CN104977284A - Capture and identification method for fetal nucleated red blood cells - Google Patents
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Abstract
The invention relates to a capture and identification method for fetal nucleated red blood cells. The method comprises the following steps: step 1, preparing a biotin-labeled antibody, which is used for recognizing one or a plurality of surface specific proteins of fetal nucleated red blood cells; step 2, bonding the antibody obtained in the step 1 with a chemically-modified nanometer substrate membrane through affinity, then adding a fetal nucleated red blood cell suspension and standing the obtained mixture in a constant-temperature cell incubator so as to allow the antibody on the nanometer substrate membrane to capture fetal nucleated red blood cells; and step 3, with fetal red blood cell hemoglobin antibody and fetal nucleated red blood cell membrane protein antibody respectively labeled by fluorescent dyes with different excitation wavelengths as double antibodies, identifying the fetal nucleated red blood cells captured in the step 2 by using double-positive fluorescent staining. The method provided by the invention has high sensitivity and specificity and is simple to operate.
Description
Technical field
The present invention relates to catching and authentication method of a kind of fetal nucleated red blood.
Background technology
Fetal nucleated red blood is a characteristic mark of fetal hemopoiesis system and fetus peripheral blood circulation, and it does not exist only in the blood circulation of fetus, and can enter maternal peripheral blood by placental barrier.Fetal nucleated red blood has various features: 1. do not have nucleated red blood cell in normal human peripheral blood, if occur, belongs to pathological phenomenon; 2. mononuclearcell is belonged to, containing fetus full gene group; 3. surface has more relatively special antigen composition as CD71, CD36 and glycophorin (GPA) etc.; 4. in gravid woman's peripheral blood, content is less, between 1: 105 ~ 1: 109; 5. life cycle is short, and about 90 day postpartum disappeared very soon in female blood, not by previous gestation effect during for diagnosing.The special nature of erythroblast, for the prediction of some pathological pregnancy and diagnosis provide new approaches, wherein, the fetal nucleated red blood examination fetal genetic disease in the female blood of antenatal utilization, has become the Non-invasive Prenatal Diagnosis technology that new development is in recent years got up.
In normal human blood, erythroblast and leucocyte belong to monocyte, and leucocyte diameter is about 7-20um, and erythroblast diameter is about 10-20um, and form is more or less the same, but leukocytic number is approximately 4-10 * 10
6/ ml, and the number of erythroblast is only several to hundreds of, quantitatively differ huge, therefore, how to realize high-level efficiency and highly purified sub-elect from millions of leucocytes required several to a hundreds of erythroblast, namely from blood, capturing erythroblast and identify it, is an important research topic.
At present, conventional cell sorting techniques is flow cytometry, but this technology use apparatus expensive, bulky, need professional to operate, and the large usage quantity of cell, is difficult in laboratory and hospital's penetration and promotion.The operation of Magnetic activated cell sorting method is relatively simple, and expense relative moderate, the used time is short, and its principle is under the effect of externally-applied magnetic field, by the cell separation of the labeling of monoclonal antibody in conjunction with magnetic particle out.The antibody used can be divided into positive selectable marker and negative selection marker, positive mark comprises some specific antigen markers of fetal nucleated red blood surface as TfR (transferrin receptor, CD71), glycophorin A (glycophorin A, GPA), the responsive plain acceptor (thromboxane receptor, CD36) of thrombus; Negative selection marker comprises CD45, CD14, CD32 etc.But the purity that the method is separated is not high, except the target cell needing to be separated, lymphocyte and some monocytes also may be activated, so the selection of antibody seems most important during sorting.Unicellular micromanipulation partition method identifies target cell from morphology under the microscope, and with micromanipulator by its one by one picking go out, then analyze.This technology separating purity is high, but technical difficulty is large, and instrument and equipment requires high, not easily promotes.
