CN106932579A - A kind of kit of the liver cancer detection based on liquid biopsy - Google Patents
A kind of kit of the liver cancer detection based on liquid biopsy Download PDFInfo
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Abstract
The invention provides a kind of kit of the liver cancer detection based on liquid biopsy, including：Dyeing for strengthening Color strengthens liquid and the specific antibody with fluorescent staining mark；Wherein described specific antibody includes：ASGPR1 antibody, CD45 antibody and CPS1 antibody；The dyeing enhancing liquid includes surfactant that concentration is 0.001~1mg/mL.Kit of the invention can effectively be enriched with target cell, and confirm whether the target cell of enrichment derives from the early stage patient of liver cancer.Meanwhile, the present invention ensures the accuracy for detecting by double knurl marks detection increase nodule detection sensitivities and further by the detection of CEP8.Additionally, the present invention strengthens Color by dyeing enhancing liquid, various antibody with fluorescent staining mark is combined with target cell, target cell is dyeed, more preferably, fluorescence is stronger, and sharpness of border for Color.
Description
Technical field
The present invention relates to liquid biopsy field, the kit of specifically a kind of liver cancer detection based on liquid biopsy.
Background technology
Used as a kind of common cancer, compared with other cancers, its incidence of disease in worldwide occupies the 5th to liver cancer,
And the death rate occupies the 3rd.In China, the morbidity and mortality of liver cancer are higher than world standard.According to statistics, per annual
Every 100,000 population just has nearly 30 people to be diagnosed as liver cancer patient, and just has 26 people to die from liver cancer in every 100,000 population of every annual,
It is only below the lung cancer death number for ranking the first.Thus, for the detection in the early screening and disease progression of liver cancer
Just it is particularly important.In this regard, the method based on circulating tumor cell (CTC) detection has numerous traditional detection methods
Unexistent advantage, including noninvasive, quick and suitable for early screening etc..
CTC refers to spontaneous or discharges into the tumour of Peripheral Circulation by solid tumor primary tumor or MET because of operation of diagnosis and treatment
Cell.Due to transfer be cancer related mortality main cause, and CTC as transfer seed, in new knubble biological flag
The discovery of thing, tumor prognosis judge and individualized treatment aspect has very big application potential, are the heat of domestic and international tumor research
One of point.According to statistics, 40% liver cancer patient will be recurred in 1 year after the treatment, and the positive rate of CTC in blood is pointed out in research
It is related to the prognosis recurrence of liver cancer and low life cycle.So, the death rate higher and risk of recurrence just more highlight CTC morning
The importance of phase detection.
Up to the present, the detection for liver cancer CTC depends on the CTC captures of EpCAM, enrichment analysis platform, base
About 40% can be reached in this CTC positive rate.Although the CTC capture enrichments based on EpCAM are still by as CTC in liver cancer
The goldstandard of detection, but the detection for having the CTC with EpCAM as biomarker for generation EMT still suffers from false negative very high.
Additionally, the epithelium biomarker of EpCAM this wide spectrum is not suitable for the detection of tumour early stage not only, nor with group
Knit specificity, it is impossible to review the specific tissue-derived of CTC, and this point is significant for follow-up oncotherapy.Cause
This, finds and the biomarker using some CTC tissue specificities carries out the early screening of CTC and detection and analysis seems particularly
It is important.
According to display has been studied, there are numerous biomarkers such as including EpCAM, CK family protein, MUC1 to be used for liver cancer
The detection and analysis of CTC, wherein using asialoglycoprotein-receptors 1 (ASGPR1) and carbamyl phosphate synthetase 1 (CPS1) as
Mark not only has CTC positive rates higher, but also with liver tissue-specific very high.There is research to be based on
ASGPR1- magnetic beads are used for the capture of liver cancer CTC, the CTC that as a result there is ASGPR1+ in liver cancer patient of the display higher than 80%, connection
After closing CPS1, its CTC positive detections rate can reach 91%.And CPS1 is directed to, the CTC positive rates for combining CK are
89%, and it also has specificity higher in hepatic tissue.
Accordingly, biomarkers of the ASGPR1 and CPS1 as liver cancer CTC early screenings is combined, not only can be by detection
Sensitivity is maximized, but also can accordingly judge that CTC's in early screening sample is specific tissue-derived.
