagnetic beads |
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| CN103756967B (en) * | 2013-12-31 | 2018-09-21 | 卢英 | Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting |
| CN105087493B (en) * | 2014-09-26 | 2018-09-21 | 卢英 | Three kinds of monoclonal antibody coupling immunomagnetic beads of combination are applied in tumour cell sorting |
| US20190119385A1 (en) * | 2015-03-27 | 2019-04-25 | University Of Southern California | Hla-g as a novel target for car t-cell immunotherapy |
| CN106350485B (en) * | 2016-08-24 | 2020-02-21 | 杭州百凌生物科技有限公司 | Method for quickly and efficiently separating single antigen specific B cells |
| CN106366198A (en) * | 2016-08-31 | 2017-02-01 | 上海美吉生物医药科技有限公司 | Immunomagnetic bead and preparation method thereof |
| CN106366197B (en) * | 2016-08-31 | 2021-03-30 | 上海美吉生物医药科技有限公司 | HER2, EGFR, EpCAM and MUC1 multiple antibody immunomagnetic beads and preparation method thereof |
| CN108548920A (en) * | 2018-02-28 | 2018-09-18 | 江苏医诺万细胞诊疗有限公司 | A kind of detection method for the kit detecting circulating tumor cell using immunomagnetic beads negative sense absorption joint flow cytometry |
| CN109111525B (en) * | 2018-05-24 | 2021-10-29 | 卢英 | HLA-G chimeric antigen receptor, coding sequence, expression vector and application |
| JP2022511203A (en) * | 2018-11-08 | 2022-01-31 | 世奇 廖 | Target-Tumor Serum Aptamer Complex Detection Methods and Kits |
| CN110468187A (en) * | 2019-07-18 | 2019-11-19 | 江苏宏微特斯医药科技有限公司 | The method and kit be enriched with before high-flux sequence to the pathogenic microorganism infected in host |
| CN110456076A (en) * | 2019-09-12 | 2019-11-15 | 太仓极光克赛生物技术有限公司 | The method of multiple antibody immune magnetic beads enrichment detection CTCs |
| CN110456077A (en) * | 2019-09-12 | 2019-11-15 | 太仓极光克赛生物技术有限公司 | A method of the immunomagnetic beads enrichment detection CTCs of coupling anti-EpCAM antibody |
| CN110687280A (en) * | 2019-10-14 | 2020-01-14 | 河南省商业科学研究所有限责任公司 | Preparation method of immunomagnetic beads for detecting ethylenediamine tetraacetate and immunomagnetic beads prepared by same |
| CN111474356A (en) * | 2020-04-16 | 2020-07-31 | 江西省达臻医疗科技有限公司 | Double-immunomagnetic-bead sorting reagent, preparation method thereof and application thereof in enrichment of humoral exosomes |
| CN113832100A (en) * | 2021-10-26 | 2021-12-24 | 江苏大学附属医院 | Method for obtaining immune cells of tumor liver metastasis tissues |
| WO2023224923A2 (en) * | 2022-05-16 | 2023-11-23 | The Regents Of The University Of California | Engineered cells and methods of use |
| CN118146376B (en) * | 2024-05-09 | 2024-07-05 | 成都微芯新域生物技术有限公司 | HLA-G antibodies, methods of making and uses thereof |
| CN118184783B (en) * | 2024-05-09 | 2024-07-09 | 成都微芯新域生物技术有限公司 | HLA-G antibodies, methods of making and uses thereof |
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Effective date of registration: 20210126 Address after: Room 601, No.3 workshop, No.32 Zhujiang Road, Yantai Development Zone, Yantai area, China (Shandong) pilot Free Trade Zone, Yantai City, Shandong Province 264000 Patentee after: Yantai Panya Biotechnology Co.,Ltd. Address before: Room 1-301, No. 322, Shunfeng Road, Overseas Students Pioneer Park, Jinan hi tech Zone, Shandong Province, 250101 Patentee before: Lu Ying |
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