In addition, carry out qualification institute employing method to erythroblast to comprise: immuno-chemical method, immunofluorescence protein labeling method.Immuno-chemical method main detection nucleus and cytoplasmic ratio judge cell type, but during working expenditure and False Rate is higher; Immunofluorescence protein labeling method uses the antibody labeled cells with fluorophor, such as TfR CD71 or γ globin (gamma-hemoglobin-chain) etc., but antibody specificity is not high, and Detection results is bad; In addition pcr amplification carried out to Y chromosome distinguished sequence in male fetus cell in addition or carry out fluorescence in situ hybridization (FISH) with the probe of Y chromosome distinguished sequence, if detect, Y chromosome is thought of as the erythroblast of tire source property, and this method is simple but limit by sex.Therefore, this area method of catching in the urgent need to the erythroblast of a kind of novel high sensitivity, high specific and identifying.
Summary of the invention
In order to overcome the defect that prior art exists, the invention provides catching and authentication method of a kind of highly sensitive, high specificity, fetal nucleated red blood easy and simple to handle.
Particularly, said method comprising the steps of:
Step one: prepare biotin labeled antibody, described antibody is the antibody identifying one or more fetal nucleated red blood surface specific albumen;
Step 2: by affine for the step one gained antibody nano based counterdie after chemical modification, then add fetal nucleated red blood suspending liquid, leaves standstill, makes fetal nucleated red blood by the antibody capture on nano based counterdie in constant temperature cell culture incubator;
Step 3: the fetal erythrocyte hemoglobin antibodies marked respectively using the fluorescent dye that excitation wavelength is different and fetal nucleated red blood memebrane protein antibody, as double antibody, adopt " two positive " immunofluorescence staining to identify the fetal nucleated red blood of catching through step 2.
In step one of the present invention:
Described antibody is the antibody of fetal nucleated red blood surface specific albumen, described specific proteins is as antigen markers, comprise CD14, CD32, CD35, CD36, CD45, CD47, CD71, CD147, GPA or EPO-r, above-mentioned specific proteins except can except erythroblast is expressed, also has expression to a certain degree at the lymphocyte of activation, granulophilocyte, platelet surface.When using single antibody, the fetal nucleated red blood of sorting gained keeps higher degree, and described antibody is preferably CD147 antibody protein; Conbined usage CD36 and GPA or CD71 and GPA antibody protein can realize the concentration effect that erythroblast is caught.
Obtain after the antibody protein that described antibody can be bought by market conventionally combines with biotin; Or the antibody protein mother liquor after directly buying biotin labeling obtains after dilution, concrete steps are: get biotin labeled antibody protein mother liquor, use aseptic PBS by the concentration dilution 10 times of antibody, save backup under-20 DEG C of conditions.
In step 2 of the present invention:
Described nano based counterdie is raw material with nano particle, is prepared from a glass substrate, and described glass primary backing surface should be smooth, smooth, clean, enables nano particle at its surface filming.Concrete, the step of preparation nano based counterdie is: use butyl titanate in deionized water hydrolysis particle diameter is 300 ~ 500nm nano particle, by the nanoparticulate dispersed of synthesis in organic solvent, preferred organic solvent consist of 0.05g lauric acid, 0.2g ethyl cellulose, 10ml terpinol and 10ml ethanol, evenly be coated to clean glass surface, 450 ~ 550 DEG C of high annealings 10 ~ 20 minutes, be preferably 500 DEG C of high annealings 15 minutes, obtain the nano based counterdie that surfaceness is 40 ~ 100nm.Nano material provided by the invention catches substrate can strengthen the interaction with cell surface compound, and improve the effect of catching, this is the advantage that other fluorescent activation methods and immunomagnetic beads method catch not available for erythroblast.The present invention can reach the best capture effect of erythroblast by the roughness of the nano particle and control nanometer basement membrane surface that change preparation condition synthesis different-grain diameter, as most preferably scheme, nano particle diameter used in the present invention is 400nm, and the surfaceness of nano based counterdie is 85nm.