The day for announcing is on January 15th, 2014, and notification number passes through to disclose one kind in the Chinese patent of CN102313813B
The method that immunofluorescence dyeing is detected to rare cell, the method carries out three colors to leucocyte, rare cell and nucleus
Dyeing, then by fluoroscopic examination.But the method is only capable of by a kind of monoclonal antibody come the specific recognition rare cell, is easily caused
Missing inspection.Simultaneously as the heterogeneity of cell, antigen (antibody) amount of each cell expression differs, and causes fluorescence intensity sometimes non-
It is often faint, it is difficult to differentiate under fluorescence microscope.
The content of the invention
The deficiency that the main object of the present invention is directed to prior art presence provides a kind of liver cancer inspection based on liquid biopsy
The kit of survey, the quantity of CTC in peripheral blood can be detected using the kit, judge that whether the CTC, from liver cancer, is distinguished
The type of the early liver cancer CTC of detection, and testing result is accurate, and error is small.
In one aspect of the invention, the present invention is achieved through the following technical solutions：
A kind of kit of the liver cancer detection based on liquid biopsy, the kit includes：For strengthening Color
Dyeing enhancing liquid and the specific antibody with fluorescent staining mark；
Wherein described specific antibody includes：ASGPR1 antibody, CD45 antibody and CPS1 bodies；
The dyeing enhancing liquid includes surfactant that concentration is 0.001~1mg/mL.
The solvent of the dyeing enhancing liquid is biological buffer.
Preferably, the surfactant can be Triton X-100, dimethyl sulfoxide (DMSO), NP-40, dodecyl sulphate
Any one in sodium (SDS).It is furthermore preferred that the surfactant is Triton X-100 or SDS.Most preferably, the dye
Color enhancing liquid includes the SDS that concentration is 0.01~1mg/mL, and the solvent of dyeing enhancing liquid is conventional biological buffer, such as PBS
Buffer solution etc..
Preferably, lymphocyte separation medium and the immunomagnetic beads for removing leucocyte are also included in the kit.Drench
Bar cell separation liquid and immunomagnetic beads can be used in removing the interference of red blood cell and leucocyte, be easy to preferably be enriched with CTC.
It is furthermore preferred that the immunomagnetic beads for removing leucocyte is surface coupling antibody CD45, CD14 and CD15
Immunomagnetic beads in one or more, most preferably including whole above-mentioned immunomagnetic beadses.
Preferably, the fluorescent staining mark of the specific ASGPR1 antibody, CD45 antibody and CPS1 antibody has respective
Different launch wavelengths.According to well known to a person skilled in the art in order to distinguish different specific antibodies, the present invention should be adopted
Marked with different fluorescent stainings.Labeling dye can be using any commonly employed fluorescent dye in this area, as long as being swashed by regulation
Emission wavelength can be distinguished.Because the launch wavelength of fluorescent staining mark is each different, therefore by fluorescence microscope not
Can be distinguished completely under same optical filter.Present invention preferably employs fluorescent staining labeled as Alexa Fluor Series Molecules,
Cyanine dye.More preferably Alexa594, CY5 and Alexa488, wherein Alexa594 launch wavelengths are 618nm, CY5 transmitted waves
A length of 670nm, Alexa488 launch wavelength are 519nm.CD45 antibody wherein with fluorescent staining mark is preferably CD45-
Alexa594 (red), the ASGPR1 antibody with fluorescent staining mark can be ASGPR1-Alexa488 (green) or ASGPR1-
(naked eyes are not for CPS1-CY5 for CY5 (naked eyes are invisible, and micro- scarnning mirror is assigned to purple), the CPS1 antibody with fluorescent staining mark
It can be seen that, micro- scarnning mirror is assigned to purple) or CPS1-Alexa488 (green).
The fluorescence probe for FISH can be the probe for chromosome fluorescence in-situ hybridization, such as
CEP8 fluorescence probes.
Preferably, also include accounting for the poly- second two of dyeing enhancing liquid weight/mass percentage composition 0.1~3% in the dyeing enhancing liquid
Alcohol.Polyethylene glycol coordinates the Color that can further strengthen dyeing liquor with surfactant.
