CN110275025A - PD-L1 immunohistochemical kit - Google Patents
PD-L1 immunohistochemical kit Download PDFInfo
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Abstract
The present invention relates to the immunohistochemistry reagent of PD-L1 protein expression in detection people tissue especially human tumour tissue and kits and its preparation method and application.
Description
Technical background
Programmed death 1 (PD-1) is mainly expressed in T lymphocyte, bone-marrow-derived lymphocyte and the macrophage table of activation
Face.The ligand that Programmed death 1 ligand -1 (PD-L1), also known as B7 homolgous molecule 1 (B7-H1) are PD-1, belong to I type across
Memebrane protein.After PD-L1 is in conjunction with PD-1, inhibition signal is transmitted by PD-1, is able to suppress the proliferation and activation of lymphocyte.Closely
The tumor cell surface height of several Nian Faxian, some patients express PD-L1, pass through the PD-1 with tumor infiltrating lymphocyte surface
Molecule combines, and inhibits the function of lymphocyte, and immunologic escape occurs so as to cause tumour.Clinical and experimental study shows to block PD-
1 or PD-L1 can effectively treat or inhibit tumour growth.Therefore, easy-to-use detection means is clinically needed to filter out
The tumor patient of tumor tissues or cell height expression PD-L1, to carry out that the treatment of PD-1 or PD-L1 is blocked to provide personalizedly
Guidance and foundation.
Brief description
Fig. 1 is to carry out immunohistochemical staining result to human placenta with 6E8 and MS-1215 combination.
Fig. 2 is to carry out immunohistochemical staining result to human placenta with the combination of 6E8 and 859643.
Fig. 3 is to carry out immunohistochemical staining result to human placenta with 6E8 and ENS004 combination.
Fig. 4 is to carry out immunohistochemical staining result to human placenta with 6E8 and KIT-5010 combination.
Fig. 5 is to carry out immunohistochemical staining result to human placenta with RIgG and MS-1215 combination.
Fig. 6 is to carry out immunohistochemical staining result to people's lung squamous cell carcinoma cancers with 6E8 and MS-1215 combination.
Fig. 7 is to carry out immunohistochemistry negative control coloration result to people's lung squamous cell carcinoma cancers with Fig. 6.
Brief summary of the invention
The present invention relates to a kind of PD-L1 immunohistochemical kits, for detecting in people's tissue, especially in human tumour tissue
The expression of PD-L1 albumen.Immunohistochemical kit generally include at least primary antibody for specifically binding purpose labelled protein and
In conjunction with the secondary antibody with the label that can be detected of primary antibody, the combination pairing of both antibody is most important, finds
One optimal combination is the key that one high-quality immunohistochemistry product of exploitation.Inventor for anti human PD-L 1 antibody (primary antibody) and
Secondary antibody has carried out a large amount of test, has screened countless combinations, have passed through failure repeatedly, has finally found several best groups
It closes, and then has developed the PD-L1 immunohistochemical kit of high specificity, high sensitivity.
Therefore, on the one hand, PD-L1 immunohistochemical kit of the invention includes at least (1) anti human PD-L 1 antibody；(2)
Secondary antibody being combined with the anti human PD-L 1 antibody and with the label that can be detected.Wherein anti human PD-L 1
It is mostly anti-that antibody can be mostly anti-mouse monoclonal antibody, mouse, rabbit monoclonal antibody or rabbit.The known anti human PD-L 1 antibody that can be selected includes Abcam
Anti-PD-L1antibody 28-8 article No. ab205921, AbcamAnti-PD-L1antibody article No. ab58810,
AbcamAnti-PD-L1antibody article No. ab153991, AbcamAnti-PD-L1 antibody MIH2 article No. ab109052,
Thermo Fisher PD-L1/CD274Antibody article No. PA5-20343, Thermo Fisher PD-L1/
CD274Antibody article No. PA5-30937, Thermo Fisher PD-L1/CD274Antibody article No. PA5-28115,
AbDSerotec Rabbit anti Human CD274 article No. AHP1703, AbDSerotec Goat anti Human
CD274 (C-Terminal) article No. AHP2128, MicroSpin (micro- rotation) Anti-PD-L1 6E8 (article No. MS-1028),
Spring bioscience PD-L1/CD274SP142 article No. M4420, Cell Signaling TechnologyPD-L1Rabbit mAb article No. 13684, Cell Signaling Technology PD-L1 (405.9A11)
Mouse mAb article No. 29122, Dako 28-8 and Dako 22C3.
Secondary antibody can combine with the anti human PD-L 1 antibody and with the label that can be detected can be selected
From: Shenzhen Xin Bosheng ELIAS secondary antibody article No. ENS004, Foochow step new ELIAS secondary antibody article No. KIT-0015, Foochow steps new ELIAS secondary antibody
Article No. KIT-0016, Foochow step new ELIAS secondary antibody article No. KIT-5010, Foochow steps new ELIAS secondary antibody article No. KIT-9921,
MicroSpin (micro- rotation) ELIAS secondary antibody article No. MS-1215, Life Technologies ELIAS secondary antibody article No. 859643, middle China fir gold
Bridge ELIAS secondary antibody article No. ZB-2301, eBioscience ELIAS secondary antibody article No. 18-4818, Abcam ELIAS secondary antibody article No.
