CN104122285A - 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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- CN104122285A CN104122285A CN201410326420.9A CN201410326420A CN104122285A CN 104122285 A CN104122285 A CN 104122285A CN 201410326420 A CN201410326420 A CN 201410326420A CN 104122285 A CN104122285 A CN 104122285A
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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 the detection method of rare cell in a kind of biological fluid samples.
Background technology
Rare cell refers to some atypical cells in biological fluid samples (comprising blood, hydrothorax, ascites, urine, cerebrospinal fluid etc.), quantity research shows greatly, and the detection to rare cell and qualification have great importance for pathomechanism and the targeted drug exploitation of relevant disease.Therefore the rare cell detection method of, finding accurately and rapidly will become problem demanding prompt solution.But the concentration of rare cell in biological fluid is very low, with the ratio of non-target cell be approximately 1:10
7, cannot count with conventional art, thus in the urgent need to one simply quickly and accurately method solve this difficult problem.
Because the content of circulation rare cell in body fluid is little, could in follow-up experiment, identify qualification through effective enriching step.The method that is widely used in the market rare cell detection research mainly contains density－gradient centrifuga－tion method, membrane filter method and immunomagnetic isolation technology.
Density gradient zonal centrifugation method is called again zonal centrifugation, is sample is added in inertia gradient media and carries out centrifugal settling or sedimentation equilibrium, under certain centrifugal force, particle is assigned to gradient on some ad-hoc location, form the not separation method of same district band.Use this method can make the whole components of several work in sample separate simultaneously, there is good resolution.The advantage of this method is: 1. good separating effect, can once obtain purer particle; 2. wide accommodation, can separate as the differential centrifugation and have the poor particle of sedimentation coefficient, can separate again the poor particle of certain buoyant density; 3. particle can crimp, can keep seed activity, and prevent that established district band is because convection current causes mixing.The shortcoming of this method is: 1. centrifugation time is longer; 2. need to prepare inertia gradient media solution.In research, often utilize the principle of density gradient centrifugation, by the separation of Ficoll liquid and purifying human or animal PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC).
Thereby membrane filtering method is to be greater than granulocyte in peripheral blood according to some rare cell volume to make rare cell separation and concentration from peripheral blood out, then utilize immunofluorescence technique to carry out antidiastole to rare cell.In the method, membrane filtration cell enrichment process is relatively easy, granulocytic but not all rare cell volume is all greater than in peripheral blood, and the volume of a lot of rare cells and granulocyte size are similar, even less than granulocyte volume.Based on above understanding, membrane filtering method is abandoned gradually.
Summary of the invention
The object of the present invention is to provide a kind of can be easy, quick, the low-field nuclear magnetic resonance determination method of high specific and the rare cell that content is lower in detection of biological body fluid sample in high sensitivity.
For solving the problems of the technologies described above, the invention provides a kind of low NMR rare cell detection method based on magnetic micro-beads, comprise following steps:
(1) sample preparation
By the clone of the target rare cell of pure culture, carry out different gradient dilutions with damping fluid, make the rare cell suspending liquid master sample of series concentration;
Biological fluid samples to be measured is carried out to Ficoll density gradient centrifugation and obtain candidate's mixed cellularity group, pass through washing the high heart resuspendedly, make rare cell suspending liquid sample to be checked;
(2) the affine enrichment of mri contrast agent
Get above-mentioned master sample, add coupling to have the mri contrast agent of target rare cell specifically expressing antibody, make the rare cell generation immune response enrichment in mri contrast agent and master sample, then remove unreacted mri contrast agent, obtain the master sample of mri contrast agent enrichment;
Get above-mentioned sample to be checked, add coupling to have the mri contrast agent of target rare cell specifically expressing antibody, make the rare cell generation immune response enrichment in mri contrast agent and sample to be checked, then remove unreacted mri contrast agent, obtain the sample to be checked of mri contrast agent enrichment;
(3) NMR (Nuclear Magnetic Resonance) relaxation time detecting
Get the master sample of above-mentioned mri contrast agent enrichment, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
sample; Using damping fluid as blank, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
blank;
Get the sample to be checked of above-mentioned mri contrast agent enrichment, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
test;
(4) standard curve making and result draw
Calculate the relaxation time change amount Δ T2 of master sample, described Δ T2=T
sample-T
blank, taking Δ T2 as ordinate, taking the target rare cell content in master sample as horizontal ordinate, be depicted as typical curve;
Calculate the relaxation time change amount Δ T2 of sample to be checked
test, described Δ T2
test=T
test-T
blank, Δ T2
testbe not 0, show to contain target rare cell in sample to be checked, draw the content of target rare cell in sample to be checked simultaneously according to above-mentioned typical curve.
