CN106834511B - Breast cancer detection kit based on liquid biopsy - Google Patents
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Abstract
The invention provides a kit for breast cancer detection based on liquid biopsy, which comprises a staining enhancement solution for enhancing a staining effect and a specific antibody with a fluorescent staining marker, wherein the specific antibody comprises an ER α antibody, a VIM antibody, a CD45 antibody and an HER2 antibody, and the staining enhancement solution comprises a surfactant with the concentration of 0.001-1 mg/mL.
Description
Technical Field
The invention relates to the field of liquid biopsy, in particular to a kit for breast cancer detection based on liquid biopsy.
Background
Breast cancer is a disease that severely threatens the health of women, with an incidence of 23% of all tumors, with a median prevalence in women and a second-degree mortality in all cancers. According to statistics, 30-40 breast cancer patients are diagnosed in every hundred thousand population in China, and the average age of the breast cancer patients diagnosed is 45-55 years old, which is younger than western women. Therefore, it is very important for early screening of breast cancer and detection in the process of disease development.
At present, aiming at early screening of breast cancer, the method for detecting Circulating Tumor Cells (CTC) based on liquid biopsy has the advantages which are not possessed by a plurality of traditional detection methods, including the advantages of no wound, rapidness, high accuracy and the like. CTCs are tumor cells that are released into the peripheral blood circulation from solid tumor primary or metastatic foci, either spontaneously or as a result of a diagnostic procedure. Since metastasis is the main cause of cancer-related death, and CTC is regarded as a seed for metastasis, detection of CTC has great application potential in discovery of new tumor biomarkers, prognosis of tumors and personalized treatment, and is one of the hotspots of tumor research at home and abroad. To date, most of the breast cancer CTC-related studies have focused on Metastatic Breast Cancer (MBC) patients, and some studies have shown that CTCs are also present in Early Breast Cancer (EBC) patients with a positive rate of 9.4-48.6%. The positive rate of CTCs in blood is also associated with early recurrence and low survival of breast cancer. In fact, studies have shown that CTCs can be detected in blood when tumors are 1mm, and cancer can be detected at an early stage by detecting the presence of CTCs in peripheral blood circulation. For this reason, efficient CTC capture, enrichment analysis methods are important for CTC-based early screening.
Although the EpCAM-based CTC capture enrichment analysis platform is still used as a gold standard for detecting CTCs in MBC so far, whether the method is suitable for detecting EBC still remains controversial, and EpCAM is used as a biomarker, so that the detection of CTCs subjected to EMT has high false negative, the broad-spectrum epithelial biomarker has no tissue specificity, and the specific tissue source of CTCs cannot be traced, which is of great significance for subsequent tumor treatment. Therefore, it is important to find and apply some biomarkers specific to breast cancer tissues for the detection and analysis of CTCs.
Chinese patent No. CN102313813B, publication No. 1/15/2014, discloses a method for detecting rare cells by immunofluorescence staining, which comprises performing trichrome staining on leukocytes, rare cells and cell nuclei, and then detecting by fluorescence. However, this method can only specifically recognize the rare cell by a single monoclonal antibody, which is likely to cause missed detection. Meanwhile, due to the heterogeneity of cells, the amount of antigen expressed by each cell is different, so that the fluorescence intensity is sometimes very weak and is difficult to distinguish under a fluorescence microscope.
Disclosure of Invention
The invention mainly aims to provide a liquid biopsy-based breast cancer detection kit aiming at the defects in the prior art, the kit can be used for detecting the number of CTCs in peripheral blood, carrying out traceability analysis, tumor typing and early screening on the CTCs, and simultaneously, the fluorescence display cell membrane boundary is clear and the detection result sensitivity is high.
The invention is realized by the following technical scheme:
a kit for breast cancer detection based on liquid biopsy, the kit comprising: a staining enhancement solution for enhancing the staining effect and a specific antibody with a fluorescent staining mark;
wherein the specific antibodies include ER α antibody, VIM antibody, CD45 antibody and HER2 antibody;
the dyeing enhancement liquid comprises a surfactant with the concentration of 0.001-1 mg/mL.
