CN108507992A - The detection method of circulating tumor cell surface markers PD-L1 - Google Patents
The detection method of circulating tumor cell surface markers PD-L1 Download PDFInfo
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- CN108507992A CN108507992A CN201810312287.XA CN201810312287A CN108507992A CN 108507992 A CN108507992 A CN 108507992A CN 201810312287 A CN201810312287 A CN 201810312287A CN 108507992 A CN108507992 A CN 108507992A
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Abstract
The invention discloses a kind of detection methods of circulating tumor cell surface markers PD L1.It includes the following steps:(1) whole blood is handled with erythrocyte cracked liquid, isolates karyocyte, formaldehyde is used in combination to be fixed;(2) tumour immunity fluorescent marker anti-cytokeratin Ab anti CK are first passed through and carry out positive-selecting, it is incubated all cells with PD L1 primary antibodies, then it is incubated with the PD L1 secondary antibodies for being marked with FITC fluorophors, then all cells is marked with nucleus fluorescent dye DAPI;(3) using high-throughput polychrome imaging analysis, the optical filter of CY5, FITC and DAPI are selected, observes channel surface fluorescence color, the final detection realized to circulating tumor cell surface markers PD L1.Detection method is easy, quick, economical, noninvasive, high sensitivity, and specificity is good.
Description
Technical field
The invention belongs to technical field of molecular biology, specifically a kind of circulating tumor cell surface markers
The detection method of PD-L1.
Background technology
Programmed death receptor -1 (PD-1) is main immunologic test point receptor, by combining its ligand programmed death
Ligand -1 (PD-L1), makes the downward of T cell effector function, to help to maintain the tolerance to tumour cell.By anti-
PD-1 and anti-these accesses of PD-L1 antibody blockings potentially contribute to prevent this downward to make T cell maintain its antitumor spy
The ability of property and mediate tumor cell death.There are mainly three types of the inhibitor for being directed to PD-1 and PD-L1 currently on the market,
Pembrolizumab (trade names:Keytruda), Nibolumab (trade names:) and Atezolizumab (trade names Opdivo:
Tecentriq), it can be used for the treatment of the kinds cancers such as melanoma, non-small cell lung cancer, carcinoma of urinary bladder.But it is not all
Patient can be benefited during PD-1/PD-L1 inhibitor for treating, and PD-1/PD-L1 inhibitor is at present only in least a portion of cancer
Patient can generate the effect of persistently control tumour with it.Therefore, whether detection patient has PD-L1 positive expressions, can be effective
Help the suitable drug of patient selection for treating.Currently, the detection method of PD-1/PD-L1 is mainly based upon cell protein water
Flat detection, in clinic, the main method for using immunohistochemistry is cut using the tumor tissues obtained after operation or puncture
Piece dyes.The result of immunohistochemistry and the experience of Pathology Doctors ' are closely related.Other than staining technique, the specificity of antibody is also outstanding
Its is important.And the detection of China PD-L1 at present is more chaotic, first, the disunity of staining technique and condition;Second is that staining antibodies
It is various;Third, pathological evaluation standard is not yet unified, it is horizontal using immunohistochemistry evaluation PD-L1 that these have resulted in domestic patient
Value reduce.In addition, be detected since immunohistochemistry must obtain tissue, the either hand by performing the operation or puncturing
Method is all a kind of invasive operation, can be damaged to patient.Therefore, we there is an urgent need to a kind of new noninvasive, evaluation methods
The PD-L1 detection method relatively stable with standard.
Circulating tumor cell is fallen off by primary tumor, and into after blood circulation, organ or primary internal organs are settled down a long way off, are formed
Transfer stove.Circulating tumor cell CTC detects the popular domain as liquid biopsy, in diagnosing tumor, treatment and monitoring etc.
Clinical manifestation gradually show up prominently, be current most potential tumour non-invasive diagnosis and real-time curative effect monitoring means,
Clinical value is extremely notable.Therefore, if a kind of method of detection circulating tumor cell surface PD-L1 can be provided, will have
Very important meaning.