From above-mentioned detecting step: the present invention utilize mri contrast agent can with the characteristic of rare cell combination, use immunomagnetic isolation technology, under the effect of additional downfield by the hydrogen atom signal of hydrone in the inspection magnetic bead-cell suspending liquid rare cell that quantitatively circulates.
Specifically, first the present invention carries out the fast enriching of rare cell in sample pre-treatments; Then, based on specific antibody antigen immune reaction, utilize low-field nuclear magnetic resonance analyser to detect the variation in the relaxation time of the mri contrast agent that is bonded to rare cell surface, realize the qualitative and quantitative detection fast and delicately to rare cell.The variation that finally detects the relaxation time characterisitic parameter of evaluation method based on nuclear magnetic resonance technique of the target rare cell described in the present invention, described relaxation time characteristic, refers to spin-lattice relaxation time and spin spin relaxation time.
Preferably, in detection method of the present invention, the mri contrast agent using in step (2) is paramagnetism nanometer magnetic bead or super-paramagnetism nano magnetic bead, and described paramagnetism nanometer magnetic bead or super-paramagnetism nano magnetic bead are preferably one or more in the magnetic liposome of anti-EpCAM immune nanometer magnetic bead, anti-CD45 immune nanometer magnetic bead, Streptavidin nanometer magnetic bead, modified with folic acid.The high degree of specificity of distinctive solidified reagents advantage and immunological response is incorporated into one by above-mentioned immunomagnetic beads, catches and the object that separates rare target cell thereby realize rapidly from mixed cellularity group.Gained rare cell can be used for counting or other aspect research application.
Preferably, in detection method of the present invention, make mri contrast agent with the reaction conditions of the rare cell generation immune response enrichment in master sample, sample to be checked be respectively: after mixing, under 4～8 DEG C of low temperature, to hatch 10～15min.
Under above-mentioned reaction conditions, in mri contrast agent and sample, to carry out the background signal of antigen-antibody immune response generation minimum for rare cell, and accuracy is better.
Preferably, in detection method of the present invention, in described step (2), adding before coupling has the mri contrast agent of target rare cell specifically expressing antibody, also comprise the steps: that non-target cell is carried out magnetic mark by the mri contrast agent that adds coupling to have anti-CD45, removes the non-target cell of above-mentioned mark by externally-applied magnetic field mode.
Immunomagnetic beads enrichment method in the present invention, except the described mri contrast agent that directly adds coupling to have target rare cell specifically expressing antibody of step (2) in foregoing invention content, make outside mri contrast agent and rare cell generation target immunity (antigen-antibody reaction) enrichment (positive enrichment), also can adopt the method for the two enrichments of feminine gender+positive to reach the object of target cell magnetite gathering.Concrete operation method is: adding before coupling has the mri contrast agent of target rare cell specifically expressing antibody, first utilize mri contrast agent (magnetic micro-beads) that coupling has an anti-CD45 that non-target cell (being mainly more leucocyte of content in epithelial cell etc.) is carried out to magnetic mark, by externally-applied magnetic field mode, leucocyte is got rid of from the cell mass potpourri that contains target rare cell (the non-target cell of negative marker); Then, above-mentioned acquisition cell subsets potpourri, select suitable coupling to have the mri contrast agent (magnetic micro-beads) of target cell specifically expressing antibody to carry out immune response enrichment according to subgroup mark, thereby obtain the mri contrast agent (positive mark's target cell) that is marked with target cell.By the method for operating of the two enrichments of above-mentioned feminine gender+positive, can more accurately reach reliably the object of target cell magnetite gathering.
Preferably, in detection method of the present invention, in described step (2), mri contrast agent addition is preferably: in every 200 target rare cells, add 1 microlitre mri contrast agent, the method of removing unreacted mri contrast agent is: slowly siphon away supernatant, then apply externally-applied magnetic field or stablize centrifugally, the sample of enrichment mri contrast agent is washed with damping fluid.The mri contrast agent of this ratio not only can obtain rare cell in optimization ground, can also reduce because mri contrast agent adds the background enhanced too much causing, and impact detects effect.