The solvent of the staining enhancement solution is biological buffer solution.
Preferably, the surfactant may be any one of Triton X-100, dimethyl sulfoxide, NP-40, Sodium Dodecyl Sulfate (SDS). More preferably, the surfactant is Triton X-100 or SDS. Most preferably, the staining enhancement solution comprises SDS with the concentration of 0.01-1 mg/mL, and the solvent of the staining enhancement solution is a commonly used biological buffer solution, such as PBS buffer solution and the like.
Preferably, the kit further comprises a lymphocyte separation solution and immunomagnetic beads for removing leukocytes. The lymphocyte separation liquid and the immunomagnetic beads can be used for removing the interference of red blood cells and white blood cells, and are convenient for enriching CTC better.
More preferably, the immunomagnetic beads for removing leukocytes are one or more of immunomagnetic beads with antibodies CD45, CD14 and CD15 coupled to the surface, and most preferably comprise all of the immunomagnetic beads.
Preferably, the specific antibodies ER α, VIM and HER2 carry fluorescent staining markers of at least two different emission wavelengths, and the CD45 antibody carries a fluorescent staining marker different from all the fluorescent staining markers described above, as is well known to those skilled in the art, different fluorescent staining markers should be used in order to distinguish between different specific antibodies, but due to the number of fluorescent microscopic channels, two of the ER α, VIM and HER2 antibodies are usually made to carry the same fluorescent staining marker, preferably the ER 7 and HER2 antibodies carry the same fluorescent staining marker, the fluorescent staining marker can be any fluorescent dye commonly used in the art, as long as it can be distinguished by adjusting the excitation light wavelength, the fluorescent staining markers can be completely distinguished under different filters by a fluorescent microscope due to their different emission wavelengths, wherein the fluorescent staining markers preferably used in the present invention are Alexa Fluor series molecules, cyanine dyes, more preferably Alexa, CY 685 594 and Alexa488, wherein the fluorescent staining markers are Alexa 618, CY α, 18 a, 18 a-18 nm, and the fluorescent staining markers are red fluorescent staining markers with a-18 nm fluorescence staining marker, and are preferably invisible fluorescence staining markers with a wavelength of Alexa-18 nm.
Preferably, the fluorescent probe for fluorescence in situ hybridization may be a probe for fluorescence in situ hybridization of chromosomes, such as CEP8 fluorescent probe.
Preferably, the dyeing enhancement liquid also comprises polyethylene glycol accounting for 0.1-3% of the dyeing enhancement liquid by mass. The dyeing effect of the dyeing liquid can be further enhanced by matching the polyethylene glycol with the surfactant.
As is well known to those skilled in the art, the process of incubating cells with a specific antibody labeled with a fluorescent dye to perform fluorescent staining may be liquid staining or solid staining. And if liquid staining is carried out, cells needing cell staining are firstly resuspended into cell suspension, then staining pretreatment and cell staining are carried out in sequence, and then the cells are transferred onto a glass slide for cell fixation and glass slide mounting. If solid staining is carried out on the glass slide, cells are firstly fixed on the glass slide, then staining pretreatment and cell staining are carried out in sequence, and then the glass slide is mounted.
The cell fixation is performed by a cell fixation solution, which may be a cell fixation solution commonly used in the art, such as one or a combination of several of paraformaldehyde, glutaraldehyde, formalin, ethanol, and acetone.
The method for detecting CTC by using the breast cancer detection kit based on liquid biopsy comprises the following steps:
enriching for target cells in peripheral blood;
carrying out enhanced staining pretreatment on the target cells by using a staining enhancement solution;
carrying out fluorescent staining on the target cells by using a specific antibody with a fluorescent staining marker;
fixing the target cells;
performing fluorescence in situ hybridization by using a fluorescence probe;
DAPI mounting and microscopic examination.
The target cell after the enhanced staining pretreatment has better combination of the specific antibody and the target cell, more obvious fluorescent staining and clear cell membrane boundary.