As well known to a person skilled in the art, by the specific antibody marked with fluorescent staining and cell be incubated from
And the process for carrying out fluorescent staining can be liquid dyeing or solid dyeing.To first need to carry out carefully if carrying out liquid and dyeing
The cell of born of the same parents' dyeing is resuspended into cell suspension, dyeing pretreatment, cell dyeing is then carried out successively, then cell is transferred into load glass
Carried out on piece cell fix, slide mounting.And if carrying out solid dyeing on slide, then first by cell on slide
Carry out cell to fix, after then carrying out dyeing pretreatment, cell dyeing successively, then to slide mounting.
Above-mentioned cell is fixed and carried out by cell fixer, and the cell fixer can be commonly used in the art thin
The combination of one or more in born of the same parents' fixer, such as paraformaldehyde, glutaraldehyde, formalin, ethanol, acetone.
A kind of method that kit detected using the above-mentioned liver cancer based on liquid biopsy carries out CTC detections includes following step
Suddenly：
Target cell in enrichment peripheral blood；
Enhancing dyeing pretreatment is carried out to target cell using dyeing enhancing liquid；
Fluorescent staining is carried out to target cell using the specific antibody marked with fluorescent staining；
Target cell is fixed；
FISH (FISH) is carried out with fluorescence probe；
DAPI mountings and microscopy.
By the target cell after enhancing dyeing pretreatment, the combination of specific antibody and target cell is more preferable, fluorescence
Dyeing becomes apparent from, cell membrane sharpness of border.
As well known to a person skilled in the art, when tumor cell specific antigen is present in intracellular, should be to thin
After after birth padding and fixed target cell, then by cell membrane rupture of membranes and using the specific antibody pair with fluorescent staining mark
Target cell carries out intracellular Fluorescence dyeing.
Preferably, the target cell in the enrichment peripheral blood is comprised the following steps：
(1) by peripheral blood centrifugal separation plasma and haemocyte, and blood plasma is removed；
(2) to Cell Buffer and lymphocyte separation medium centrifugation layering is added in step (1), red blood cell layer is removed；
(3) to the immunomagnetic beads that is added in step (2) for removing leucocyte and incubation obtains suspension；
(4) the suspension Magneto separate in step (3) is removed into leucocyte and remaining red blood cell, then obtains richness after washing
Target cell after collection.
Described lymphocyte separation medium can be lymphocyte separation medium commonly used in the art, for according to Density Separation
Red blood cell, currently preferred use Ficoll separating liquids.
Enrichment of the present invention to target cell includes removing red blood cell twice, can be more clean to red blood cell removal, and
And by immunomagnetic beads, leucocyte and remaining red blood cell can be disposably removed, bioaccumulation efficiency is higher.Processed by above-mentioned enrichment
Interference of other cells for fluorescent staining can be excluded afterwards, be conducive to preferably being observed by fluorescence microscope.
Preferably, it is described using dyeing enhancing liquid target cell is carried out the process time of enhancing dyeing pretreatment for 5~
20 minutes.The final concentration of 0.2 μ g/mL~1mg/mL of surfactant after dyeing enhancing liquid addition.Wherein, dyeing enhancing liquid is excellent
The addition of choosing is 1~20 μ L.When the addition of dyeing enhancing liquid is more, the intensity for strengthening fluorescence is higher, but may
Cell membrane is caused to damage, stiffening effect is not again too obvious when addition is very few.
Preferably, it is described when carrying out fluorescent staining to target cell using the specific antibody marked with fluorescent staining, first
The specific antibody that will be marked with fluorescent staining is according to 1:The volume ratio of (100~200) is diluted with buffer solution, then by after dilution
Specific antibody mixes incubation with target cell.
Preferably, final concentration of 2~10 μ of specific antibody after the specific antibody with fluorescent staining mark is added
g/mL.Wherein, the specific antibody with fluorescent staining mark is according to 1:200 volume ratio is diluted with buffer solution.What is added is special
Property AC it is higher, more easily cause unspecific staining, influence the effect of fluorescence.
Above-mentioned buffer solution can be general biological cell buffer solution, such as PBS.It is furthermore preferred that PBS
It is the phosphate buffer of 0.01M, wherein Na2HPO4、NaCl、KH2PO4With the concentration of KCl be respectively 8mM, 136mM, 2mM and
2.6mM, pH value is 7.2-7.4.