Preferably, in detection method of the present invention, in described step (3), the magnetic field intensity of controlling low-field nuclear magnetic resonance analyser is 20～25MHz, and magnet temperature is 30～35 DEG C, repetition time 3～5s.More preferably, the magnetic field intensity of controlling low-field nuclear magnetic resonance analyser 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, it is more accurate to make to detect, and controllability is stronger, and is beneficial to comparison and the Quality Control of parallel laboratory test.
Preferably, in detection method of the present invention, in described step (4), calculate the relaxation time change amount Δ T2 of master sample and the relaxation time change amount Δ T2 of sample to be checked
testtime, get respectively and repeatedly measure the mean value in relaxation time and calculate, thereby can make testing result of the present invention more accurately and reliably.
From above-mentioned discussion, the present invention is by combining mri contrast agent (as super suitable immune micro-magnetic bead) enrichment and the detection of low-field nuclear magnetic resonance instrument, by the separation of rare cell together with qualification organic combination, thereby fast realize and from mixed cellularity group, catch and separate rare target cell.Any rare cell existing in utilizing in the present invention immunity magnetic micropearls selection and separating highly enriched and concentrated humoral sample, caught cell can be detected and is marked with the specific expressed antibody of rare cell, and be coated with by superparamagnetism microballon, can differentiate and count and in nuclear magnetic resonance analyser, distinguish clearly with contaminative non-target cell the rare cell of being caught making.Owing to thering is the extremely strong susceptibility that micro-rare cell can be detected in every 7.5ml body fluid, it is a kind of separation scheme with extremely strong susceptibility and specific wide spectrum.
The existing detection method for rare cell all relates to immunofluorescence technique, need carry out quantitatively the number of the rare cell that circulates in body fluid by fluorescent microscope microscopy, generally there is complicated operation, sensitivity is low, proving time is long, price is high, it is high that operating personnel are required, be difficult for the drawback (as operating personnel's single of iFISH detection method maturation only can operate 6 samples simultaneously, 2 days consuming time of whole process) repeating.Compared with prior art, the present invention has coupling the rare target cell technology of mri contrast agent enrichment of specifically expressing antibody to combine with the low-field nuclear magnetic resonance instrument with quick and high-sensitivity detection technology, thereby realize the object that fast, efficiently and with sensitivity detects rare cell in peripheral blood, tool has the following advantages: (1) can make target rare cell directly from biological fluid, separate, and has easy, feature fast; (2) compare with methods such as centrifugal, filtrations, the shearing force that rare cell is subject in the time of magnetic resolution is little, can avoid the inactivation of cell; (3) there is very high selectivity; (4) externally-applied magnetic field can not exert an influence to the motion of the ion in material liquid and ionic sample; (5) instrument and equipment is simple, and operation cost is low.Method of the present invention can realize the rare cell fast detecting of extensive sample, for all having great importance undoubtedly in industrialization or customer experience.The method, for being suggested first, has no relevant report at present, and the method is applicable to the various rare cells in biological fluid samples detection, qualification and the quantitative test of (comprising the rare epithelial cell of all circulations).
Brief description of the drawings
Fig. 1 is the process flow diagram of low NMR rare cell detection method of the present invention;
Fig. 2 is taking the relaxation time change amount Δ T2 of master sample as ordinate, taking the target rare cell content in master sample as the made typical curve of horizontal ordinate in embodiment 1.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.But, persons of ordinary skill in the art may appreciate that in the each embodiment of the present invention, in order to make reader understand the application better, many ins and outs are proposed.But, even without these ins and outs and the many variations based on following embodiment and amendment, also can realize the each claim of the application technical scheme required for protection.
Embodiment 1
(1) sample preparation
Sample preparation to be checked:
1.1 obtain anti-freezing blood sample (～10ml) from breast cancer model mouse, and described sample comprises suspects the mixed cellularity group that contains target rare cell.The sample gathering should be processed on the same day! The room temperature lucifuge retention time should not exceed 24 hours;
1.2Ficoll density－gradient centrifuga－tion method obtains candidate's mixed cellularity group
1) add appropriate cell layering liquid (Ficoll solution preparation forms) to aseptic centrifuge tube A bottom, then above-mentioned anticoagulation test specimens is done to suitably dilution rear (2:1) with PBS liquid, above tube wall is added in layering liquid gently, make both form an interface clearly.