The chemical modification of described nano based counterdie specifically comprises the following steps: use washes of absolute alcohol nanometer basement membrane surface; Nano based counterdie being dipped into concentration is in 3-mercaptopropyi trimethoxy silane (MPTM) solution of 4% 1 ~ 1.5 hour, uses washes of absolute alcohol 3 times, re-uses dimethyl sulfoxide (DMSO) (DMSO) and clean 3 times; Nano based counterdie being dipped into concentration is in N-maleimide succinimide ester (GMBS) solution of 1mg/ml 45 ~ 60 minutes, uses DMSO to clean 3 times, re-uses phosphate buffer (PBS) and clean 3 times; In nano based counterdie surface coverage, the Streptavidin (SA) of 50ug/ml, spends the night 4 DEG C of placements, obtains the nano based counterdie after chemical modification.
Described antibody is affine to the method on nano based counterdie is standing, and time of repose is 1 ~ 3 hour, is preferably 2 hours.
The preparation method of described fetal nucleated red blood suspending liquid is conventional density－gradient centrifuga－tion method, and wherein, the density of parting liquid is preferably 1.097-1.099, and centrifugal force is preferably 400g.
The described time left standstill in constant temperature cell culture incubator is preferably 1 ~ 1.5 hour.
Described step 2 also comprises: wash fetal nucleated red blood cell at large on nano based counterdie off with PBS.
In step 3 of the present invention:
The fetal erythrocyte hemoglobin antibodies that " two positive " immunofluorescence staining adopts the different fluorescent dye of excitation wavelength to mark respectively and fetal nucleated red blood memebrane protein antibody are as double antibody, fetal nucleated red blood is identified, is labeled as the positive standard as fetal nucleated red blood qualification using double antibody simultaneously; In the memebrane protein of numerous fetal nucleated red bloods, be combined memebrane protein CD71 and cytoplasm protein ε-HbF and identify that fetal nucleated red blood specificity is higher.
Concrete, step 3 comprises the following steps: carry out pre-service to fetal nucleated red blood, fixes with 4% paraformaldehyde solution, and the set time is preferably 10 minutes; With the Triton X-100 solution punching of 0.1%, the punching time is preferably 10 minutes; Use confining liquid is closed, and is preferably 10 minutes off-period; Add the mixed liquor of CD71 antibody and the ε-HbF antibody marked respectively containing the fluorescent dye that excitation wavelength is different, the configuration proportion of described mixed liquor is ε-HbF antibody: CD71 antibody: confining liquid (w%)=0.5-1.5:0.5-1.5:6-10, the allocation ratio of preferred described mixed liquor is ε-HbF:CD71: confining liquid (w%)=1:1:8, keeps in Dark Place and spend the night at 4 DEG C; Working concentration is that the nucleus dyestuff DAPI of 0.1ug/ml redyes nucleus, and the time of redying is preferably 10 minutes; Be placed on fluorescence microscopy Microscopic observation by washed with de-ionized water, and use CCD shooting results.
The concentration preferably 0.09 ~ 0.11mol/L of phosphate buffer of the present invention.
The standard of perfection of fetal nucleated red blood of the present invention is: fetal nucleated red blood fluorescence results be anti-ε-HbF bright/anti-CD71 is bright/DAPI is bright, and anti-ε-HbF and DAPI fluorescent brightness form complementation, and cell size is 10-30um; Leukocytic fluorescence results be anti-ε-HbF do not work/anti-CD71 is slightly bright/DAPI is bright, cell size is less than 15um.
Compared with prior art, method provided by the invention has remarkable advantage, is mainly manifested in: the correlative study that (1) uses the realization of novel antibodies albumen to catch for novel antibodies albumen the identification of special cells is significant; (2) utilize novel antibodies to carry out specific recognition to fetal nucleated red blood to catch, effectively can improve the identification capture rate of target cell, the correlative study for fetal nucleated red blood is significant; (3) use " two positive " immunofluorescence albumen to carry out identification and analysis to fetal nucleated red blood, reliability and the sensitivity of discriminating can be improved, contribute to the correlative study of fetal nucleated red blood.
Accompanying drawing explanation
Fig. 1 shows the process flow diagram of the method for the invention.
Fig. 2 shows the pictorial diagram of nano based counterdie.
Fig. 3 shows the schematic diagram that novel antibodies is caught fetal nucleated red blood, and wherein, 100 is glass substrate, and 200 is nano particle, and the surface antigen of fetal nucleated red blood 300 is identified by specific antibody 201 catches.
