CN108982874A - It is a kind of detect human prostate cancer antigen PSA, AR-V7 immunofluorescent reagent box and application - Google Patents
It is a kind of detect human prostate cancer antigen PSA, AR-V7 immunofluorescent reagent box and application Download PDFInfo
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Abstract
The present invention relates to a kind of immunofluorescent reagent box for detecting human prostate cancer antigen PSA, AR-V7 and applications, the method of the invention, testing principle is as follows: being enriched with non-antibody mediated rare karyocyte first, then according to antigen-antibody reaction principle, using immunofluorescent detection method, expression of the destination protein in cell in the cell of enrichment is detected.This kit is by carrying out fluorescent marker to target cell and leukocyte common antigen CD45, positive, CD45 feminine gender the cell by screening destination protein, so that the non-antibody mediated rare karyocyte positive to specific protein in blood carries out interpretation and counting.
Description
Technical field:
The present invention relates to a kind of medical detection methods, the in particular to detection of the tumour cell in body fluid, and in particular to people
Prostate cancer antigen PSA, androgen receptor splicing variant AR-V7 immunofluorescence detection agent box.Suitable for inhomogeneity pattern
This PSA, AR-V7 detection further relate to purposes of the kit in detection PSA, AR-V7.
Background technique:
Prostate cancer refers to the epithelial malignancy occurred in prostate, is the common malignant tumour of male, is listed in complete
Ball causes one of five big cancers of deaths in men.The important means for the treatment of prostate cancer is Androgen deprivation therapy at present, but more
Number patient can develop in two years after the treatment as castration-resistant prostate cancer (castration-resistant prostate
Cancer, CRPC), prognosis is poor.
Prostate-specific antigen (Prostate specific antigen, PSA) is a kind of serine protease.
1987, PSA was approved as prostate cancer monitoring index by FDA.1991, PSA was proved to can be applied to prostate cancer sieve for the first time
It looks into.The current prostate cancer that PSA becomes detects most commonly seen one of marker, and the detection of PSA improves prostate cancer and examines in early days
Disconnected efficiency reduces mortality (Catalona WJ.Med Clin North Am.2018).
AR-Vs (androgen receptor splice variants, AR-Vs) refers in Androgen receptor mRNA
Montage generating during aberrant splicing or genetic recombination, in the case where no androgen remaining to that target gene is promoted to transcribe is prominent
Variant.In castration-resistant prostate cancer patient, the appearance of AR-Vs, so that patient generates drug resistance to antiprostate cancer
Property, increase and cures difficulty.AR-V7 (androgen receptor spice variant 7, AR-V7) is the table being currently known
Up to most commonly used androgen receptor splicing variant.TAXYNERGY studies (Antonarakis ES, et a1.J Clin
Oncol.2017 it) shows, is especially in the positive patient for being positioned at nucleus in the AR-V7 positive, taxane chemotherapy is male than targeting
Drug (the enzalutamide or abiraterone) treatment of hormone receptor is more efficient；And in AR-V7 negative patient, it is right
Target treatment more sensitive (the Antonarakis ES, et of the drug enzalutamide and abiraterone of androgen receptor
al.N Engl J Med.2014)。
Clinic depends on three kinds of pathology, iconography and serology methods to the monitoring of oncotherapy at present.Pathology
Be the goldstandard of diagnosing tumor, but sample of tissue has centainly traumatic, and is limited to materials position, simultaneously because swollen
The heterogeneity of tumor tissue, single-point sample of tissue are not enough to react whole neoplastic state, and are difficult to repeat to sample dynamic monitoring.Image
Method is using wide, but generally there are radiation injuries for iconography.Moreover, iconography usually only reacts the letter such as tumor size
Breath, it is difficult to judge the grade malignancy of tumour.More important point, iconography is typically only capable to the tumor tissues of detection 2mm or more, right
Poor in lesser tumor tissues diagnostic sensitivity, there are hysteresis qualitys.Serologic detection sampling it is more convenient, but sensitivity and
Specificity is poor and poor with pathologic, physiologic correlation.Likewise, clinically there is also certain limitations at present in terms for the treatment of
Property.For tumor patient, the patient especially shifted usually only judges treatment side according to the information of primary tumor at present
Case has ignored to the transfer stove even detection and treatment of micrometastasis stove.Judged in therapeutic process by iconography or serology
Curative effect is treated, there are certain limitations in terms of curative effect judges.
