CN104122285B - Magnetic-bead-based low field NMR (nuclear magnetic resonance) rare cell detection method - Google Patents
Magnetic-bead-based low field NMR (nuclear magnetic resonance) rare cell detection method Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology and discloses a magnetic-bead-based low field NMR (nuclear magnetic resonance) rare cell detection method which comprises the following steps: (1) a standard sample and a to-be-tested sample of a target rare cell suspension are respectively prepared; (2) a nuclear magnetic resonance contrast medium coupled with a specific expression antibody of target rare cells is respectively added, the nuclear magnetic resonance contrast medium is enriched in the rare cells in the samples by immunoreactions, and the unreacted nuclear magnetic resonance contrast medium is removed to obtain the nuclear magnetic resonance contrast medium-enriched standard sample and to-be-tested sample; (3) the relaxation time of the standard sample and the to-be-tested sample are respectively detected by use of a low field nuclear magnetic resonance analyzer, and sterile deionized water or a buffer solution is used as a blank control; and (4) the change amount of the relaxation time is calculated for drawing a standard curve to obtain the content of the target rare cells in the to-be-tested sample. The detection method can be used for convenient, fast, high specificity and high sensitivity detection of low content of rare cells in biological fluid samples.
Description
Technical field
The present invention relates to molecular Biological Detection technology, particularly to a kind of detection of rare cell in biological fluid samples
Method.
Background technology
Rare cell refers to that some in biological fluid samples (including blood, hydrothorax, ascites, urine, cerebrospinal fluid etc.) are non-
Typical cells, numerous studies show, the detection to rare cell and identification are for the pathomechanism of relevant disease and targeted drug
Exploitation has great importance.Therefore, the rare cell detection method found accurately and rapidly will become urgently to be resolved hurrily asking
Topic.But concentration in biological fluid for the rare cell is very low, it is about 1 with the ratio of untargeted cells：107, with tradition
Technology cannot count, so solving this difficult problem in the urgent need to a kind of simple quickly and accurately method.
Due to circulation content in body fluid for the rare cell seldom, could be in follow-up reality through effective enriching step
Test middle identification identification.Be widely used in the market rare cell detection research method mainly have density-gradient centrifugation method,
Membrane filter method and immunomagnetic isolation technology.
Density gradient zonal centrifugation method is also called zonal centrifugation, is sample to be added in inertia gradient media be centrifuged
Sedimentation or sedimentation equilibrium, are assigned to particle on some ad-hoc locations in gradient under certain centrifugal force, form different zone
Separation method.The whole Component seperation of several work in sample can be made using this method simultaneously, there is good resolution ratio.This method
Advantage is：1. good separating effect, can once obtain purer particle；2. wide accommodation, can separate tool as differential centrifugation
There is the particle of sedimentation coefficient difference, can separately have the particle of certain buoyant density difference again；3. particle will not crimp, can keep
Grain activity, and prevent established zone from causing mixing due to convection current.The shortcoming of this method is：1. centrifugation time is longer；2. need
Prepare inertia gradient media solution.It is frequently utilized that the principle of density gradient centrifugation in research, separated with Ficoll liquid and purify
Human or animal's PMNC (PBMC).
Membrane filtering method is so that rare cell is from outer according to some rare cell volumes more than granulocyte in peripheral blood
In all blood, separation and concentration out, then carries out antidiastole using immunofluorescence technique to rare cell.In the method, membrane filtration
Cell enrichment process is relatively easy, but granulocytic in not all rare cell volume both greater than peripheral blood, much dilute
There are volume and the granulocyte size of cell similar or even less than granulocyte volume.Based on above understanding, membrane filtering method
Gradually abandoned.
Content of the invention
It is an object of the invention to provide one kind can easy, quick, high specific and detect biological fluid in high sensitivity
The low-field nuclear magnetic resonance determination method of the relatively low rare cell of content in sample.