As is well known to those skilled in the art, when a tumor cell specific antigen exists in a cell, after staining the surface of a cell membrane and fixing a target cell, the cell membrane is broken and the target cell is subjected to intracellular fluorescent staining using a specific antibody labeled with fluorescent staining.
Preferably, the method for enriching target cells in peripheral blood comprises the following steps:
(1) centrifuging peripheral blood to separate plasma and blood cells, and removing plasma;
(2) adding a cell buffer solution and a lymphocyte separation solution into the step (1), centrifuging and layering to remove a red blood cell layer;
(3) adding immunomagnetic beads for removing leukocytes into the step (2) and incubating to obtain a suspension;
(4) and (4) magnetically separating the suspension liquid obtained in the step (3) to remove white blood cells and residual red blood cells, and washing to obtain enriched target cells.
The lymphocyte separating medium can be a lymphocyte separating medium commonly used in the field and is used for separating red blood cells according to density, and the Ficoll separating medium is preferably adopted in the invention.
The enrichment of the target cells comprises twice red blood cell removal, so that the red blood cells can be removed more cleanly, white blood cells and residual red blood cells can be removed at one time through the immunomagnetic beads, and the enrichment efficiency is higher. The interference of other cells on fluorescent staining can be eliminated after the enrichment treatment, and the better observation can be carried out through a fluorescent microscope.
Preferably, the treatment time for performing the enhanced staining pretreatment on the target cells by using the staining enhancement liquid is 5-20 minutes. The final concentration of the surfactant after the dyeing enhancement solution is added is 0.2 mu g/mL-1 mg/mL. Wherein the preferable adding amount of the dyeing enhancement liquid is 1-20 mu L. When the amount of the dye enhancing solution is too large, the intensity of the enhanced fluorescence is high, but the cell membrane may be damaged, and when the amount is too small, the enhancing effect is not so significant.
Preferably, when the specific antibody with the fluorescent staining marker is used for carrying out fluorescent staining on the target cells, the specific antibody with the fluorescent staining marker is diluted by a buffer solution according to the volume ratio of 1 (100-200), and then the diluted specific antibody is mixed with the target cells for incubation.
Preferably, the final concentration of the specific antibody with the fluorescent staining mark after the specific antibody is added is 2-10 mug/mL. Wherein, the specific antibody with the fluorescent staining label is diluted by a buffer solution according to the volume ratio of 1: 200. The higher the concentration of the added specific antibody, the more easily non-specific staining is caused, and the accuracy of the result is influenced.
The buffer may be a general biological cell buffer, for example, PBS buffer. More preferably, the PBS buffer is 0.01M phosphate buffer, whichIn Na2HPO4、NaCl、KH2PO4And KCl at concentrations of 8mM, 136mM, 2mM and 2.6mM, respectively, and pH 7.2-7.4.
Principle of the invention
In primary breast cancer, about 75-80% of patients with primary breast cancer show estrogen receptor α positivity (ER α +), the status of ER α plays an important role in the classification and guiding of breast cancer, the status of ER α determines whether breast cancer patients are suitable for hormone therapy, and the therapy is still the main adjuvant therapy for patients with ER α + breast cancer, therefore, the CTC detection based on ER α not only has the ability of tissue-specific localization, but also has a certain guiding significance in the treatment of breast cancer, in addition, human epidermal growth factor receptor 2(HER2) is also an important breast cancer prediction marker, about 10-30% of primary breast cancers show HER2+, and is associated with poor prognosis and high invasion, in addition, about 11% of breast cancers show ER α -/HER2+, so that the detection sensitivity of the combination of ER α + and HER2+ can reach about 80%, and part of primary breast cancer patients with HER 2-may also develop HER2+ HER 2.
Although TNBC accounts for about 15% of breast cancer, TNBC has higher metastatic capacity and poorer prognosis, so that the TNBC cannot be ignored, and the Vimentin (VIM) is widely expressed in breast cancer tissues independent of hormone and is early used for detecting hormone receptor negative basal breast cancer, the detection sensitivity of the TNBC in the breast cancer tissues can reach about 94%, so the TNBC can be used as a good biomarker of TNBC breast cancer CTC.