Invention content
A kind of circulating tumor cell surface mark is provided present invention aim to solve above-mentioned the deficiencies in the prior art
The detection method of will molecule PD-L1, the detection method is easy, quick, economical, and noninvasive, high sensitivity, specificity is good, can solve
The certainly deficiency of prior art detection PD-L1.
Technical scheme of the present invention is specifically described as follows.
A kind of detection method of circulating tumor cell surface markers PD-L1, includes the following steps:
(1) whole blood is handled with erythrocyte cracked liquid, isolates karyocyte, formaldehyde is used in combination to be fixed;
(2) it first passes through tumour immunity fluorescent marker anti-cytokeratin Ab anti-CK and carries out positive-selecting, use PD-L1
Primary antibody is incubated all cells, is then incubated with the PD-L1 secondary antibodies for being marked with FITC fluorophors, then with nucleus fluorescent dye
DAPI marks all cells;
(3) using high-throughput polychrome imaging analysis, the optical filter of CY5, FITC and DAPI are selected, observes channel surface fluorescence
Color, the final detection realized to circulating tumor cell surface markers PD-L1.
In the present invention, the specific reaction condition of step (2) is as follows:
A. the PBS solution of the skimmed milk power containing 4-6% is added in the culture dish after being fixed to cell, 4 DEG C are protected from light incubation 40-
70min;
B. after absorbing skimmed milk power PBS solution, PBST cleanings are added several times along wall;
C. tumour immunity fluorescent marker anti-cytokeratin Ab anti-CK and PD-L1 primary antibody is added into confining liquid, resists
Body source is abcam companies, is added in culture dish along wall after mixing, 4 DEG C are protected from light overnight incubation;
D. mixed liquor is absorbed, the PD-L1 secondary antibodies for being marked with FITC fluorophors are added into confining liquid, antibody sources are
Abcam is public, is added in culture dish along wall after mixing, and 4 DEG C are protected from light incubation 40-70minh;
E. antibody is absorbed, is cleaned several times with PBST, DAPI is added, room temperature, which is protected from light, is incubated 20-40min.
In the present invention, in condition b, the PBS containing 0.02wt%Tween-20 in PBST.
In the present invention, in step (3), channel surface fluorescence color is observed, shows the then DAPI+ of blue, not aobvious blue is then
DAPI- shows green then PD-L1+, and not aobvious green then PD-L1- shows red then CK+, not aobvious red then CK-;According to fluorescence developing,
The point of DAPI+ and CK+ are considered circulating tumor cell CTC, if there are PD-L1+ on circulating tumor cell CTC, then should
Circulating tumor cell CTC is PD-L1+CTC.
The present invention compared with the existing technology, for the first time combines CTC detections and PD-L1 marker detections, by blood
Liquid carries out erythrocyte splitting, makes remaining karyocyte (mainly CTC and lymphocyte) all tiling enrichments using nanotechnology
It is fixed in nanometer substrate, all cells then are marked with nucleus fluorescent dye DAPI, pass through tumour immunity fluorescence later
Marker anti-cytokeratin Ab anti-CK carries out positive-selecting, since the surfaces CTC are specific expressed with PD-L1, uses
PD-L1 primary antibodies are incubated all cells, then are incubated with the secondary antibody for being marked with FITC fluorophors, are swept finally by high-throughput techniques
It retouches, the final detection realized to circulating tumor cell surface markers PD-L1, the detection method is easy, quick, economical, nothing
Wound, high sensitivity, specificity is good, solves the deficiency of prior art detection PD-L1.
Description of the drawings
Fig. 1 is the schematic diagram for detecting circulating tumor cell PD-L1 positive expressions.
Specific implementation mode
The present invention is made with reference to specific embodiment further explained below.
Embodiment 1
Fig. 1 is the schematic diagram for detecting circulating tumor cell PD-L1 positive expressions.Fig. 1 a are blue-fluorescence channel, are used
DAPI marks nucleus, shows the then DAPI+ of blue, and explanation is a complete cell, does not show blue then DAPI-, is not complete
Cell;Fig. 1 b are green fluorescence channel, mark PD-L1 albumen, show green then PD-L1+, do not show green then PD-L1-;Fig. 1 c are
Red fluorescence channel marks CK albumen, shows red then CK+, not aobvious red then CK-.Fig. 1 d are the synthesis in three channels.