For solving above-mentioned technical problem, the invention provides a kind of low field NMR rare cell detection based on magnetic micro-beads
Method, comprises the steps of：
(1) sample preparation
By the clone of the target rare cell of pure culture, different gradient dilutions, prepared series concentration are carried out with buffer solution
Rare cell suspension master sample；
Biological fluid samples to be measured are carried out with Ficoll density gradient centrifugation and obtains candidate's mixed cellularity group, pass scrubbed height
The heart is resuspended, prepared rare cell suspension sample to be checked；
(2) the affine enrichment of mri contrast agent
Take above-mentioned standard sample, add and be coupled the mri contrast agent having target rare cell specifically expressing antibody, make
There is immune response enrichment with the rare cell in master sample in mri contrast agent, then remove unreacted nuclear magnetic resonance
Contrast preparation, obtains the master sample of mri contrast agent enrichment；
Take above-mentioned sample to be checked, add and be coupled the mri contrast agent having target rare cell specifically expressing antibody, make
There is immune response enrichment with the rare cell in sample to be checked in mri contrast agent, then remove unreacted nuclear magnetic resonance
Contrast preparation, obtains the sample to be checked of mri contrast agent enrichment；
(3) NMR relaxation time detecting
Take the master sample of above-mentioned mri contrast agent enrichment, carry out relaxation time survey with low-field nuclear magnetic resonance analyzer
Fixed, obtain relaxation time values Tsample；Using buffer solution as blank, carry out relaxation time survey with low-field nuclear magnetic resonance analyzer
Fixed, obtain relaxation time values Tblank；
Take the sample to be checked of above-mentioned mri contrast agent enrichment, carry out relaxation time survey with low-field nuclear magnetic resonance analyzer
Fixed, obtain relaxation time values Ttest；
(4) standard curve making and result draw
Calculate the relaxation time knots modification Δ T2, described Δ T2=T of master samplesample-Tblank, with Δ T2 as ordinate,
With the target rare cell content in master sample as abscissa, it is depicted as calibration curve；
Calculate the relaxation time knots modification Δ T2 of sample to be checkedtest, described Δ T2test=Ttest-Tblank, Δ T2testIt is not
0, then show to contain target rare cell in sample to be checked, show that in sample to be checked, target is rare according to above-mentioned standard curve simultaneously
The content of cell.
From above-mentioned detecting step：The present invention utilizes the characteristic that mri contrast agent can combine with rare cell,
With immunomagnetic isolation technology, pass through to check the hydrogen of hydrone in magnetic bead-cell suspending liquid in the presence of additional downfield
Atom signals are carried out quantitation and are circulated rare cell.
Specifically, the present invention carries out the fast enriching of rare cell first in sample pre-treatments；Then, based on special
Property antibody antigen immune response, be bonded to the nuclear magnetic resonance on rare cell surface using the detection of low-field nuclear magnetic resonance analyzer and make
The change in the relaxation time of shadow agent, realizes the quickly and delicately qualitative and quantitative detection to rare cell.Institute in the present invention
The change based on the relaxation time characterisitic parameter of nuclear magnetic resonance technique for the final detection evaluation method of the target rare cell stated, institute
State relaxation time characteristic, refer to spin-lattice relaxation time and spin spin relaxation time.
Preferably, in the detection method of the present invention, the mri contrast agent used in step (2) is paramagnetic nanoparticles
Magnetic bead or super-paramagnetism nano magnetic bead, and described paramagnetic nanoparticles magnetic bead or super-paramagnetism nano magnetic bead are preferably anti-EpCAM
In immune nanometer magnetic bead, anti-CD45 immune nanometer magnetic bead, Streptavidin nanometer magnetic bead, the magnetic liposome of modified with folic acid one
Plant or several.The high degree of specificity of distinctive for solidified reagents advantage and immunological response is incorporated into one by above-mentioned immunomagnetic beads
Body, thus rapidly realize capturing and separate the purpose of rare target cell from mixed cellularity group.Gained rare cell can use
In counting or other aspect researchs application.
Preferably, in the detection method of the present invention, make mri contrast agent respectively with master sample, sample to be checked in
Rare cell occur immune response enrichment reaction condition be：It is incubated 10～15min under 4～8 DEG C of low temperature after mixing.