The chromosome 8 abnormality exists in various solid tumors, including prostate cancer, ovarian cancer, renal cancer, breast cancer, colorectal cancer and the like, and the condition of detecting the chromosome 8 abnormality in cells or tissues by using a Fluorescence In Situ Hybridization (FISH) method is widely used for clinical diagnosis. The CEP8 probe is a fluorescent probe labeled with Spectrumorange, and is used for selecting cells with abnormal number of chromosome 8 in all cells as target cells. Through observation by a fluorescence microscope, when the wavelength of excitation light is adjusted to be 559nm, emission light of the Spectrumorange is 588nm orange light, the fluorescence can be simultaneously detected with the tumor marker and the leucocyte marker, and the number of chromosomes of a target cell No. 8 is determined through detection of the number of specific orange light probe points. The invention can select FISH detection verification assisted with CEP8 while biopsy is carried out based on tumor markers, and the detection specificity is further ensured by the detection of CEP8 while the detection sensitivity of CTC is increased by three-tumor-marker detection.
According to the method, the detection of the breast cancer CTC by combining three biomarkers including ER α, HER2 and VIM has high sensitivity, and the CTC of the breast cancer can be classified according to the fact that ER α + is Luminal breast cancer (Luminal), HER2+/ER α -is HER2 positive breast cancer (HER2+), ER α -/HER2-/VIM + is Basal breast cancer (Basal), the detected CTC can be judged whether the CTC is derived from the breast cancer according to the expression of ER α and HER2, and the breast cancer can be classified and traced by combining the expression of the VIM, and the early screening of the breast cancer can be realized.
According to the invention, CD45 antibody with an Alexa594 fluorescent staining marker is used for specifically staining the surface of leukocytes, ER α antibody and HER2 antibody with an Alexa488 fluorescent staining marker and VIM antibody with a CY5 fluorescent staining marker are used for specifically staining captured circulating tumor cells, when the excitation wavelength is 591nm, Alexa emission light is 618nm orange red light, when the excitation wavelength is 650nm, CY5 emission light is 670nm far-red light (invisible to the naked eye, and purple color is given by microscopic scanning), when the excitation wavelength is 499nm, Alexa emission light is 519nm green light, so that different fluorescence of the markers on different cells can be observed by adjusting different excitation wavelengths and exposure time, and further, the target cells can be distinguished, namely CD45+ is leukocytes, HER2+ or CTC α + is tumor cells, wherein HER2+/ER 733 + can be determined for patients derived from breast cancer, and further, the target cells can be classified as HER2+ or HER α + HER2 type breast cancer (HER2) based on the expression of different specific antigens, and the HER 6342 + or HER2 is determined as HER + type breast cancer (HER).
The selection of multiple antibodies with fluorescent staining markers is beneficial for identifying specific types of target cells, however, when the types of the antibodies are more than one, the combination of the target cells and the antibodies is easily interfered, so that the fluorescent staining is poor, and the fluorescent microscope observation is influenced. The staining enhancement solution of the invention enables the antibody with the fluorescent staining label to be more easily combined on the target protein on the cell surface by exposing the membrane protein antigenic determinant on the cell surface, thereby enhancing the fluorescence intensity of the labeled cell membrane and enabling the cell membrane boundary to be clear.
The kit for detecting the breast cancer based on the liquid biopsy can effectively enrich target cells, confirm whether the enriched target cells are from early-stage patients of the breast cancer and carry out tumor typing. Meanwhile, the tumor detection sensitivity is increased through the detection of the three tumor markers, and the detection accuracy is further ensured through the detection of CEP 8. In addition, the invention enhances the dyeing effect through the dyeing enhancement liquid, so that various antibodies with fluorescent dyeing marks can be combined with target proteins of target cells, the target cells are dyed, the dyeing effect is better, the fluorescence is stronger, and the boundary is clear. Therefore, the surface specific fluorescent staining effect of the blood-derived cells and the fluorescent staining effect of the tumor cell specific marker can be enhanced simultaneously, so that CTC and the blood-derived cells are easier to distinguish, the false positive of detection is reduced, and the specificity of detection is improved. In addition, the method is simple, low in cost and high in practicability.