Fig. 4 shows the light field design sketch that specific recognition is caught; Wherein 200 represent nano based counterdie, and 300 expressions capture suprabasil cell.
Fig. 5 shows the fluorescent effect figure that method utilizes " two positive " immunofluorescence protein staining qualification fetal nucleated red blood; Wherein a, e are haemoglobin fluorescence (redness), and b, f are transferrins fluorescence (green), and c, g are nucleus (blueness), and d, h are fluorescence composite diagram, and a, b, c all have fluorescence to be expressed as fetal nucleated red blood; And e, f do not work, only g has fluorescence to be expressed as leucocyte.
Fig. 6 shows the fluorescence composite diagram utilizing " two positive " immunofluorescence protein staining qualification fetal nucleated red blood according to the inventive method; According to " two positive " standard of perfection, wherein white circle acceptance of the bid is depicted as fetal nucleated red blood, and other bright spots are leucocyte.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
The present invention utilize novel antibodies albumen to catch for fetal nucleated red blood specific recognition and utilize " two positive " immunofluorescence staining to identify that the method for fetal nucleated red blood relates to parameter is more; therefore specific embodiment is only as the exemplary illustration to implementation of the present invention, and does not form limiting the scope of the invention.Below by using provided by the invention utilize novel antibodies albumen be used for fetal nucleated red blood specific recognition catch and utilize " two positive " immunofluorescence staining to identify the specific operation process of fetal nucleated red blood method is described further as embodiment.
Wherein, PBS represents phosphate buffer, and pH=7.4 is isotonic with blood of human body, and principal ingredient is potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and potassium chloride;
Biotin labeled CD147 antibody (biotinylatedanti-CD147) is bought from Sino Biological Inc company;
3-mercaptopropyi trimethoxy silane (MPTMS) and N-maleimide succinimide ester (GMBS) all available from Sigma, Streptavidin (SA) is purchased from Invitrogen company;
Confining liquid is formulated by 3% bovine serum albumin(BSA) (BSA)+5% sheep blood serum+0.1% Tween-20;
Red fluorescence ε-HbF antibody is the anti-ε-HbF antibody that phycoerythrin (PE) marks, 0.2mg/ml, purchased from BD Biosciences company, green fluorescence CD71 antibody is the anti-CD71 antibody that fluorescein isothiocynate (FITC) marks, purchased from BD Biosciences company.
embodiment 1: the preparation of nano based counterdie and modification
1) preparation of nano based counterdie: the nano particle of the butyl titanate of use hydrolysis particle diameter 400nm in deionized water, by the nanoparticulate dispersed of synthesis in the organic solvent be made up of 0.05g lauric acid, 0.2g ethyl cellulose, 10ml terpinol and 10ml ethanol, evenly be coated to clean glass surface, 500 DEG C of high annealings 15 minutes, obtain the nano based counterdie that surfaceness is 85nm; Gained nano based counterdie pictorial diagram as shown in Figure 2;
2) modification of nano based counterdie: the surface using washes of absolute alcohol step 1) gained nano based counterdie; Nano based counterdie being dipped into concentration is in the MPTMS solution of 4% 1 hour, uses washes of absolute alcohol 3 times, re-uses DMSO and clean 3 times; Nano based counterdie being dipped into concentration is in the GMBS solution of 1mg/ml 45 minutes, uses DMSO to clean 3 times, re-uses PBS and clean 3 times; In nano based counterdie surface coverage, 20ul concentration is the SA of 50ug/ml, and places 6 hours at 4 DEG C, obtains the nano based counterdie after chemical modification.
embodiment 2: the preparation of the suspending liquid containing fetal nucleated red blood
Get Cord blood, adopt Conventional density gradients centrifuge method to be separated, obtain the suspending liquid containing fetal nucleated red blood; Wherein, the density of parting liquid is 1.098, and centrifugal force is 400g.