Under the above-described reaction conditions, mri contrast agent and rare cell in sample carry out antigen-antibody immune response product
Raw background signal is minimum, and accuracy is more preferable.
Preferably, in the detection method of the present invention, in described step (2), there is target rare cell special adding coupling
Before the mri contrast agent of expression antibody, also comprise the steps：Add and be coupled the mri contrast agent having anti-CD45
Untargeted cells is carried out magnetic mark, by externally-applied magnetic field mode, the untargeted cells of above-mentioned mark is removed.
Immunomagnetic beads enrichment method in the present invention, except being directly added into coupling described by step (2) in foregoing invention content
There is the mri contrast agent of target rare cell specifically expressing antibody, so that mri contrast agent is targetted with rare cell
Immune (antigen-antibody reaction) enrichment (positive enrichment) outward, also can reach target cell using the method for negative+positive double enrichments
The purpose of magnetite gathering.Concrete operation method is：It is coupled the nuclear-magnetism having target rare cell specifically expressing antibody adding altogether
Shake before contrast preparation, first with being coupled the mri contrast agent (magnetic micro-beads) having anti-CD45 by untargeted cells (predominantly
More leucocyte of content etc. in epithelial cell) carry out magnetic mark, by externally-applied magnetic field mode by leucocyte from containing target
Get rid of (negative marker untargeted cells) in the cell mass mixture of rare cell；Then, above-mentioned acquisition cell subsets mixing
Thing, (magnetic is micro- to select suitable coupling to have the mri contrast agent of target cell specifically expressing antibody according to subgroup mark
Pearl) carry out immune response enrichment, thus obtaining the mri contrast agent (positive mark's target cell) being marked with target cell.
By the method for operating of above-mentioned negative+positive double enrichments, can more accurately, reliable reach the purpose of target cell magnetite gathering.
Preferably, in the detection method of the present invention, in described step (2), mri contrast agent addition is preferably：Often
1 microlitre of mri contrast agent, the method removing unreacted mri contrast agent is added in 200 target rare cells
For：Slowly siphon away supernatant, then apply externally-applied magnetic field or stable centrifugation, by the sample buffering of enrichment mri contrast agent
Liquid is washed.The mri contrast agent of this ratio not only most optimally can obtain rare cell additionally it is possible to reduce due to
The background enhanced that mri contrast agent addition excessively leads to, affects Detection results.
Preferably, in the detection method of the present invention, in described step (3), control the magnetic field of low-field nuclear magnetic resonance analyzer
Intensity is 20～25MHz, and magnet temperature is 30～35 DEG C, repetition time 3～5s.It is highly preferred that controlling low-field nuclear magnetic resonance to divide
The magnetic field intensity of analyzer is preferably 20.18MHz, and magnet temperature is preferably 35 DEG C, and the repetition time is preferably 5s.
To above-mentioned nuclear magnetic resonance system magnetic field intensity, magnet temperature and the control of repetition time, detection can be made more accurate,
Controllability is higher, and the comparison beneficial to parallel laboratory test and Quality Control.
Preferably, in the detection method of the present invention, in described step (4), calculate the relaxation time knots modification of master sample
The relaxation time knots modification Δ T2 of Δ T2 and sample to be checkedtestWhen, take the mean value in multiple measurement relaxation time to be counted respectively
Calculate, so as to make the present invention testing result more accurately and reliably.
From above-mentioned discussion, the present invention passes through to be enriched with and low mri contrast agent (as super suitable immune micro- magnetic bead)
The detection of field nuclear magnetic resonance instrument combines, by the separation of rare cell together with identification organic combination, thus quickly realizing from mixed
Close capture and the rare target cell of separation in cell mass.Selected using immunity magnetic micropearls in the present invention and separate highly rich
Any rare cell present in collection and concentration humoral sample, the cell being captured detectably is marked with rare cell specificity
Expression antibody, and is coated by superparamagnetic microbeads so that the rare cell being captured can be differentiated and counted and
Clearly distinguish on NMR with contaminative non-target cell.Due to have can detect that in every 7.5ml body fluid micro-
The extremely strong sensitiveness of amount rare cell, is a kind of separation scheme of the wide spectrum with extremely strong Sensitivity and Specificity.