It is directed to castration-resistant prostate cancer, the directive significance that the detection of simple PSA selects its therapeutic scheme at present
And it is indefinite, and the detection of PSA, AR-V7, which are faced with detection, in organizing is carried out on different tissues slice, due to tumour
The reason of cell heterogeneity, PSA, AR-V7 can not reflect PSA, AR-V7 expression of patient's entirety on individual piece.Cause
This needs PSA, AR-V7 expression in the relevant non-antibody mediated rare karyocyte of a kind of new method pair and palindromia, transfer
Level is detected, for predicting that therapeutic effect of the certain drug to patient, the selection for disease treatment scheme provide reference.
The detection of body fluid tumour cell based on circulating tumor cell (Circulating Tumor Cell, CTC) can
Some solutions are provided for above-mentioned limitation.CTC refers to de- from primary tumor or transfer stove in tumour formation or progression
It drops into sanguimotor tumour cell, the detection of CTC prompts presence or the intraepithelial neoplasia cells of internal primary tumor or transfer stove
Presence, be that metastatic lesion forms " seed " cell, be the positive evidence of tumor development.It constantly shows on evidence in recent years
Show: early stage tumor development, the pre-neoplastic of the even visible lesion formation of iconography is just sent out into blood, this to send out
It is likely to be present in during the entire process of tumor development.So carrying out detection to CTC is conducive to early detection tumour, auxiliary diagnosis
Or reference is provided for selection therapeutic scheme.Meanwhile dynamic detection is carried out to CTC and can be appreciated that the sensibility of patient for treatment's drug,
Drug resistance signal is found early.Antibody mediated karyocyte refers to the karyocyte contained in biological fluid, also includes these cells in disease
Exudation or leakage in the case of reason, such as the various leucocytes in blood, inflammatory exudate；Non- antibody mediated rare karyocyte refers to
The improper rare karyocyte occurred in body fluid includes CTC, circulating tumor stem cell (circulating tumor
Cell, CSC) and chest/seroperitoneum, irrigating solution, hydropericardium, cerebrospinal fluid, urine, drainage-fluid, sputum, prostatic fluid, essence
The tumour cell and the fetal cell in blood in the sources such as liquid, marrow, Cord blood, amniotic fluid.To it is non-it is antibody mediated it is rare have core these
Cell is detected, and can be diagnosed for patient, treatment provides more fully information.
Goal of the invention:
Kit in the present invention can detect the expression water of PSA, AR-V7 in the non-antibody mediated rare karyocyte of prostate cancer
It puts down the medication to patients with prostate cancer and the opinion of directiveness is provided.By the specific antibody for PSA, AR-V7 come to trouble
Person is non-, and antibody mediated rare karyocyte carries out parting, passes through the expression water of quantity and its AR-V7 to PSA, AR-V7 positive cell
Flat, intracellular targeting carrys out the medication to prostate cancer and provides informative opinion.
The pathological examination of tumor patient at present, mainly progress histology are faced with sampling difficulty repeatedly, inactive
State monitoring, single tissue samples are not enough to the deficiencies of reflecting whole tumor load, cannot effectively reflect in real time into body fluid
Non- antibody mediated rare karyocyte variation.By immunofluorescence dyeing to the non-antibody mediated rare karyocyte of patient in the present invention
The protein expression level of middle PSA, AR-V7 are detected, its dynamic change of real-time monitoring can more accurately reflect the disease of patient
Sick load, thus to take therapeutic scheme to provide more foundations.
The present invention can be simultaneously to PSA, AR-V7 in such cell on the basis of to non-antibody mediated rare Other nucleated cells differential count
Protein expression level detected, and analyze the cellular localization of AR-V7.The present invention can to include tissue, blood, cell line,
The non-antibody mediated rare karyocyte in the sources such as hydrops, irrigating solution, prostatic fluid, urine, drainage-fluid is detected.
Summary of the invention:
In view of the deficiencies of the prior art, the present invention provide it is a kind of detect human prostate cancer antigen PSA, AR-V7 method and
Its kit.
The prior art, which there is no, detects what PSA, AR-V7 in non-antibody mediated rare karyocyte were expressed using immunofluorescence method
Report.