Ab20043, Thermo Fisher ELIAS secondary antibody article No. A16110, Sigma-Aldrich ELIAS secondary antibody article No. A0168 and
Sigma-Aldrich ELIAS secondary antibody article No. SAB3701066.
Optionally, kit also for example can show red AEC (3- ammonia as color developing agent comprising at least one enzyme base number of a tender object
DAB (the benzidine ammonia of base -9- ethyl carbazole (3-amino-9-ethyl-carbazole) or aobvious brown color
Diaminobenzidin,DAB).DAB can be Guangzhou as deep as DAB article No. KS003, doctor's moral DAB article No. AR1000, Bo Shide
DAB article No. AR1026, Zhong Shan Golden Bridge DAB article No. ZLI-9017, Zhong Shan Golden Bridge DAB article No. ZLI-9031, new DAB article No. DAB- is stepped
0031, new DAB article No. DAB-2031, SantaCruzBio DAB article No. sc-24982 or Abcam DAB article No. are stepped
ab103723。
Kit can also be used for nuclear targeting comprising counterstain to show cell outline.Applicable counterstain can be with
It is that haematoxylin (hematoxylin, blue after dyeing), methyl green (methyl green, in green after dyeing) or core are red fastly
(nuclearfast red, take on a red color).
Suitable color developing agent+counterstain combination can be following any: DAB+ haematoxylin；DAB+ methyl green；DAB+ core
It is fast red；AEC+ haematoxylin；Or AEC+ methyl green.
In one embodiment, anti human PD-L 1 antibody (primary antibody) is Dako28-8.
In one embodiment, anti human PD-L 1 antibody (primary antibody) is Dako22C3.
In one embodiment, anti human PD-L 1 antibody (primary antibody) is 6E8 (MS-1028).
In one embodiment, secondary antibody is sheep anti mouse/rabbit secondary antibody MS-1215.
In one embodiment, secondary antibody is sheep anti mouse/rabbit secondary antibody Life Technologies article No. 859643.
In one embodiment, secondary antibody is Abcam ELIAS secondary antibody article No. ab20043.
In one embodiment, secondary antibody is Thermo Fisher ELIAS secondary antibody article No. A16110.
In one embodiment, secondary antibody is Sigma-Aldrich ELIAS secondary antibody article No. A0168.
In one embodiment, primary antibody is selected from 6E8,28-8 and 22C3；Secondary antibody is selected from sheep anti mouse/rabbit secondary antibody MS-1215
With sheep anti mouse/rabbit secondary antibody 859643；Color developing agent is DAB；Counterstain is haematoxylin.
In application kit of the invention, combined using antibody and target antigen site (PD-L1), then with it is labeled
Secondary antibody is reacted, and the target antigen site in histotomy is presented finally by chromogenic reaction, can be true by optical microscopy
Set the goal the distribution and content of antigen site, and result not only serves as the diagnosis basis of clinician, and can be PD-L1
Inhibitor/antibody drug screens clinical target user and provides clinical application guidance.
Embodiment
Compare the secondary antibody of different reagent producer productions, preferably best pairing scheme.
Immunohistochemical experiment step:
1) it dewaxes: being placed in 60 DEG C of oven preheating 30min, be then respectively put into dimethylbenzene I&II each 15 minutes；
2) aquation: each 5 minutes in dehydrated alcohol I&II, 95%, 85%, 75% ethyl alcohol each 2 minutes；
3) endogenous peroxydase inactivates: 3%H is added dropwise2O2, it is stored at room temperature 5 minutes；
4) PBS is washed 3 times, every time 3 minutes；
5) it closes: confining liquid is added dropwise, 37 DEG C are incubated for 30 minutes, remove surplus liquid；
6) primary antibody: the primary antibody covering tissue of debita spissitudo is added, is incubated at room temperature 1 hour
7) PBS is washed 3 times, every time 3 minutes；
8) secondary antibody: ELIAS secondary antibody is added and covers tissue, is incubated at room temperature 30 minutes；
9) PBS is washed 3 times, every time 3 minutes；
10) it develops the color: prepared DAB developing solution is added, is stored at room temperature 1-5 minutes, observation color terminates in due course；
11) tap water rinses 3 minutes；
12) it redyes: haematoxylin is added, is stored at room temperature 5-60 seconds, tap water rinses；
13) it is dehydrated: 75%, each 2 minutes in 85%, 95% and dehydrated alcohol I&II；
14) transparent: each 2 minutes in dimethylbenzene I&II；
15) appropriate mountant mounting is used, microscopically observation is simultaneously taken pictures.