The existing detection method for rare cell all refers to immunofluorescence technique, need to pass through fluorescence microscopy
To carry out quantitation to the number circulating rare cell in body fluid, generally have that complex operation, sensitivity be low, Check-Out Time length, valency
Lattice are high, operating personnel are had high demands, and are difficult the drawbacks of repeat (as the ripe operating personnel's single of i FISH detection method is only capable of
Operate 6 samples, whole process takes 2 days) simultaneously.Compared with prior art, coupling is had specifically expressing antibody by the present invention
Mri contrast agent is enriched with rare target cell technology and has the low-field nuclear magnetic resonance of quick and high-sensitivity detection technology
Instrument combines, it is achieved thereby that detecting the purpose of rare cell in peripheral blood quickly, efficiently and with sensitivity, has excellent as follows
Point:(1) target rare cell can be made directly to separate from biological fluid, there is simplicity, quick feature；(2) and centrifugation,
The methods such as filtration are compared, and the shearing force that rare cell is subject in Magnetic Isolation is little, can avoid the inactivation of cell；(3) have very
High selectivity；(4) externally-applied magnetic field will not produce impact to the motion of the ion in material liquid and ionic sample；(5) instrument sets
Standby simple, operating cost is low.The method of the present invention can realize the rare cell quick detection of extensive sample, no matter for product
All undoubtedly have great importance in industry or customer experience.The method is to be suggested first, has no relevant report at present, and
The method be applied to the detection to the various rare cells (include all of circulate rare epithelial cell) biological fluid samples,
Identification and quantitative analysis.
Brief description
Fig. 1 is the flow chart of the low field NMR rare cell detection method of the present invention；
Fig. 2 is with the relaxation time knots modification Δ T2 of master sample as ordinate, with the mesh in master sample in embodiment 1
Mark rare cell content is the calibration curve made by abscissa.
Specific embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with each reality to the present invention for the accompanying drawing
The mode of applying is explained in detail.However, it will be understood by those skilled in the art that in each embodiment of the present invention,
In order that reader more fully understands that the application proposes many ins and outs.But, even if there is no these ins and outs and base
In following embodiment many variations and modification it is also possible to realize each claim of the application technical side required for protection
Case.
Embodiment 1
(1) sample preparation
Sample preparation to be checked：
1.1 obtain anti-freezing blood sample (～10ml) from breast cancer model mouse, and it is rare containing target that described sample comprises suspection
The mixed cellularity group of cell.The sample of collection should be processed on the same day！The room temperature lucifuge retention time is not to be exceeded 24 hours；
1.2Ficoll density-gradient centrifugation method obtains candidate's mixed cellularity group
1) add appropriate cell layering liquid (Ficoll solution is formulated) to sterile centrifugation tube A bottom, then will be above-mentioned
Anticoagulation test specimens are made (2 after suitably dilution with PBS liquid:1), gently it is added in above layering liquid along tube wall, make both form one
Clearly interface.
2) (increase of centrifuge speed and minimizing will uniformly, steadily, to ensure to be formed clearly for 400 × g horizontal centrifugal 30min
Clear interface).
3) final visible three-layer-liquid body in pipe after being centrifuged, upper strata is yellow liquid, and intermediate layer is transparency liquid, and bottom is palm fibre
(red blood cell and granulocyte density are more than layering liquid to red sedimentary red blood cell, simultaneously the aggegation bunchiness because red blood cell runs into Ficoll
Money shape and be deposited on ttom of pipe.Blood platelet is then suspended in because density is little in blood plasma, has the single core suitable with being layered liquid-tight degree only
Cell intensive plasma layer and layering liquid interface in, in tunica albuginea shape), draw this confluent monolayer cells pass the scrubbed high heart resuspended (300 ×
G10min washes twice) obtain the cell suspending liquid sample to be checked that can be used for the micro- enrichment with magnetic bead of next step immunity in B pipe.