Drawings
FIG. 1 is a graph of fluorescent staining for Merge of MCF7 human breast cancer cell line;
FIG. 2 is a graph of the fluorescent staining detection for Merge of an SK-BR-3 human breast cancer cell line;
FIG. 3 is a photograph of the fluorescence staining detection of Merge of MDA-MB-231 human breast cancer cell line;
FIG. 4 is a graph of fluorescence staining for Merge after tumor cell capture.
Detailed Description
The invention is further illustrated by the following specific examples: the experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Lymphocyte isolates were obtained from Ficoll-Paque PLUS from GE healthcare.
CD45 immunomagnetic beads from Thermo Fisher Scientific
CD45。
Alexa594, Alexa488, and CY5 fluorescent dyes were from Thermo Fisher Scientific.
The CEP8 Probe was obtained from the CEP 8Spectrum orange DNA Probe Kit of Yapei corporation.
Example 1 enrichment of target cells in peripheral blood
(1) Peripheral blood was centrifuged to remove plasma proteins: 8.5mL of peripheral blood was centrifuged at room temperature for 7min at 800g in a horizontal centrifuge, and the supernatant was discarded.
(2) And (2) adding 5-6 mL of PBS buffer solution and 3mL of lymphocyte separation solution into the plasma in the step (1), and centrifuging the mixture in a centrifuge for 7min at normal temperature at 450 g. The red blood cell layer is separated into three layers after centrifugation, the red bottom layer is the red blood cell layer, the slightly white middle layer is mainly white blood cells, CTC and the like, the yellow upper layer is blood plasma, all liquid above the red blood cell layer is absorbed, and the red blood cell layer at the bottom is removed.
(3) And (3) dropwise adding 200mL of immunomagnetic beads with CD45 antibody coupled on the surface into the step (2), and incubating on a horizontal shaking table to obtain a suspension, wherein the rotation speed of the horizontal shaking table is set at 120-150rpm, the inclination is 20-30 ℃, and the horizontal shaking table is horizontally shaken for 20min at normal temperature.
(4) And (4) adsorbing the suspension liquid in the step (3) for 2min by using a large magnetic frame. Carefully sucking the liquid not attached to the tube wall, removing white blood cells and residual red blood cells, washing with PBS buffer solution and re-suspending to obtain enriched target cells.
Example 2 fluorescent staining of enriched target cells
(1) Enhancing dyeing pretreatment: about 50. mu.L of the enriched target cells was added with 2. mu.L of the staining enhancement solution and allowed to stand at room temperature for 10 min. The staining enhancement solution is SDS or PBS buffer solution of Triton X-100, and the concentration of SDS is 0.1 mg/mL.
(2) Cell surface staining: after diluting CD45-Alexa 5941. mu.L with 199. mu.L of PBS buffer, the diluted solution was added to the cell suspension after the pretreatment in step (1), and the cell suspension was incubated for 20min in the absence of light. After incubation, the cell fluid was washed with PBS buffer, centrifuged at 950g for 4min and the supernatant removed to 100. mu.L.
(3) Cell fixation: and (3) transferring and smearing the cells in the step (2) on a glass slide, then adding fixing liquid paraformaldehyde, fixing for 10min, and washing the glass slide for 2 times by PBS (phosphate buffer solution) for 5min each time.
(4) Cell membrane rupture: after the cells are fixed, 200 mu L of cell membrane-breaking liquid is dripped into the cell area on the glass slide, after incubation for 10min, PBS buffer is added to wash the glass slide for 2 times, 5min each time. The cell membrane-disrupting solution is a PBS (phosphate buffer solution) solution of Triton X-100, and the concentration of the Triton X-100 is 0.5%.