Primary antibody selects 6E8 (MS-1028), 28-8,23C3, and secondary antibody selects micro- rotation (MicroSpin) MS-1215, Life
Technologies (859643), Xin Bosheng (ENS004), new (KIT-0015) is stepped.Fig. 1 is with 6E8 and MS-1215 combination pair
Human placenta carries out immunohistochemical staining result.Fig. 2 is to carry out immunohistochemistry to human placenta with the combination of 6E8 and 859643
Coloration result.Fig. 3 is to carry out immunohistochemical staining result to human placenta with 6E8 and ENS004 combination.Fig. 4 be with 6E8 and
KIT-5010 combination carries out immunohistochemical staining result to human placenta.Fig. 5 is with RIgG and MS-1215 combination to Human plactnta
Tissue carries out immunohistochemical staining result.Fig. 6 is to carry out immunohistochemistry dye to people's lung squamous cell carcinoma cancers with 6E8 and MS-1215 combination
Color result.Fig. 7 is to carry out immunohistochemistry negative control coloration result to people's lung squamous cell carcinoma cancers with Fig. 6.
As a result as shown, MS-1215 and Life Technologies (859643) effect is substantially better than other secondary antibodies:
Cell membrane dyeing is most obvious, and background is minimum, and eucaryotic cell structure shows clearest.
The experimental result answers clear, the pairing (primary antibody: 6E8 of scheme one；Secondary antibody: MS-1215 or Life
Technologies (859643)), and remaining pairing scheme experimental result is indefinite, background has heavier unspecific staining, or
Person's dyeing is fuzzy, unintelligible, even without dyeing, therefore matching scheme one is best pairing scheme.
Claims (6)
1. a kind of immunohistochemical kit, comprising:
(1) anti human PD-L 1 antibody is selected from MicroSpin Anti-PD-L1 6E8 (article No. MS-1028), Abcam Anti-PD-
L1 antibody 28-8 (article No. ab205921) or Cell Signaling Technology PD-L1 (405.9A11)
Mouse mAb (article No. Dako 22C3)；
(2) secondary antibody can combining with the anti human PD-L 1 antibody and with the label that can be detected, is selected from
MicroSpin sheep anti mouse/rabbit secondary antibody (article No. MS-1215) and Life Technologies sheep anti mouse/rabbit secondary antibody (article No.
859643)；And
(3) color developing agent.
2. kit according to claim 1, wherein anti human PD-L 1 antibody is MicroSpin Anti-PD-L1 6E8
(article No. MS-1028).
3. kit according to claim 1, the kit is used to detect the expression of PD-L1 in human tumour tissue.
4. kit according to claim 3, the tumor tissues are lung squamous cell carcinoma cancers.
5. kit according to claim 1 to 3 further includes counterstain.
6. kit according to claim 5, color developing agent+counterstain combination is selected from benzidine ammonia+haematoxylin, diamino
The fast red, 3-amino-9-ethyl carbazole+haematoxylin of base biphenyl ammonia+methyl green, benzidine ammonia+core or 3- amino -9- ethyl click
Azoles+methyl green.
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| CN109975554A (en) * | 2019-03-04 | 2019-07-05 | 宁波美晶医疗技术有限公司 | The detection method and its special agent of cell PD-L1 protein expression in a kind of body fluid sample |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101104640A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human PD-L1 monoclonal antibody and application thereof |
| CN104470949A (en) * | 2012-05-15 | 2015-03-25 | 百时美施贵宝公司 | Cancer immunotherapy by disrupting pd-1/pd-l1 signaling |
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| KR102291355B1 (en) * | 2012-11-30 | 2021-08-19 | 에프. 호프만-라 로슈 아게 | Identification of patients in need of pd-l1 inhibitor cotherapy |
| KR102158924B1 (en) * | 2013-03-15 | 2020-09-22 | 제넨테크, 인크. | Biomarkers and methods of treating pd-1 and pd-l1 related conditions |
| US20160084839A1 (en) * | 2013-04-02 | 2016-03-24 | Marisa Dolled-Filhart | Immunohistochemical assay for detecting expression of programmed death ligand 1 (pd-l1) in tumor tissue |
| EP3149042B1 (en) * | 2014-05-29 | 2019-08-28 | Spring Bioscience Corporation | Pd-l1 antibodies and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104640A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human PD-L1 monoclonal antibody and application thereof |
| CN104470949A (en) * | 2012-05-15 | 2015-03-25 | 百时美施贵宝公司 | Cancer immunotherapy by disrupting pd-1/pd-l1 signaling |
Non-Patent Citations (2)
| Title |
|---|
| THERESE PHILLIPS, MA, ET AL.: "Development of an Automated PD-L1 Immunohistochemistry (IHC) Assay for Non–Small Cell Lung Cancer.", 《APPL IMMUNOHISTOCHEM MOL MORPHOL》 * |
| 陈茜，等: "抗PD-1/PD-L11治疗在晚期难治性肺鳞癌中的疗效和安全性。", 《循证医学》 * |
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Application publication date: 20190924 |