The method of the invention, testing principle are as follows:
It adopts first and is enriched with non-antibody mediated rare karyocyte by known method, wherein during the enrichment method can use
Method described in state's patent 200810097889.4,201310057307.0,2015104411229.Then anti-according to antigen
Precursor reactant principle detects the expression of destination protein in the cell of enrichment using known immunofluorescent detection method.This kit
By carrying out fluorescent marker to target cell and leukocyte common antigen CD45, CD45 feminine gender positive by screening destination protein
Cell, so that the non-antibody mediated rare karyocyte positive to specific protein in blood carries out interpretation and counting.It is basic herein
On, target protein is positive, CD45 the is negative non-antibody mediated rare karyocyte of candidate is according to destination protein fluorescence intensity by its table
Classify up to according to high, medium and low expression.
But it is used to detect PSA, AR-V7 expression in non-antibody mediated rare karyocyte for immunofluorescent detection method practicing
In encounter many difficulties, comprising: cell is easy to fall off from slide, fluorescence is weaker, non-specific dyeing, substrate background height etc..
The present invention passes through multiple screening study, finally obtains a kind of suitable method, solves above-mentioned difficulties, while in reality
It during trampling, is found surprisingly that, compared to the prior art detection accuracy and accuracy rate greatly improve testing result of the invention.
Detection method of the invention, steps are as follows:
1) enrichment of rare karyocyte
Blood and other humoral samples are taken, non-antibody mediated rare karyocyte therein can be enriched with, wherein described
Enrichment, method can be selected the method for separating and concentrating of any rare karyocyte, be selected from: red blood cell removal, negative enrichment, sun
Property enrichment, density gradient separation, membrane filter method, microfluidic method, Flow Cytometry etc.；It can not also be enriched by blood specimen
It is applied to multiple glass slides.
The wherein rare karyocyte: it is the rare karyocyte occurred under a kind of improper physiological status, usually makes
It is enriched with to obtain with selected by flow cytometry apoptosis, magnetic bead positive method for separating, preferred to obtain using negative enrichment method, the present invention is most
Best method is as follows:
The abnormal cell of various hypotypes is obtained using the negative enrichment method of removal blood plasma, red blood cell, leucocyte；Use room
Temperature or conditions of similarity spontaneously dry slide；Use the fixed cell of fixative and antigen
Sample after enrichment is fixed using fixer, and the fixer is selected from: ethyl alcohol, methanol, formalin, acetone, ice
(periodates-lysine-paraformaldehyde is fixed for acetic acid, formaldehyde, paraformaldehyde, glutaraldehyde, methacrylaldehyde, picric acid, PLP liquid
Liquid), ECD-G liquid (carbon two sub- amide-glutaraldehyde) etc..It is solid after can also being carried out using these reagents in following detection step
It is fixed.
2) the rare karyocyte of enrichment is subjected to Immunofluorescence test, steps are as follows for immunofluorescent detection method:
2.1 CYP1 wash glass slide 3min × 3 time, every time 100~150 μ L, it is ensured that cover full entire sample area；
2.2 suck surplus liquid on glass slide, and CYPP is added and acts on 5min, CYP1 is same as above 3min × 1 time of developing a film；It sucks more
Extraction raffinate body, adds 200 μ l ice acetone: methanol (7: 3) acts on 5min, and CYP1 develops a film 3min × 3 time, sucks excessive moisture；
2.3 plus 100~150 μ l confining liquid room temperatures closing, 20~30min.Extra confining liquid is sucked, adds 100~150 μ l dilute
PSA antibody, AR-V7 antibody and the CD45 antibody released are protected from light 1~2h of incubation in room temperature wet box；(direct labelled antibody can also be
2.5 are added).
2.4 are protected from light, and take the μ L/ piece of CYP2100~150 to develop a film 3min × 3 time, suck excessive moisture；
The fluorescence secondary antibodies that have diluted of 2.5 plus 100~150 μ L, be protected from light in wet box incubation (37 DEG C, 25~30min or room temperature 30
~60min).CYP2 washes 3min × 4 time, sucks excessive moisture.
2.6 redye: taking 10 μ L DAPI dye liquors, add to sample area.
2.7 covereds, and suck peripheral liquid.Under the microscope or it is placed under 2~8 DEG C or -20 DEG C of environment and is protected from light guarantor
It deposits.