Inventive principle
ASGPR1 is a kind of transmembrane receptor protein, and it passes through endocytosis and lyase body function utilizes the exposed end of glycoprotein
End galactolipin or N- acetyl galactose amine degradation glycoprotein, it is vital so as to be played in the regulation of seroglycoid stable state
Effect.Research shows that ASGPR may promote the liver including the various viruses including hepatitis type B virus to infect, while being also liver
One Effective target site of dirty specific drug conveying, this also demonstrates specificity of the ASGPR1 in liver expression from side, is liver
Tracing to the source for cancer provides the foundation.There is expression high in liver cancer tissue and normal liver tissue there are some researches show ASGPR1, it is positive
Rate is respectively 75% and 88%.More there is research to combine CPS1 by ASGPR1 to detect CTCs in liver cancer patient blood, examine
The sensitivity of survey is up to 91%.Wherein, CPS1 is the rate-limiting enzyme in a kind of urea cycle special positioned at mitochondrial liver, its
Played an important role during unnecessary urea in excluding cell.For the detection of liver cancer, it has hepatic tissue higher
Specificity, can strengthen knurl mark and trace to the source ability, and also have for CTCs after its joint ASGPR1 or pan-cytokeratin
Detection sensitivity higher.
The various antibody with fluorescent staining mark of selection are conducive to confirming the particular type of target cell, but when antibody kind
When class is more than one, the combination of target cell and antibody is easy for being disturbed, and causes luciferase expression not good, influences fluorescence microscope
Observation.And the Membrane protein antigen determinant that dyeing enhancing liquid of the invention passes through exposed cell surface, mark band fluorescent staining
Antibody be easier to combine on the cell membrane of cell surface, so as to strengthen the fluorescence intensity of mark cell membrane, and make cell
Membrane boundary is clear.
Therefore, it can with the CD45 antibody marked with Alexa594 fluorescent stainings to leukocyte surface specific stain, use
The ASGPR1 antibody and CPS1 antibody of mark Alexa488 and CY5 carry out specific stain to the CTC for capturing respectively.By fluorescence
Micro- sem observation, Alexa594 launching lights are 618nm feux rouges when regulation excitation wavelength is 591nm；When excitation wavelength is 650nm
CY5 launching lights are 670nm far-red lights (naked eyes are invisible, and micro- scarnning mirror is assigned to purple)；When excitation wavelength is 499nm
Alexa488 launching lights are 519nm green glows.So by adjusting different excitation wavelength and the time for exposure, it can be observed that no
With the different fluorescence marked on cell, and then target cell can be made a distinction：CD45+ is leucocyte；ASGPR1+ or CPS1+
It is tumour CTC cells, wherein ASGPR1+/CPS1+ can determine that patient of the target cell from liver cancer.
No. 8 chromosome abnormalities are present in various solid tumors, including prostate cancer, oophoroma, kidney, breast cancer, liver cancer
Deng, using FISH (FISH) method detection cell or tissue in No. 8 chromosome abnormalities situation it is extensive
For in clinical diagnosis, the present invention to be aided with No. 8 X chromosome centrics domain (CEP8) while based on tumor markers biopsy
FISH detection checkings, inspection is further ensured by the detection of CEP8 while the detection of double knurl marks increases nodule detection sensitivity
The specificity of survey.Wherein, CEP8 probes are the fluorescence probe for being marked with SpectrumOrange, for selecting 8 in whole cells
The cell of number numerical abnormalities of chromosomes is target cell.By fluorescence microscope, during a length of 559nm of regulation excitation light wave
SpectrumOrange launching lights are 588nm orange lights, can be carried out simultaneously with above-mentioned tumor markers and leucocyte mark fluorescence
Detection, the detection counted out by specific orange light probe determines No. 8 numbers of chromosome of target cell.The present invention is based on swollen
While tumor markers biopsy, the FISH for the being aided with CEP8 detection checkings that can be selected increase CTC detection spirits in the detection of double knurl marks
Detection while sensitivity further by CEP8 ensures the specificity of detection.