(5) Staining cell intracellular marker, diluting 1 μ L each of ER α -Alexa488, Her2-Alexa488 and VIM-CY5 with 198 μ L PBS buffer solution, adding to the cell area on the above slide, incubating for 20min in the dark, and washing the slide 2 times with PBS buffer solution for 5min each.
(6) Fluorescence in situ hybridization: 10 μ L of CEP8 fluorescent probe was added dropwise to the slide, the cover slip was covered, and the coverslip was sealed with a sealing gel on the periphery. Prehybridization at 76 ℃ for 10min, followed by hybridization at 37 ℃ for 4 h.
(7) DAPI mounting and microscopic examination: the mounting gel was torn off, washed 2 times (5 min/time) with PBS buffer and the cover slips were removed, after natural drying 10 μ L of mounting agent was added and the cover slips were covered (where the content of the mounting agent is DAPI: glycerol ═ 1:9), and finally observed with a Nikon DS-U3 fluorescence microscope under the microscopic conditions: when the wavelength of the excitation light is 591nm, the Alexa594 emits light with 618nm red light, and the exposure time is 100 ms; when the excitation wavelength is 650nm, the emission light of CY5 is 670nm far-red light (invisible to naked eyes, purple is given by microscope scanning), and the exposure time is 100 ms; when the wavelength of the excitation light is 499nm, the Alexa488 emission light is 519nm green light, and the exposure time is 100 ms; when the excitation light wavelength is 345nm, the DAPI emitted light is 455nm blue light, and the exposure time is 10-20 ms; the excitation light wavelength is 559nm, the emission light of the Spectrumorange is 588nm orange light, the exposure time is 100ms, the result shows that most of white blood cells separated from normal human peripheral blood show positive staining of CD45 and negative staining of tumor markers, the FISH result shows that the white blood cells are diploid of CEP8, and the white blood cells have no tumor markers or abnormal number of CEP8 cells.
Example 3 fluorescent staining assay of cell lines
Subjecting MCF7(ER α +/HER2-) or SKBr3(ER α -/HER2+) or MDA-MB-231(ER α -/HER2-/VIM +) cell line (purchased from cell bank of Chinese academy of sciences) to enzymolysis and digestion, and collecting 105Cells, approximately 50. mu.L, were stained with fluorescent light and examined by fluorescent microscope according to the procedure of example 2. The microscopic examination conditions are as follows: when the wavelength of the excitation light is 591nm, the Alexa594 emits light with 618nm red light, and the exposure time is 100 ms; when the excitation wavelength is 650nm, the emission light of CY5 is 670nm far-red light (invisible to naked eyes, purple is given by microscope scanning), and the exposure time is 100 ms; when the wavelength of the excitation light is 499nm, the Alexa488 emission light is 519nm green light, and the exposure time is 100 ms; when the excitation light wavelength is 345nm, the DAPI light emission is 455nm blue light, and the exposure time is 10-20 ms, and the result is shown in FIGS. 1-3.
As can be seen from FIGS. 1-3, the cells of each cell line showed a blue-emitting nucleus under 358nm excitation light, indicating the presence of the cell; no red color was shown under 591nm excitation, indicating negative CD45 and the absence of leukocytes.
Under 499nm excitation light, MCF7 cells and SKBr3 cells showed green color, indicating ER α or HER2 positive, and MDA-MB-231 cells did not show green color, indicating ER α and HER2 negative.
Under 650nm excitation light, MDA-MB-231 cells showed purple color, indicating that VIM was positive, MCF7 cells and SKBr3 cells did not show purple color, indicating that VIM was negative.