3) microscopy observation is carried out to sample
Sample is placed under fluorescence microscope, entire sample area is scanned.If it was found that being switched to when sheet or round signal
It is further identified under high power lens.The coordinate of each positive cell is recorded, and 40 × object lens are taken pictures under different channels respectively.It must
Oil mirror observation can be used when wanting.
4) result judges
Positive cell: destination protein fluorescence signal is positive and has core thin without haematogenous Swine lymphocyte antigen fluorescence signal
Born of the same parents are denoted as a positive cell.The cellular localization of positive signal is recorded simultaneously.Negative cells: cell has haematogenous leucocyte table
Face antigen fluorescence signal, no matter whether destination protein has fluorescence to be denoted as feminine gender.
In above step, need to be marked directly on using to fluorescein on antibody or by fluorescent marker
Secondary antibody is detected；The fluorescein of labelled antibody has different emission spectrum, preferably green fluorescence (such as Alexa
Fluor 488), orange fluorescence (such as Alexa Fluor 555), red (such as AlexaFluor 594) or crimson fluorescent (such as
AlexaFluor 647) combination；The fluorescein of labelled antibody is not limited to Alexa Fluor line fluorescent element, can equally adopt
With Dylight, China green dyestuff, fluorescein isothiocynate (Fluorescein isothiocynate, FITC), biotin
(biotin), the series such as quantum dot, Texas Red, phycoerythrin (PE), phycocyanin (APC), rhodamine (rhodamine)
Fluorescein labelled antibody or different series fluorescein labelled antibody combination.
CD45 antibody is can also to be selected from following for the antibody of the general antigen of leucocyte for excluding blood borne cell
The antibody of cell surface antigen: CD3, CD11b, CD14, CD16, CD19, CD34, CD35, CD36, CD38, CD41, CD58,
CD61,CD66b,CD235a；Immunofluorescence test is carried out on tissue or cell line can be omitted CD45 antibody.
Detection method of the invention includes the operation with automation equipment by hand.
Detection method of the invention can be combined with fluorescence in situ hybridization (FISH) technology, and step is following any:
(i): first carrying out fluorescence in situ hybridization again with Immunofluorescence test；(ii): first carrying out immunofluorescence inspection again with fluorescence in situ hybridization
It surveys；(iii): while carrying out Immunofluorescence test and fluorescence in situ hybridization.
Contain surfactant and protein protective agent in buffer CYP1, CYP2, CYPP that this detection process uses, these
Surfactant can use 0~10% Triton X-100, saponin, Tween, NP-40, SDS, polyethylene glycol；Albumen
0~10% BSA, trehalose (Trehalose), sucrose can be used in protective agent.
As well known to a person skilled in the art the confining liquid that this detection process uses can also be selected: animal blood serum,
0.5%~10%BSA, Fc receptor antibody.
As well known to a person skilled in the art restorative procedure can be used to handle sample for this detection process, include
Enzymic digestion reparation, EDTA reparation, Pressure method, Microwave method etc..
The core dyestuff that this detection process uses is not limited to DAPI, equally can be using Hoechst, propidium iodide etc..
As well known to a person skilled in the art the incubation process of specific antibody and cell can be divided into Immunofluorescence test
Liquid dyeing and solid dyeing.Liquid dyeing is that cell is prepared into cell suspension, carries out cell permeabilization in suspension, antibody is incubated
The operation such as educate, then by cell be transferred to glass slide fix, mounting；And solid dyeing is that cell is first transferred to glass slide to consolidate
It is fixed, then the operation such as carry out cell permeabilization, antibody incubation.
The present invention is to carry out above-mentioned Immunofluorescence test, devises a kind of kit convenient for operation, in the kit
It include the main agents in above-mentioned Immunofluorescence test step, such as following reagent:
Buffer CYP1, CYP2, CYPP, confining liquid, the specific antibody for detecting PSA, AR-V7 is at least one, accordingly
Fluorescence secondary antibody, the CD45 antibody of fluorescent marker, antibody diluent, nuclear targeting liquid, control involved in operating process are thin
Born of the same parents may include or be not included in kit.
The preferred following components of kit of the present invention:
The explanation of title term is carried out to reagent of the present invention below:
PSA antibody is directed to PSA sequence in this kit, belongs to known array, is published in Logozzi M, Angelini
DF, et al.Increased PSA expression on prostate cancer exosomes in vitro
It is entitled in condition and in cancer patients.Cancer Lett.2017,403:318-329. document
PSA；The AR-V7 sequence that AR-V7 antibody is directed in this kit belongs to known array, is disclosed in Guo Z, et al.A novel
androgen receptor splice variant is up-regulated during prostate cancer
progression and promotes androgen depletion-resistant growth.Cancer
Res.2009Mar 15；69 (6): entitled AR3 in 2305-13. document.