The kit of the liver cancer detection based on liquid biopsy of the invention, can effectively be enriched with target cell, and really
Whether the target cell for recognizing enrichment derives from the early stage patient of liver cancer.Meanwhile, the present invention detects that increasing tumour examines by double knurl marks
Survey sensitivity and the accuracy for further being detected by the detection guarantee of CEP8.Additionally, the present invention is added by dyeing enhancing liquid
Strong Color, makes various antibody with fluorescent staining mark to be combined with target cell, target cell is dyeed, dyeing
More preferably, fluorescence is stronger, and sharpness of border for effect.Can so strengthen simultaneously the fluorescent staining of blood borne cell surface specific and
The fluorescent staining effect of tumor cell specific mark so that CTC is easier to distinguish with blood borne cell, reduces the vacation of detection
The positive, improves the specificity of detection.Also, the inventive method is simple, low cost, with very strong practicality.
Brief description of the drawings
Fig. 1 is the fluorescent staining detection Merge figures of Hep3B Bel7402s；
Fig. 2 is the fluorescent staining detection Merge figures after tumour cell capture.
Specific embodiment
By the following specific examples further illustrate the invention：The experiment of unreceipted actual conditions in the following example
Method, conventionally and condition, or selects according to catalogue.
Ficoll-Paque PLUS of the lymphocyte separation medium from GE healthcare companies.
CD45 immunomagnetic beadses are from Thermo Fisher Scientific's CD45。
Alexa 594, Alexa 488 and CY5 fluorescent dyes come from Thermo Fisher Scientific.
CEP 8 SpectrumOrange DNA Probe Kit of the CEP8 probes from Abbott.
Target cell in the enrichment peripheral blood of embodiment 1
(1) peripheral blood is centrifuged to remove plasma protein：By 8.5mL peripheral bloods in horizontal centrifuge 800g, normal temperature from
Heart 7min, supernatant discarded.
(2) to the PBS and the lymphocyte separation medium of 3mL that 5~6mL is added in the blood plasma of step (1), in centrifugation
450g in machine, normal temperature centrifugation 7min.It is divided into three layers after centrifugation, red bottom is red blood cell layer, the intermediate layer master of whitish
To be leucocyte and CTC etc., the upper strata of yellow is blood plasma, draws whole liquid more than red blood cell layer, remove the red thin of bottom
Born of the same parents' layer.
(3) there is the immunomagnetic beads of CD45 antibody to being added dropwise over the coupling of 200mL surfaces in step (2), on horizontal shaker
Incubation obtains suspension, and horizontal shaker rotating speed is arranged on 120~150rpm, inclines 20~30 °, normal temperature, level shake 20min.
(4) suspension in step (3) is adsorbed into 2min with big magnetic frame.Careful absorption upper strata and middle level liquid, go
Except leucocyte and remaining red blood cell, with PBS wash and it is resuspended after be enriched with after target cell.
Target cell after 2 pairs of enrichments of embodiment carries out fluorescent staining
(1) enhancing dyeing pretreatment：Enhancing liquid, normal temperature are dyeed to 2 μ L are added in the target cell after the enrichment of about 50 μ L
Stand 10min.The dyeing enhancing liquid is the PBS solution of SDS or Triton X-100, and SDS concentration is 0.1mg/mL.
(2) cell surface dyeing：Each 1 μ L of CD45-Alexa 594 and ASGPR1-Alexa 488 are delayed with the PBS of 198 μ L
After fliud flushing dilution, it is added in the cell suspension after the completion of step (1) pretreatment, then lucifuge is incubated 20min.Add PBS after incubation
Buffer solution for cleaning cellular liquid, 950g centrifugation 4min, removes supernatant to 100 μ L.
(3) cell is fixed：Cell transfer in step (2) is applied on slide, fixer poly first is subsequently adding
Aldehyde, fixed 10min, PBS washings slide 2 times, each 5min.
(4) cell rupture of membranes：Cell compartment is added dropwise the μ L of cell rupture of membranes liquid 200 on slide after cell fixation, is incubated 10min
Afterwards plus PBS cleaning slide 2 times, each 5min.The cell rupture of membranes liquid is molten for the PBS of Triton X-100
Liquid, Triton X-100 concentration is 0.5%.
(5) cell intracellular mark dyeing：After CPS1-CY51 μ L are diluted with the PBS of 199 μ L, it is added to
Cell compartment on slide is stated, then lucifuge is incubated 20min, then adds PBS cleaning slide 2 times, each 5min.
(6) FISH：To the CEP8 fluorescence probes that 10 μ L are added dropwise on slide, covered, surrounding is sealed up
Piece glue mounting.The prehybridization 10min at 76 DEG C, then hybridizes 4h at 37 DEG C.