The present embodiment expresses an example to detect clinical liver cancer sample loops tumor cell surface PD-L1, specifically includes following
Step:
1) whole blood is handled:
A. 200 μ 1 × erythrocyte cracked liquids of L are added into 2mL whole bloods, is placed at room temperature for 15min, during which uniformly rocks.
B.200RCF it centrifuges 5 minutes, absorbs supernatant, retain cell.
C. 2mL 1%FPBS (V (FBS) are added into centrifuge tube:V (PBS)=1:99) 5, are centrifuged in 200RCF after mixing
Minute, remove supernatant.
2) cell is enriched with entirely:
A. it is transferred in the culture dish handled well after 1mL FPBS mixings being added, culture dish is placed in 37 DEG C of shaking tables and is cultivated
45min。
B. culture dish is placed in 4 DEG C of refrigerators after cultivating, stands 1min.
3) cell is fixed:
A. FPBS is absorbed, 4% formaldehyde is added into culture dish and is placed on 4 DEG C of standing 1min.
B. formaldehyde is absorbed, 1mL methanol is added, is placed in -20 DEG C of 20min.
C. methanol is absorbed, is cleaned three times with 2mL PBS every time.(note:When liquid being added every time, it is added, keeps away along culture dish wall
Exempt to rush in cell.
4) closing and antibody incubation:
A. the addition skimmed milk power (being prepared with PBS) of 1mL 5% (m/V) containing 0.02%Tween-20 into culture dish, 4 DEG C
It is protected from light and is incubated 1h.
B. after absorbing skimmed milk power, 2mL PBST (PBS containing 0.02%Tween-20) cleanings 4 times are added along wall, every time
5min。
C. 1 μ L anti-CK (being purchased from abcam companies) are added into 500 μ L confining liquids, add 2 μ L PD-L1 primary antibodies
(being purchased from abcam companies), is added along wall in culture dish, 4 DEG C are protected from light overnight incubation after mixing.
D. mixed liquor is absorbed, 5 μ L are added into 500 μ L confining liquids are marked with the PD-L1 secondary antibodies of FITC fluorophors and (be purchased from
Abcam companies), it is added along culture dish along wall after mixing, 4 DEG C are protected from light and are incubated 1h.
E. antibody is absorbed, is cleaned three times with PBST, 1mL nucleus dyestuffs DAPI (4,6- connection narrow -2-phenylindone) is added,
Room temperature, which is protected from light, is incubated 30min.
5) it scans:Antibody is absorbed, is cleaned three times with 2mL PBST, 1mL PBS scannings are added.
The optical filter of CY5, FITC and DAPI are selected using high-throughput polychrome imaging analysis to above-mentioned clinical liver cancer sample,
Channel surface fluorescence color is observed, the then DAPI+ of blue is shown, not aobvious blue then DAPI- shows green then PD-L1+, not aobvious green
Then PD-L1- shows red then CK+, and " point " of DAPI+ and CK+ is considered CTC by not aobvious red then CK- according to fluorescence developing,
Otherwise it is not then for PD-L1+CTC (Fig. 1) if there are PD-L1+, the CTC on the CTC.The detection method is easy, fast
Speed, economic, noninvasive, high sensitivity, specificity are good.
The present invention is simultaneously not limited to the embodiments described above, other any Spirit Essences and principle without departing from the present invention
Changes, modifications, substitutions, combinations, simplifications made by lower, should be equivalent substitute mode, be included in the protection model of the present invention
Within enclosing.
Claims (4)
1. a kind of detection method of circulating tumor cell surface markers PD-L1, which is characterized in that include the following steps:
(1) whole blood is handled with erythrocyte cracked liquid, isolates karyocyte, formaldehyde is used in combination to be fixed;
(2) it first passes through tumour immunity fluorescent marker anti-cytokeratin Ab anti-CK and carries out positive-selecting, with PD-L1 primary antibodies
All cells are incubated, are then incubated with the PD-L1 secondary antibodies for being marked with FITC fluorophors, then marked with nucleus fluorescent dye DAPI
Remember and all cells;
(3) using high-throughput polychrome imaging analysis, the optical filter of CY5, FITC and DAPI are selected, observes channel surface fluorescence face
Color, the final detection realized to circulating tumor cell surface markers PD-L1.