2) 400 × g horizontal centrifugal 30min (it is even, steady that the increase of centrifuge speed and minimizing are wanted, to ensure to form interface clearly).
3) final visible three layers of liquid in centrifugal rear pipe, upper strata is yellow liquid, middle layer is transparency liquid, and bottom is that (red blood cell and granulocyte density are greater than layering liquid to brownish red deposition red blood cell, are deposited on the pipe end because red blood cell runs into Ficoll aggegation bunchiness money shape simultaneously.Blood platelet is suspended in blood plasma because density is little, have the mononuclearcell suitable with the liquid-tight degree of layering only intensive in the interface of plasma layer and layering liquid, be tunica albuginea shape), draw this confluent monolayer cells and pass and obtain the cell suspending liquid sample to be checked that can be used for next step immune micro-enrichment with magnetic bead through washing the high heart resuspended (300 × g10min washed twice) in B pipe.
Master sample preparation:
By pure culture mouse mastopathy cell, carry out different gradient dilutions (0,10,10 with PBS
2, 10
3, 10
4, 10
5, 10
6, 10
7cells/ml), thus obtain the rare cell suspending liquid master sample of series concentration;
(2) the affine enrichment of magnetic micro-beads immunity
Get respectively above-mentioned master sample and sample cell suspending liquid standard series to be checked in 96 porocyte culture plates, add respectively 100 μ L couplings to have the superparamagnetic immunomagnetic beads of rare cell specifically expressing antibody, in 4-8 DEG C of incubation reaction 15min, suitably can select gentle shaking table to carry out blending incubation, obtain serial Rare cell-Microbeads-Ab compound titer.In addition,, for preventing the non-specific binding of cell surface antibody, operation must be rapidly, cell need be placed on ice, required solution needs 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, and 35 DEG C of magnet temperatures, repetition time 5s, averages after each DATA REASONING 3 times.
The master sample relaxation time measures and standard curve making:
Get respectively the above-mentioned serial Rare cell-Microbeads-Ab compound titer of 180 μ L to nmr tube, measure sample to be tested T2 with low-field nuclear magnetic resonance detector, obtain relaxation time values T
sample; Using aseptic deionized water or damping fluid as blank, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
blank, taking relaxation time change amount (Δ T2) as ordinate, taking the content of rare cell in master sample as horizontal ordinate, be depicted as typical curve.Wherein, Δ T2 represents according to formula (1).
ΔT2＝T
sample-T
blank.......................................(1)
In formula: T
sample: series standard sample relaxation time three horizontal survey mean values; T
blank: negative sample relaxation time three horizontal survey mean values.
In master sample, circulate rare cell quantitatively and qualification result statistical form:
| Number of cells | 5 | 10 | 20 | 30 | 40 | 50 |
| ΔT2(ms) | 60 | 67 | 82 | 105 | 162 | 211 |
The typical curve of drawing according to the data in upper table as shown in Figure 2.
The relaxation time of sample to be tested is measured with result and draws:
Get the sample to be checked of magnetic micro-beads enrichment, measure identical method with the above-mentioned master sample relaxation time, it is carried out to relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
test, calculate the relaxation time change amount Δ T2 of sample to be checked
test, described Δ T2
test=T
test-T
blank, Δ T2
testbe not 0, show to contain in sample to be checked target rare cell.Described T
blankand T
testneed measure respectively three times and average.From above-mentioned typical curve, derive the content of rare cell in sample to be checked.