(7) DAPI mountings and microscopy：Tear mounting glue off, cleaned with PBS 2 times (5min/ times), and slough lid glass
Piece, 10 μ L mountants mountings is added after natural drying and (wherein the content of mountant is DAPI to covered：Glycerine=1:
9), finally with fluorescence microscope, microscopy condition is：Alexa594 launching lights are that 618nm is red during excitation light wave a length of 591nm
Light, time for exposure 100ms；CY5 launching lights are 670nm far-red lights (invisible, the microscope of naked eyes during excitation light wave a length of 650nm
Scanning is assigned to purple), time for exposure 100ms；Alexa488 launching lights are 519nm green glows, exposure during excitation light wave a length of 499nm
Time 100ms；DAPI launching lights are 455nm blue lights, 10~20ms of time for exposure during excitation light wave a length of 345nm；Excitation light wave
SpectrumOrange launching lights are 588nm orange lights during a length of 559nm, and as a result time for exposure 100ms shows, Normal human peripheral
Blood system from leucocyte it is most CD45 positive stainings are presented, knurl mark negative staining, FISH results are CEP8 dliploids, are not had
The cell of the knurl mark positive or CEP8 numerical abnormalities.
The fluorescent staining detection of embodiment 3Hep3B Bel7402s
By Hep3B human liver cancer cells (source Chinese Academy of Sciences cell bank is bought), 10 are taken after enzymolysis, digestion5Individual cell, greatly
About 50 μ L, carry out cell fluorescence dyeing and fluorescence microscopy the step of according to embodiment 2.Microscopy condition is：Excitation light wave
Alexa594 launching lights are 618nm feux rouges, time for exposure 100ms during a length of 591nm；CY5 transmittings during excitation light wave a length of 650nm
Light is 670nm far-red lights (naked eyes are invisible, and micro- scarnning mirror is assigned to purple), time for exposure 100ms；Excitation light wave is a length of
Alexa488 launching lights are 519nm green glows, time for exposure 100ms during 499nm；DAPI launching lights during excitation light wave a length of 345nm
It is 455nm blue lights, 10~20ms of time for exposure, as a result as shown in Figure 1.
Result shows that Hep3B human liver cancer cells show that blue light, for nucleus, illustrates there is cell under 358nm exciting lights；
Show that green explanation ASGPR1 is positive under 499nm exciting lights；Show purple explanation CPS1 in sun under 650nm exciting lights
Property；And do not show red under 591nm exciting lights, and illustrate that CD45 is negative, selected SMMC-7721 knurl mark positive expression is illustrated,
And negative sense screening can be carried out by CD45.
The tumour cell capture rate of embodiment 4 is detected
Collection healthy volunteer's 8.5ml blood samples, add 27 Hep3B human liver cancer cells.Then according to embodiment 1 the step of
Tumor cell enrichment is carried out, fluorescent staining and the fluorescence microscopy of tumour cell are carried out the step of according still further to embodiment 2.Mirror
Inspection condition is：Alexa594 launching lights are 618nm feux rouges, time for exposure 100ms during excitation light wave a length of 591nm；Excitation light wave
CY5 launching lights are 670nm far-red lights (naked eyes are invisible, and micro- scarnning mirror is assigned to purple), time for exposure during a length of 650nm
100ms；Alexa488 launching lights are 519nm green glows, time for exposure 100ms during excitation light wave a length of 499nm；Excitation light wave is a length of
DAPI launching lights are 455nm blue lights, 10~20ms of time for exposure during 345nm；During a length of 559nm of excitation light wave
SpectrumOrange launching lights be 588nm orange lights, time for exposure 100ms, as a result as shown in Figure 2.
Result shows that tumour cell (ASGPR1+ or CPS1+) captures number 20, capture rate 74.07%.ASGPR1 sun
Property (ASGPR1+) cell number 20, positive rate 74.07%；The CPS1 positive (CPS1+) cell numbers 13, positive rate
48.15%；Double positive tumor cells number (ASGPR1+/CPS1+) 13 of knurl mark, positive rate 48.15%；The non-dliploids of CEP8
(CEP8 ≠ 2) tumor cell number 10, non-dliploid rate 37.04%.
It can be seen from the results above that using kit of the invention carry out based on liquid biopsy CTC detection relative to
For existing single knurl mark detection, the tumour cell sensitivity in capture blood is higher.Simultaneously according to the detection of CEP8, Ke Yizeng
Plus the specificity of detection, meet different testing goals.