Prepared by master sample：
By pure culture mouse mastopathy cell, different gradient dilutions (0,10,10 are carried out with PBS2,103,104,105,106,
107Cells/ml), thus obtaining the rare cell suspension master sample of series concentration；
(2) magnetic micro-beads affine in immunity enrichment
Take above-mentioned standard sample and sample cell suspending liquid standard series to be checked respectively in 96 porocyte culture plates, respectively
Adding 100 μ L to be coupled has the superparamagnetic immunomagnetic beads of rare cell specifically expressing antibody, in 4-8 DEG C of incubation reaction 15min, suitably
Gentle shaking table may be selected and carry out blending incubation, obtain serial Rare cell-Microbeads-Ab compound titer.Additionally,
For preventing the non-specific binding of cell surface antibodies, operation must rapidly, cell need to be placed on ice, required solution need precooling.
Obtain the master sample of magnetic micro-beads enrichment and the sample to be checked of magnetic micro-beads enrichment.
(3) magnetic resonance relaxation time test
Instrument parameter is as follows：Magnetic field intensity 20.18MHz, 35 DEG C of magnet temperature, repetition time 5s, each DATA REASONING 3 times
After average.
The measurement of master sample relaxation time and standard curve making：
Take 180 μ L above-mentioned series Rare cell-Microbeads-Ab compound titer respectively to nmr tube,
Measure sample to be tested T2 with low-field nuclear magnetic resonance detector, obtain relaxation time values Tsample；With aseptic deionization
Water or buffer solution, as blank, carry out relaxation time mensure with low-field nuclear magnetic resonance analyzer, obtain relaxation time values Tblank,
With relaxation time knots modification (Δ T2) as ordinate, with master sample, the content of rare cell, as abscissa, is depicted as standard
Curve.Wherein, Δ T2 represents according to formula (1).
Δ T2=Tsample-Tblank.......................................(1)
In formula：Tsample：Three horizontal survey mean values of series standard sample relaxation time；Tblank：During negative sample relaxation
Between three horizontal survey mean values.
Circulation rare cell quantitation and qualification result statistical form in master sample：
| Number of cells | 5 | 10 | 20 | 30 | 40 | 50 |
| ΔT2(ms) | 60 | 67 | 82 | 105 | 162 | 211 |
The calibration curve drawn according to the data in upper table is as shown in Figure 2.
The relaxation time of sample to be tested is measured and is drawn with result：
Take the sample to be checked of magnetic micro-beads enrichment, identical method is measured with the above-mentioned master sample relaxation time, uses low field
Magnetic nuclear resonance analyzer carries out relaxation time mensure to it, obtains relaxation time values Ttest, calculate sample to be checked relaxation time change
Variable Δ T2test, described Δ T2test=Ttest-Tblank, Δ T2testIt is not 0, then show rare thin containing target in sample to be checked
Born of the same parents.Described TblankAnd TtestNeed to measure respectively three times and average.Derive dilute in sample to be checked from above-mentioned standard curve
There is the content of cell.
The relaxation time measurement result of sample to be checked is：
| 1 | 2 | 3 | Mean value | |
| Tblank | 2721 | 2778 | 2579 | 2692.6 |
| Ttest | 2786 | 2851 | 2638 | 2758.3 |
| ΔT2test | 69 | 78 | 58 | 68.3 |
By formula computing formula：Y=49.353e^ (0.0284X), can be calculated rare cell number in testing sample is：
11.46.
It will be understood by those skilled in the art that the respective embodiments described above are to realize the specific embodiment of the present invention,
And in actual applications, can to it, various changes can be made in the form and details, without departing from the spirit and scope of the present invention.