Core of the invention technology is that the rare karyocyte of enrichment is carried out Immunofluorescence test by step 2), therein
Smear methods are that the present inventor obtains by many experiments and data screening, and the screening process in relation to condition is as follows:
In step 2.2, surplus liquid on glass slide is sucked, CYPP is added and acts on 5min, 100~150 μ l is added to close liquid chamber
20~30min of temperature closing；
Screening process is as follows: test different types and various concentration gradient penetrate reagent, different rear fixating reagents, envelope
Closing the time finally screens suitable reaction condition.
In step 2.3, PSA, AR-V7 antibody and CD45 antibody for adding 100~150 μ l to dilute are protected from light in room temperature wet box
It is incubated for 1~2h；
Screening process is as follows: test different antibodies concentration, incubation temperature, incubation time, liquid of developing a film finally screen and are suitble to
Reaction condition.
In step 2.5 plus the fluorescence secondary antibody that has diluted of 100~150 μ L, be protected from light in wet box incubation (37 DEG C, 25~30min or
30~60min of room temperature).
Screening process is as follows: test different antibodies concentration, incubation temperature, incubation time, liquid of developing a film finally screen and are suitble to
Reaction condition.
By above-mentioned screening, smear condition and method of the invention is obtained, acquired results detection accuracy and accuracy rate are significantly
It improves.
Kit of the invention is mainly for detection of in blood, irrigating solution, prostatic fluid, sperm, urine, drainage-fluid etc.
Non- antibody mediated rare karyocyte.It can also be applied to the Immunofluorescence test etc. of cell sheet such as smear/creep plate, tissue.
Kit of the invention can be adapted for the detection of kinds cancer cell, such as breast cancer, the cancer of the esophagus, gastric cancer, colon
Cancer, lung cancer, carcinoma of endometrium, prostate cancer, carcinoma of testis, the cancer of the brain, cutaneum carcinoma, the carcinoma of the rectum, sarcoma, tracheocarcinoma, head and neck cancer, pancreas
Cancer, liver cancer, oophoroma, lymph cancer, cervical carcinoma, carcinoma of vulva, melanoma, celiothelioma, kidney, bladder cancer, thyroid cancer, osteocarcinoma,
Cancer, sarcoma and soft tissue cancer.
Beneficial effects of the present invention are further illustrated below by way of experimental data:
Beneficial effects of the present invention:
With the prior art according to mRNA expression in tumor tissues come direction of medication usage compared with, testing result of the invention can
To carry out real-time instruction to medication, it can not only reflect whole tumour (including carcinoma in situ and that may be present sightless small
Transfer stove) load, it equally can reflect the expression of PSA, AR-V7 in the tumour cell easily shifted in body fluid, thus
More accurately direction of medication usage.
Compared with the prior art carries out immunohistochemistry detection to PSA, AR-V7 in tumor tissues, antibody mediated rare have to non-
PSA+AR-V7+, PSA+AR-V7-, PSA-AR-V7+ cell count in nucleus and cellular localization distinguishes, Neng Gougeng
Accurately reflect the correlation and direction of medication usage of PSA, AR-V7 and clinical parameter.
Compared with prior art, sample of the present invention is the cell in various body fluid sources, and base is compared in materials
It is more convenient in the detection technique of tumor tissues.
Compared with the prior art detects protein expression in tumor tissues, antibody mediated rare there is core to non-using immunofluorescence
The expression of PSA, AR-V7, cellular localization are detected in cell, can carry out phenotypic analysis to these cells.
Detailed description of the invention:
Fig. 1 is 5 kinds of fluorescence display figures of same sample, the respectively general antigen of target protein AR-V7, PSA, leucocyte
CD45, nucleus dyestuff DAPI, figure is closed.
Specific embodiment:
Embodiment 1
Material: the blood sample smear of feminine gender enrichment, control cell can use 22RV1, LNCaP smear.