Embodiment described above is a kind of preferably scheme of the invention, not makees any formal to the present invention
Limitation, for those skilled in the art, is not departing from embodiment of the present invention principle and claim
Under the premise of, some improvements and modifications can also be made, these improvements and modifications are also considered as the protection domain of the embodiment of the present invention.
Claims (8)
1. the kit that a kind of liver cancer based on liquid biopsy is detected, it is characterised in that：The kit includes：Contaminated for strengthening
The dyeing enhancing liquid of color effect and the specific antibody with fluorescent staining mark；
Wherein described specific antibody includes：ASGPR1 antibody, CD45 antibody and CPS1 antibody；
The dyeing enhancing liquid includes surfactant that concentration is 0.001~1mg/mL.
2. the kit that the liver cancer based on liquid biopsy according to claim 1 is detected, it is characterised in that：The dyeing increases
The solvent of strong liquid is biological buffer.
3. the kit that the liver cancer based on liquid biopsy according to claim 1 is detected, it is characterised in that：Live on the surface
Property agent be selected from Triton X-100, dimethyl sulfoxide (DMSO), NP-40, lauryl sodium sulfate in any one.
4. the kit that the liver cancer based on liquid biopsy according to claim 1 is detected, it is characterised in that：The kit
In also include lymphocyte separation medium and the immunomagnetic beads for removing leucocyte.
5. the kit that the liver cancer based on liquid biopsy according to claim 4 is detected, it is characterised in that：It is described for going
There is one or more in the immunomagnetic beads of antibody CD45, CD14 and CD15 for surface coupling except the immunomagnetic beads of leucocyte.
6. the kit that the liver cancer based on liquid biopsy according to claim 1 is detected, it is characterised in that：The specificity
The fluorescent staining mark of antibody A SGPR1 antibody, CD45 antibody and CPS1 antibody has each different launch wavelengths.
7. the kit that the liver cancer based on liquid biopsy according to claim 1 is detected, it is characterised in that：The reagent
Also include the fluorescence probe for FISH in box.
8. the kit that the liver cancer based on liquid biopsy according to claim 1 is detected, it is characterised in that：The dyeing
Also include accounting for the polyethylene glycol of dyeing enhancing liquid weight/mass percentage composition 0.1~3% in enhancing liquid.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111624078A (en) * | 2019-09-16 | 2020-09-04 | 湖北第二师范学院 | Application of lighting fluorescent compound in preparation of liver cancer diagnostic kit and dyeing method |
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| CN111766384A (en) * | 2020-04-21 | 2020-10-13 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Non-diagnosis target method for detecting PD-L1 gene mutation of esophageal squamous carcinoma patient through peripheral blood circulation tumor cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111381042A (en) * | 2018-12-29 | 2020-07-07 | 南京大学 | Prediction method of large blood vessel invasion in hepatocellular carcinoma, kit and application thereof |
| CN111381042B (en) * | 2018-12-29 | 2021-07-30 | 南京大学 | Prediction method of large blood vessel invasion in hepatocellular carcinoma, kit and application thereof |
| CN111624078A (en) * | 2019-09-16 | 2020-09-04 | 湖北第二师范学院 | Application of lighting fluorescent compound in preparation of liver cancer diagnostic kit and dyeing method |
| CN110927369A (en) * | 2019-12-11 | 2020-03-27 | 安徽安龙基因科技有限公司 | Kit for identifying circulating tumor cells by combining TCPP (TCPP) with CEP (cytokine induced apoptosis protein) probe and application |
| CN111751530A (en) * | 2020-04-20 | 2020-10-09 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Kit and detection method for detecting expression of peripheral blood circulating tumor cells PD-L1 of hepatocellular carcinoma patient |
| CN111766384A (en) * | 2020-04-21 | 2020-10-13 | 山东省肿瘤防治研究院（山东省肿瘤医院） | Non-diagnosis target method for detecting PD-L1 gene mutation of esophageal squamous carcinoma patient through peripheral blood circulation tumor cells |
| WO2022242398A1 (en) * | 2021-05-21 | 2022-11-24 | 深圳安侣医学科技有限公司 | Pretreatment reagent, preparation method, cell staining method and pretreatment method |
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