Example 4 tumor cell Capture efficiency assay
Blood samples of 8.5ml were collected from healthy volunteers, and 31, 26 and 26 human breast cancer cell lines of MCF7, SK-BR-3 and MDA-MB-231 were added to the blood samples. Tumor cell enrichment was then performed according to the procedure of example 1, followed by fluorescence staining and fluorescence microscopy of tumor cells according to the procedure of example 2. The microscopic examination conditions are as follows: when the wavelength of the excitation light is 591nm, the Alexa594 emits light with 618nm red light, and the exposure time is 100 ms; when the excitation wavelength is 650nm, the emission light of CY5 is 670nm far-red light (invisible to naked eyes, purple is given by microscope scanning), and the exposure time is 100 ms; when the wavelength of the excitation light is 499nm, the Alexa488 emission light is 519nm green light, and the exposure time is 100 ms; when the excitation light wavelength is 345nm, the DAPI emitted light is 455nm blue light, and the exposure time is 10-20 ms; the spectrum orange emitted at 559nm excitation wavelength was 588nm orange, with an exposure time of 100ms, and the results are shown in FIG. 4.
The result shows that the number of the tumor cells (ER α + or Her2+ or VIM +) is 69, the capture rate is 83.13%, the number of the ER α or Her2 positive (ER α + or Her2+) cells is 49, the positive rate is 59.04%, the number of the VIM positive (VIM +) cells is 20, the positive rate is 24.1%, the number of the tumor markers are positive (ER α + or Her2+/VIM +) tumor cells is 0, the positive rate is 0%, the number of the CEP8 non-diploid (CEP8 ≠ 2) tumor cells is 37, and the non-diploid rate is 44.58%.
From the above results, it can be seen that the detection of CTCs based on liquid biopsy using the kit of the invention is more sensitive to capture of tumor cells in blood than the detection of single tumor markers currently available. Meanwhile, according to different detection results, the breast cancer can be classified. And according to the detection of CEP8, the specificity of the detection can be increased, and different detection purposes can be met.
The above-described embodiment is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the embodiment of the present invention and the claims, and these modifications and improvements are also considered to be within the scope of the embodiment of the present invention.
Claims (6)
1. A kit for breast cancer detection based on liquid biopsy, characterized in that: the kit comprises: a staining enhancement solution for enhancing the staining effect and a specific antibody with a fluorescent staining mark;
wherein the specific antibodies include ER α antibody, VIM antibody, CD45 antibody and HER2 antibody;
the dyeing enhancement liquid comprises a surfactant with the concentration of 0.1mg/mL, and the surfactant is sodium dodecyl sulfate;
the dyeing enhancement liquid also comprises polyethylene glycol accounting for 0.1-3% of the dyeing enhancement liquid by mass.
2. The kit for liquid biopsy based breast cancer detection according to claim 1, wherein: the solvent of the staining enhancement solution is biological buffer solution.
3. The kit for liquid biopsy based breast cancer detection according to claim 1, wherein: the kit also comprises lymphocyte separation liquid and immunomagnetic beads for removing leukocytes.
4. The kit for breast cancer detection based on liquid biopsy of claim 1, wherein the specific antibodies ER α, VIM and HER2 carry fluorescent staining markers with at least two different emission wavelengths, and the CD45 antibody carries a fluorescent staining marker different from all the fluorescent staining markers.
5. The kit for liquid biopsy based breast cancer detection according to claim 1, wherein: the kit also comprises a fluorescent probe for fluorescent in situ hybridization.
6. The kit for liquid biopsy based breast cancer detection according to claim 3, wherein: the immunomagnetic beads for removing the leucocytes are one or more of immunomagnetic beads with the surfaces coupled with antibodies CD45, CD14 and CD 15.
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| CN107505250A (en) * | 2017-06-23 | 2017-12-22 | 李彦萍 | Method, kit and the system of bone-marrow-derived lymphocyte are sorted in peripheral blood, identifies the kit and system of haematogenous lympha tumour cell |
| CN109298174A (en) * | 2018-09-26 | 2019-02-01 | 姜云瀚 | A kind of multiple-color immunofluorescence labeling method and imaging method |
| CN110257512A (en) * | 2019-05-20 | 2019-09-20 | 上海交通大学医学院 | Marker and composition for luminal type and HER2 type breast cancer diagnosis, treatment and prognosis |
| CN112979805A (en) * | 2019-12-13 | 2021-06-18 | 桐庐纳泰医学检验实验室有限公司 | VEGFR2 fluorescence labeled antibody for circulating tumor cell detection, and detection method and application thereof |
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