The relaxation time measurement result of sample to be checked is:
| ? | 1 | 2 | 3 | Mean value |
| T blank | 2721 | 2778 | 2579 | 2692.6 |
| T test | 2786 | 2851 | 2638 | 2758.3 |
| ΔT2 test | 69 | 78 | 58 | 68.3 |
By formula computing formula: y=49.353e^ (0.0284X), can be calculated rare cell number in testing sample and be: 11.46.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above are to realize specific embodiments of the invention, and in actual applications, can do various changes to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (10)
1. a low NMR rare cell detection method based on magnetic micro-beads, is characterized in that, comprises following steps:
(1) sample preparation
By the clone of the target rare cell of pure culture, carry out different gradient dilutions with damping fluid, make the rare cell suspending liquid master sample of series concentration;
Biological fluid samples to be measured is carried out to Ficoll density gradient centrifugation and obtains candidate's mixed cellularity group, pass through washing resuspended from the high heart, make rare cell suspending liquid sample to be checked;
(2) the affine enrichment of mri contrast agent
Get above-mentioned master sample, add coupling to have the mri contrast agent of target rare cell specifically expressing antibody, make the rare cell generation immune response enrichment in mri contrast agent and master sample, then remove unreacted mri contrast agent, obtain the master sample of mri contrast agent enrichment;
Get above-mentioned sample to be checked, add coupling to have the mri contrast agent of target rare cell specifically expressing antibody, make the rare cell generation immune response enrichment in mri contrast agent and sample to be checked, then remove unreacted mri contrast agent, obtain the sample to be checked of mri contrast agent enrichment;
(3) NMR (Nuclear Magnetic Resonance) relaxation time detecting
Get the master sample of above-mentioned mri contrast agent enrichment, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
sample; Using damping fluid as blank, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
blank;
Get the sample to be checked of above-mentioned mri contrast agent enrichment, carry out relaxation time mensuration with low-field nuclear magnetic resonance analyser, obtain relaxation time values T
test;
(4) standard curve making and result draw
Calculate the relaxation time change amount Δ T2 of master sample, described Δ T2=T
sample-T
blank, taking Δ T2 as ordinate, taking the target rare cell content in master sample as horizontal ordinate, be depicted as typical curve;
Calculate the relaxation time change amount Δ T2 of sample to be checked
test, described Δ T2
test=T
test-T
blank, Δ T2
testbe not 0, show to contain target rare cell in sample to be checked, draw the content of target rare cell in sample to be checked simultaneously according to above-mentioned typical curve.
2. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, is characterized in that, the mri contrast agent using in step (2) is preferably paramagnetism nanometer magnetic bead or super-paramagnetism nano magnetic bead.
3. low the NMR rare cell detection method based on magnetic micro-beads according to claim 2, it is characterized in that, described paramagnetism or super-paramagnetism nano magnetic bead are preferably one or more in the magnetic liposome of anti-EpCAM immune nanometer magnetic bead, anti-CD45 immune nanometer magnetic bead, Streptavidin nanometer magnetic bead, modified with folic acid.
4. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, it is characterized in that, in described step (2), make mri contrast agent with the reaction conditions of the rare cell generation immune response enrichment in master sample, sample to be checked be respectively: after mixing, at 4～8 DEG C of temperature, to hatch 10～15min.
5. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, it is characterized in that, in described step (2), adding before coupling has the mri contrast agent of target rare cell specifically expressing antibody, also comprise the steps: that non-target cell is carried out magnetic mark by the mri contrast agent that adds coupling to have anti-CD45, removes the non-target cell of above-mentioned mark by externally-applied magnetic field mode.
6. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, it is characterized in that, in described step (2), the method of removing unreacted mri contrast agent is: slowly siphon away supernatant, then apply externally-applied magnetic field or stablize centrifugally, the sample of enrichment mri contrast agent is washed with damping fluid.
7. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, it is characterized in that, in described step (2), mri contrast agent addition is preferably: in every 200 target rare cells, add 1 microlitre mri contrast agent.
8. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, it is characterized in that, in described step (3), the magnetic field intensity of controlling low-field nuclear magnetic resonance analyser is 20～25MHz, magnet temperature is 30～35 DEG C, repetition time 3～5s.
9. low the NMR rare cell detection method based on magnetic micro-beads according to claim 8, it is characterized in that, in described step (3), the magnetic field intensity of controlling low-field nuclear magnetic resonance analyser is preferably 20.18MHz, magnet temperature is preferably 35 DEG C, and the repetition time is preferably 5s.
10. low the NMR rare cell detection method based on magnetic micro-beads according to claim 1, is characterized in that, in described step (4), calculates the relaxation time change amount Δ T2 of master sample and the relaxation time change amount Δ T2 of sample to be checked
testtime, get respectively and repeatedly measure the mean value in relaxation time and calculate.
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| CN112611868A (en) * | 2020-12-18 | 2021-04-06 | 厦门大学 | Probe for detecting novel coronavirus by magnetic resonance analysis method and preparation method thereof |
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