2. detection method according to claim 1, which is characterized in that the specific reaction condition of step (2) is as follows:
A. the PBS solution of the skimmed milk power containing 4-6% is added in the culture dish after being fixed to cell, 4 DEG C are protected from light incubation 40-70min;
B. after absorbing skimmed milk power PBS solution, PBST cleanings are added several times along wall;
C. tumour immunity fluorescent marker anti-cytokeratin Ab anti-CK and PD-L1 primary antibody is added into confining liquid, antibody comes
Source is abcam companies, is added in culture dish along wall after mixing, 4 DEG C are protected from light overnight incubation;
D. mixed liquor is absorbed, the PD-L1 secondary antibodies for being marked with FITC fluorophors, antibody sources abcam are added into confining liquid
Company is added along culture dish along wall after mixing, and 4 DEG C are protected from light and are incubated 40-70minh;
E. antibody is absorbed, is cleaned several times with PBST, DAPI is added, room temperature, which is protected from light, is incubated 20-40min.
3. detection method according to claim 2, which is characterized in that in condition b, 0.02wt%Tween-20 is contained in PBST
PBS.
4. detection method according to claim 1, which is characterized in that in step (3), channel surface fluorescence color is observed,
The then DAPI+ of blue is shown, blue then DAPI- is not shown, shows green then PD-L1+, not aobvious green then PD-L1- shows red then CK+,
Not aobvious red then CK-;According to fluorescence developing, the point of DAPI+ and CK+ are considered circulating tumor cell CTC, if the circulating tumor
There are PD-L1+ on cell CTC, then circulating tumor cell CTC is PD-L1+CTC.
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CN109374893A (en) * | 2018-11-07 | 2019-02-22 | 国家纳米科学中心 | Based on thermophoresis to the detection system and method for PD-L1 receptor |
CN110161227A (en) * | 2019-05-22 | 2019-08-23 | 北京中科纳泰生物科技有限公司 | Human archeocyte keratin mixed antibody with fluorescent marker and preparation method thereof |
CN110361536A (en) * | 2019-07-04 | 2019-10-22 | 昆山汇先医药技术有限公司 | A kind of detection method of tumor cell surface marker molecule PD-L1 |
CN110389219A (en) * | 2019-06-12 | 2019-10-29 | 杭州华得森生物技术有限公司 | A kind of enrichment detecting method of Epithelial and stromal mixed type and PD-L1 positive circulating tumor cell |
CN111141906A (en) * | 2020-01-06 | 2020-05-12 | 中南大学湘雅医院 | Detection kit for peripheral blood circulating tumor cells and PD-L1 of small cell lung cancer patient |
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WO2021213322A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting pd-l1 expression of peripheral blood circulating tumor cells of kidney cancer patient and detection method |
WO2021213297A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence test kit for measuring pd-l1 gene mutations in circulating tumor cells in peripheral blood in non-small cell lung cancer patient, and method for same |
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CN109374893A (en) * | 2018-11-07 | 2019-02-22 | 国家纳米科学中心 | Based on thermophoresis to the detection system and method for PD-L1 receptor |
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CN111141906A (en) * | 2020-01-06 | 2020-05-12 | 中南大学湘雅医院 | Detection kit for peripheral blood circulating tumor cells and PD-L1 of small cell lung cancer patient |
CN111735955A (en) * | 2020-04-20 | 2020-10-02 | 山东省肿瘤防治研究院(山东省肿瘤医院) | Immunofluorescence detection method for PD-L1 expression on peripheral blood circulation tumor cells of hepatocellular carcinoma patient |
WO2021213322A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting pd-l1 expression of peripheral blood circulating tumor cells of kidney cancer patient and detection method |
WO2021213297A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence test kit for measuring pd-l1 gene mutations in circulating tumor cells in peripheral blood in non-small cell lung cancer patient, and method for same |
CN114441411A (en) * | 2021-12-31 | 2022-05-06 | 江苏汇先医药技术有限公司 | Method and system for interpreting capture result of tumor cell capture chip |
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