embodiment 3: the catching and identifying of fetal nucleated red blood
1, the one embodiment of the present invention according to Fig. 1 utilizes novel antibodies albumen to catch for fetal nucleated red blood specific recognition and utilizes " two positive " immunofluorescence staining to identify the method for fetal nucleated red blood, and concrete steps are:
S1100: use aseptic PBS that biotin labeled CD147 antibody protein mother liquor is diluted to concentration for 100ug/ml, and at being kept at-20 DEG C with the centrifuge tube after sterilizing;
S1200: drip on the nano based counterdie after S1100 gained antibody 10ul to embodiment 1 gained modification, leave standstill 2 hours, wash unnecessary antibody with PBS; Add the suspending liquid that embodiment 2 gained contains fetal nucleated red blood, place 1 hour in 37 DEG C of constant temperature cell culture incubators, make fetal nucleated red blood by the antibody capture on nano based counterdie; Use PBS to clean 3 times gently, do not catch or the fetal nucleated red blood of non-specific adsorption to remove; The schematic diagram of catching described in this step as shown in Figure 3;
S1300: in the fetal nucleated red blood after S1200 gained is caught, adds 4% paraformaldehyde solution 1ml, leaves standstill after 10 minutes, cleans with PBS; Add the Triton X-100 solution 1ml of 0.1% again, leave standstill after 10 minutes, clean with PBS; Add confining liquid 1ml again, leave standstill 10 minutes; Add fluorescin antibody mixed liquor, consisting of of described mixed liquor: red fluorescence ε-HbF antibody 2ul+ green fluorescence CD71 antibody 2ul+ confining liquid 16ul, keep in Dark Place at 4 DEG C 8 hours; After washing away excess dyestuff with PBS, drip the DAPI 100ul that concentration is 0.1ug/ml, redye nucleus 10 minutes, by washed with de-ionized water, lucifuge natural drying; Be placed into observation of cell immunofluorescence dyeing result under fluorescent microscope, and analyze and record;
The standard of perfection of fetal nucleated red blood is: fetal nucleated red blood fluorescence results is bright (the green)/DAPI of bright (the redness)/anti-CD71 of anti-ε-HbF bright (blueness), and anti-ε-HbF and DAPI fluorescent brightness form complementation, cell size is 10-30um; Leukocytic fluorescence results be anti-ε-HbF do not work/anti-CD71 is slightly bright/DAPI is bright, cell size is less than 15um;
S1400: observed rear deionized water and ethanol rinse substrate, so far step S1400 terminates.
2, to catch and qualification result:
1) operate the microscope light field design sketch result of catching described in S1200 as shown in Figure 4, this figure can find out that the identification that novel antibodies effectively can realize fetal nucleated red blood is caught;
2) the fluorescent microscope design sketch identified described in operation S1300 as illustrated in Figures 5 and 6; Wherein, Fig. 5 illustrates the fluorogram of utilization " two positive " immunofluorescence staining qualification fetal nucleated red blood, fetal nucleated red blood and leukocytic difference can be found out, the synthesis fluorogram of utilization " two positive " immunofluorescence staining qualification fetal nucleated red blood is shown by Fig. 6, the efficient identification and the qualification that have the inventive method can realize fetal nucleated red blood can be found out.
The present invention will be described instead of limit the invention for above-described embodiment, and those skilled in the art can design alternative embodiment when not departing from claims scope.In the claims, any reference symbol between bracket should be configured to limitations on claims.Word " comprises " not to be got rid of existence and does not arrange element in the claims or step.
Although above with general explanation, embodiment and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. the catching and an authentication method of fetal nucleated red blood, is characterized in that, said method comprising the steps of:
Step one: prepare biotin labeled antibody, described antibody is the antibody identifying one or more fetal nucleated red blood surface specific albumen;
Step 2: by affine for the step one gained antibody nano based counterdie after chemical modification, then add fetal nucleated red blood suspending liquid, leaves standstill, makes fetal nucleated red blood by the antibody capture on nano based counterdie in constant temperature cell culture incubator;
Step 3: the fetal erythrocyte hemoglobin antibodies marked respectively using the fluorescent dye that excitation wavelength is different and fetal nucleated red blood memebrane protein antibody, as double antibody, adopt " two positive " immunofluorescence staining to identify the fetal nucleated red blood of catching through step 2.