Experimental procedure:
1. extracting 3.5ml peripheral blood in ACD (sodium citrate) anticoagulant tube.WithHuman peripheral leucocytes go
Except kit feminine gender enriched tumor cell, and it is fixed on glass slide；
2.CYPl washes glass slide 3min × 3 time, every time 100~150 μ L, it is ensured that covers full entire sample area；
3. sucking surplus liquid on glass slide, CYPP is added and acts on 5min, CYPl is same as above 3min × 1 time of developing a film；It sucks more
Extraction raffinate body, adds 200 μ l ice acetone: methanol (7: 3) acts on 5min, and CYP1 develops a film 3min × 3 time, sucks excessive moisture；
4. 100~150 μ l confining liquid room temperatures is added to close 25~30min.Extra confining liquid is sucked, 100 μ l is added to dilute
PSA antibody, AR-V7 antibody and CD45 antibody are protected from light 1.5~2h of incubation in room temperature wet box；
5. be protected from light, take the μ L/ piece of CYP2100~150 to develop a film 3min × 3 time, suck excessive moisture；
6. adding the secondary antibody that has diluted of 100 μ l, incubation (37 DEG C, 25~30min) are protected from light in wet box.CYP2 washes 3min × 4 time,
Suck excessive moisture.
7. redying: after DAPI pipe brief centrifugation, 10 μ L DAPI dye liquors are taken at liquid level, add to sample area.
8. cover plate: covered, under the microscope.Positive, CD45 negative antibody the cell view by PSA or AR-V7 expression
For positive cell (Fig. 1), and record the AR-V7 albumen positioning of AR-V7 positive cell.
As a result illustrate:
Embodiment 2
Material: appropriate anticoagulated blood 1 is managed, and carries out protein measurement assays step to it after being enriched with using membrane filtering method:
1. extracting appropriate peripheral blood to be put into containing in anti-coagulants heparin tube, slight concussion is mixed.
2. suspension addition film is separated by filtration in tumour cell technique device, filter and filter membrane are slow transitted through.
3. after to be filtered, continuing that 50ml 0.01M PBS is added in film filter, by what is adhered to around tube wall
Cell suspension pours in film filter, it is allowed to pass through filter and filter membrane；
4. the cell on fixed filter membrane；
5. operating detection albumen with embodiment 1.
Embodiment 3
Material: appropriate anticoagulated blood 1 is managed, and carries out Protein Detection to it after being enriched with using microfluidic methods
Experimental procedure:
1. the appropriate blood extracted is enriched with using the micro-fluidic chip of various principles.
2. sample carries out protein immunization fluorescence detection after enrichment.
Embodiment 4
Material: frozen tissue section
Experimental procedure:
1. being loaded with the glass slide of slice by taking out in freezing microtome, it is put into wet box.
2. when glass slide reach room temperature and it is not dry when, add CYP1 in washing on slice glass slide 3-5 times；
3. sucking surplus liquid, CYPP is added to be incubated for, CYP1 develops a film；
4. sucking surplus liquid, seals up and close the closing of liquid chamber temperature；
5. sucking extra confining liquid, add the PSA antibody diluted, AR-V7 antibody, room temperature is protected from light incubation appropriate time；
6. being protected from light, CYP2 develops a film, and sucks excessive moisture；
7. adding the secondary antibody diluted, it is protected from light incubation appropriate time.
8.CYP2 developing a film, excessive moisture is sucked.
9. adding 10 μ l DAPI, mounting microscopy.Positive cell is judged whether it is according to cellular morphology and PSA, AR-V7 expression.