Claims (6)
1. a kind of low field NMR rare cell detection method based on magnetic micro-beads is it is characterised in that comprise the steps of：
(1) sample preparation
By the clone of the target rare cell of pure culture, different gradient dilutions are carried out with buffer solution, prepared series concentration dilute
There is cell suspending liquid master sample；
Biological fluid samples to be measured are carried out Ficoll density gradient centrifugation obtain candidate's mixed cellularity group, pass scrubbed from the high heart
Resuspended, prepared rare cell suspension sample to be checked；
(2) the affine enrichment of mri contrast agent
Take above-mentioned standard sample, add and be coupled the mri contrast agent having target rare cell specifically expressing antibody, make nuclear-magnetism
There is immune response enrichment with the rare cell in master sample in resonance contrast preparation, then remove unreacted magnetic resonance imaging
Agent, obtains the master sample of mri contrast agent enrichment；
Take above-mentioned sample to be checked, add and be coupled the mri contrast agent having target rare cell specifically expressing antibody, make nuclear-magnetism
There is immune response enrichment with the rare cell in sample to be checked in resonance contrast preparation, then remove unreacted magnetic resonance imaging
Agent, obtains the sample to be checked of mri contrast agent enrichment；Control low-field nuclear magnetic resonance analyzer magnetic field intensity be 20～
25MHz, magnet temperature is 30～35 DEG C, repetition time 3～5s；Make mri contrast agent respectively with master sample, treat sample
Rare cell in this occurs the reaction condition of immune response enrichment to be：After mixing at a temperature of 4～8 DEG C incubation 10～
15min；Mri contrast agent addition is：1 microlitre of mri contrast agent is added in every 200 target rare cells；
(3) NMR relaxation time detecting
Take the master sample of above-mentioned mri contrast agent enrichment, carry out relaxation time mensure with low-field nuclear magnetic resonance analyzer,
Obtain relaxation time values Tsample；Using buffer solution as blank, carry out relaxation time mensure with low-field nuclear magnetic resonance analyzer,
Obtain relaxation time values Tblank；
Take the sample to be checked of above-mentioned mri contrast agent enrichment, carry out relaxation time mensure with low-field nuclear magnetic resonance analyzer,
Obtain relaxation time values Ttest；
(4) standard curve making and result draw
Calculate the relaxation time knots modification Δ T2, described Δ T2=T of master samplesample-Tblank, with Δ T2 as ordinate, to mark
Target rare cell content in quasi- sample is abscissa, is depicted as calibration curve；
Calculate the relaxation time knots modification Δ T2 of sample to be checkedtest, described Δ T2test=Ttest-Tblank, Δ T2testIt is not 0, then
Show to contain target rare cell in sample to be checked, draw target rare cell in sample to be checked according to above-mentioned standard curve simultaneously
Content step (2) used in mri contrast agent be paramagnetic nanoparticles magnetic bead or super-paramagnetism nano magnetic bead.
2. the low field NMR rare cell detection method based on magnetic micro-beads according to claim 1 is it is characterised in that institute
Stating paramagnetism or super-paramagnetism nano magnetic bead is anti-EpCAM immune nanometer magnetic bead, anti-CD45 immune nanometer magnetic bead, Streptavidin
One or more of nanometer magnetic bead, magnetic liposome of modified with folic acid.
3. the low field NMR rare cell detection method based on magnetic micro-beads according to claim 1 is it is characterised in that institute
State in step (2), before adding coupling to have the mri contrast agent of target rare cell specifically expressing antibody, under also including
State step：The mri contrast agent that coupling has anti-CD45 is added untargeted cells to be carried out magnetic mark, by externally-applied magnetic field
The untargeted cells of above-mentioned mark is removed by mode.
4. the low field NMR rare cell detection method based on magnetic micro-beads according to claim 1 is it is characterised in that institute
State in step (2), the method removing unreacted mri contrast agent is：Slowly siphon away supernatant, then apply additional magnetic
Field or stable centrifugation, the sample of enrichment mri contrast agent is washed with buffer solution.
5. the low field NMR rare cell detection method based on magnetic micro-beads according to claim 1 is it is characterised in that institute
State in step (3), the magnetic field intensity controlling low-field nuclear magnetic resonance analyzer is 20.18MHz, and magnet temperature is 35 DEG C, during repetition
Between be 5s.
6. the low field NMR rare cell detection method based on magnetic micro-beads according to claim 1 is it is characterised in that institute
State in step (4), calculate the relaxation time knots modification Δ T2 of the master sample and relaxation time knots modification Δ T2 of sample to be checkedtest
When, take the mean value in multiple measurement relaxation time to be calculated respectively.
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