2. method according to claim 1, is characterized in that, described in step one, fetal nucleated red blood surface specific albumen is CD147.
3. method according to claim 1, it is characterized in that, described in step 2, nano based counterdie is prepared from by following steps: the nano particle adopting tetrabutyl titanate hydrolysis synthesis particle diameter 300-500nm, then be distributed in organic solvent, evenly be coated in clean glass surface,, 450 ~ 550 DEG C of high annealings 10 ~ 20 minutes, obtain the nano based counterdie that surfaceness is 40 ~ 100nm.
4. method according to claim 3, is characterized in that, described nano particle diameter is 400nm, and the roughness of gained nano based counterdie is 85nm.
5. the method according to claim 1,3 or 4, is characterized in that, the step of chemical modification described in step 2 is: use washes of absolute alcohol nanometer basement membrane surface; Nano based counterdie being dipped into concentration is in the MPTM solution of 4% 1 ~ 1.5 hour, uses washes of absolute alcohol, re-uses DMSO cleaning; Nano based counterdie being dipped into concentration is in the GMBS solution of 1mg/ml 45 ~ 60 minutes, uses DMSO cleaning, re-uses PBS cleaning; The SA of 50ug/ml in nano based counterdie surface coverage, spends the night 4 DEG C of placements, obtains the nano based counterdie after chemical modification.
6. method according to claim 1, is characterized in that, the method that antibody described in step 2 is affine is standing, and described time of repose is 1 ~ 3 hour; Described time of repose is preferably 2 hours.
7. method according to claim 1, is characterized in that, the time left standstill in constant temperature cell culture incubator described in step 2 is 1 ~ 1.5 hour.
8. method according to claim 1, is characterized in that, double antibody described in step 3 is the antibody of CD71 and the antibody of ε-HbF.
9. the method according to claim 1 or 8, is characterized in that, step 3 comprises following concrete steps: in the fetal nucleated red blood after step 2 gained is caught, and adds 4% paraformaldehyde solution and fixes, clean with PBS; Add the Triton X-100 solution punching of 0.1% again, clean with PBS; Add confining liquid again to close; Add fluorescin antibody mixed liquor, described mixed liquor is configured by the composition of following weight ratio and forms: ε-HbF antibody: CD71 antibody: confining liquid=0.5-1.5:0.5-1.5:6-10, keeps in Dark Place and spend the night at 4 DEG C; With PBS cleaning, nucleus dyestuff DAPI is used to redye nucleus, by washed with de-ionized water, lucifuge natural drying; Be placed into observation of cell immunofluorescence dyeing result under fluorescent microscope, and analyze and record, ε-HbF antibody and CD71 antibody are all labeled as the positive and the cell that nuclear targeting is positive is fetal nucleated red blood.
10. method according to claim 9, is characterized in that, described mixed liquor is configured by the composition of following weight ratio and forms: red fluorescence ε-HbF antibody: green fluorescence CD71 antibody: confining liquid=1:1:8.
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| WO2020132906A1 (en) * | 2018-12-25 | 2020-07-02 | 深圳迈瑞生物医疗电子股份有限公司 | Platelet simulation particle and preparation method therefor and quality control material or calibration material containing same |
| CN112639467A (en) * | 2018-12-25 | 2021-04-09 | 深圳迈瑞生物医疗电子股份有限公司 | Platelet simulation particle, preparation method thereof and quality control substance or calibrator containing the simulation particle |
| CN112639467B (en) * | 2018-12-25 | 2024-04-09 | 深圳迈瑞生物医疗电子股份有限公司 | Platelet-like particles, method for producing the same, and quality control substance or calibrator containing the same |
| CN116731962A (en) * | 2023-08-14 | 2023-09-12 | 天津中新科炬生物制药股份有限公司 | Kit and method for separating red blood cells from whole blood |
| CN116731962B (en) * | 2023-08-14 | 2023-11-03 | 天津中新科炬生物制药股份有限公司 | Kit and method for separating red blood cells from whole blood |
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