Claims (10)
1. a kind of method of detection human prostate cancer antigen PSA, AR-V7 expression, the method, steps are as follows:
1) enrichment of rare karyocyte
Blood and other humoral samples are taken, non-antibody mediated rare karyocyte therein is enriched with, wherein the enrichment, side
The method for separating and concentrating of the optional rare karyocyte of any one of method, is selected from: red blood cell removal, feminine gender are enriched with, the positive is enriched with,
Density gradient separation, membrane filter method, microfluidic method, Flow Cytometry etc.；Enriched blood specimen can not also be applied to more
Glass slide is opened,
Sample after enrichment is fixed using fixer, and the fixer is selected from: ethyl alcohol, methanol, formalin, acetone, glacial acetic acid,
Formaldehyde, paraformaldehyde, glutaraldehyde, methacrylaldehyde, picric acid, PLP liquid, ECD-G liquid,
2) the rare karyocyte of enrichment is subjected to Immunofluorescence test, steps are as follows for immunofluorescent detection method:
2.1) CYP1 washes glass slide 3min × 3 time, every time 100~150 μ L, it is ensured that covers full entire sample area；
2.2) surplus liquid on glass slide is sucked, CYPP is added and acts on 5min, CYP1 is same as above 3min × 1 time of developing a film；It is extra to suck
Liquid, adds 200 μ l ice acetone: methanol (7:3) acts on 5min, and CYP1 develops a film 3min × 3 time, sucks excessive moisture；
2.3) plus 100~150 μ l confining liquid room temperatures close 20~30min, suck extra confining liquid, 100~150 μ l is added to dilute
PSA antibody, AR-V7 antibody and CD45 antibody, be protected from light 1~2h of incubation in room temperature wet box；
2.4) it is protected from light, takes 100~150 μ L/ piece of CYP2 to develop a film 3min × 3 time, suck excessive moisture；
2.5) it plus the fluorescence secondary antibody that has diluted of 100~150 μ L, is protected from light incubation in wet box, CYP2 washes 3min × 4 time, and it is extra to suck
Moisture,
2.6) it redyes: taking 10 μ L DAPI dye liquors, add to sample area,
2.7) covered, and suck peripheral liquid under the microscope or is placed under 2~8 DEG C or -20 DEG C of environment and is kept in dark place,
3) microscopy observation is carried out to sample
Sample is placed under fluorescence microscope, entire sample area is scanned, if switching to high power when discovery sheet or round signal
It is further identified under mirror, records the coordinate of each positive cell, and 40 × object lens are taken pictures under different channels respectively, when necessary
Oil mirror observation can be used,
4) result judges
Positive cell: the destination protein fluorescence signal positive and the karyocyte without haematogenous Swine lymphocyte antigen fluorescence signal,
It is denoted as a positive cell, while recording the cellular localization of positive signal, negative cells: cell has haematogenous leukocyte surface anti-
Former fluorescence signal, no matter whether destination protein has fluorescence to be denoted as feminine gender.
2. the method according to claim 1, wherein being needed in detection process using to fluorescein, the fluorescence
It is plain preferentially using the combination of green fluorescence, orange fluorescence, red or crimson fluorescent, be selected from: Alexa Fluor, Dylight,
Magnificent blueness dyestuff, fluorescein isothiocynate, biotin, quantum dot, Texas Red, phycoerythrin, phycocyanin, rhodamine.
3. the method according to claim 1, wherein CD45 antibody used in detection process is for leucocyte
The antibody of general antigen can also be selected from following antibody for excluding blood borne cell: CD3, CD11b, CD14, CD16,
CD19、CD34、CD35、CD36、CD38、CD41、CD58、CD61、CD66b、CD235a。
4. the method according to claim 1, wherein the method can be with fluorescence in situ hybridization (FISH) technology
Combination, step are following any: (i): first carrying out fluorescence in situ hybridization again with Immunofluorescence test；(ii): first former with fluorescence
Position hybridization carries out Immunofluorescence test again；(iii): while carrying out Immunofluorescence test and fluorescence in situ hybridization.
5. the method according to claim 1, wherein in buffer CYP1, CYP2, CYPP that detection process uses
Containing surfactant and protein protective agent, surfactant is selected from: 0~10% Triton X-100, saponin,
Tween, NP-40, SDS, polyethylene glycol；Protein protective agent is selected from: 0~10% BSA, trehalose, sucrose.
6. the method according to claim 1, wherein the confining liquid that detection process uses is selected from: animal blood serum,
0.5%~10%BSA, Fc receptor antibody.
7. the method according to claim 1, wherein the restorative procedure that detection process uses is selected from: enzymic digestion is repaired
Multiple, EDTA reparation, Pressure method, Microwave method.
8. the method according to claim 1, wherein the core dyestuff that uses of detection process is selected from DAPI,
Hoechst, propidium iodide.
It include the main agents in claim 1 detection method step: buffer 9. a kind of kit, in the kit
CYP1, CYP2, CYPP, confining liquid, the specific antibody for detecting PSA, AR-V7 is at least one, corresponding fluorescence secondary antibody, fluorescence mark
The CD45 antibody of note, antibody diluent, nuclear targeting liquid, optional related control cell include or are not included in examination
In agent box.
10. kit according to claim 9, contains following components:
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