Validated using a knockout cell line
Recombinant
RabMAb

Anti-PD-L1 antibody [28-8] (ab205921)

Overview

  • Product name
    Anti-PD-L1 antibody [28-8]
    See all PD-L1 primary antibodies
  • Description
    Rabbit monoclonal [28-8] to PD-L1
  • Tested applications
    Suitable for: IHC-P, WB, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein corresponding to Human PD-L1 (extracellular). The immunogen contains the specific extracellular domain of huPD-L1 (Phe19-Thr239). See reference for more info - www.ncbi.nlm.nih.gov/pmc/articles/PMC4561627/
    Database link: Q9NZQ7

  • Positive control
    • Tissue: Human tonsil, head and neck squamous cell carcinoma and placenta tissues; L2987 cell line. Cell Lines: Positives: B-CPAP- high, ES-2- medium, HCC70 - low CHO-PDL1 U-87 MG For additional information, please refer to here: Programmed death-ligand 1 (PD-L1) expression in various tumor types - http://www.immunotherapyofcancer.org/content/1/S1/P53
  • General notes

     

    Additional information on positive controls:

    Tissue:
    Tonsil- with hyperreactive changes
    Recommended to screen hyper-reactive tonsils to find those with highest expression of PD-L1 in crypt epithelium, macrophages homing the germinal centers and interfollicular mononuclear leukocytes.

    Tumor tissues- prescreened for positive tumor and inflammatory infiltrates
    PD-L1 expression varies by tumor type so screening is recommended to find positive and negative tumor controls.

    The following publication is useful for suggesting suitable tumor types:
    http://www.immunotherapyofcancer.org/content/1/S1/P53

    Note: Look for specimens with high numbers of inflammatory macrophages and mononuclear leukocytes.

    Cell Lines: 
    Positive controls: B-CPAP- high, ES-2- medium, HCC70 – low,
    Negative controls: COLO205, see ab95363.  

    Primary negative control, isotype control:  RabMAb negative control antibody, ab172730

    Recombinant protein: Recombinant human PD-L1 protein, ab167713

    Recommended protocols:

    For recommended Western Blotting (WB) protocol, Flow Cytometry (FC) protocol and Immunohistochemistry (IHC) protocol, please refer to the protocol section, here.

    IHC usage:
    For IHC usage on FFPE tissues, the following antigen solution and detection kit are recommended with clone 28-8 -  Universal HIER antigen retrieval reagent (ab208572) and IHC detection kit HRP/DAB (ab209101).

    Western blot usage:
    For clone 28-8, it is recommended to use Odyssey system. This system has the advantages of a wider dynamic range and less background than chemiluminescence.

    Alternative versions available:

    Conjugated versions of the antibody available (Alexa Fluor® 488, Alexa Fluor® 647, PE, HRP) - see list.

    A low endotoxin, azide free version of the antibody is available: ab209889.

    Anti-PD-L1 antibody [28-8] has been used as detector antibody in Human PD-L1 SimpleStep ELISA® kit: ab214565.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab205921 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

For antigen buffer for FFPE tissue, it is recommended to use Universal HIER antigen retrieval reagent (ab208572)

WB Use at an assay dependent concentration. Predicted molecular weight: 33 kDa.
Flow Cyt Use at an assay dependent concentration.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF Use a concentration of 2 µg/ml.

Target

  • Function
    Involved in the costimulatory signal, essential for T-cell proliferation and production of IL10 and IFNG, in an IL2-dependent and a PDCD1-independent manner. Interaction with PDCD1 inhibits T-cell proliferation and cytokine production.
  • Tissue specificity
    Highly expressed in the heart, skeletal muscle, placenta and lung. Weakly expressed in the thymus, spleen, kidney and liver. Expressed on activated T- and B-cells, dendritic cells, keratinocytes and monocytes.
  • Sequence similarities
    Belongs to the immunoglobulin superfamily. BTN/MOG family.
    Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.
  • Cellular localization
    Cell membrane and Endomembrane system.
  • Information by UniProt
  • Database links
  • Alternative names
    • B7 H antibody
    • B7 H1 antibody
    • B7 homolog 1 antibody
    • B7-H1 antibody
    • B7H antibody
    • B7H1 antibody
    • CD 274 antibody
    • Cd274 antibody
    • CD274 antigen antibody
    • CD274 molecule antibody
    • MGC142294 antibody
    • MGC142296 antibody
    • OTTHUMP00000021029 antibody
    • PD L1 antibody
    • PD-L1 antibody
    • PD1L1_HUMAN antibody
    • PDCD1 ligand 1 antibody
    • PDCD1L1 antibody
    • PDCD1LG1 antibody
    • PDL 1 antibody
    • PDL1 antibody
    • Programmed cell death 1 ligand 1 antibody
    • Programmed death ligand 1 antibody
    • RGD1566211 antibody
    see all

Images

  • Immunohistochemical analysis of Human Tonsil tissue with ab205921 at 2 µg/ml.
    PD-L1 positive expression of the crypt epithelium (large black arrow) and cells localized within the germinal centers (small black arrow)
    Note negative staining of the stroma (red arrow), additionally, stainings of follicles and some interfollicular cells

     

    For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Ab205921 specificity testing by Immunohistochemistry (KO testing): Loss of detection on KO Cells

    Strong IHC detection with anti-PD-L1 (ab205921, clone 28-8) is seen in human lung adenocarcinoma tumor cell line L2987. PDL1 gene was edited in L2987 cells using TALEN constructs targeting exon4 of human PD-L1, transcript variant 1 (NM_014143.3) and complete knock out (K.O) confirmed by deep sequencing in clone L2-14. IHC detection is completely eliminated in the L2987 L2-14 K.O. cell line.

     

    For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).



  • Predicted band size : 33 kDa

    Primary ab Dilution 1:100 dilution, Secondary ab Goat Anti-Rabbit IgG, (H+L), HRP conjugated (ab97051), 1:20,000 dilution, Blocking and diluting buffer and concentration 5% NFDM/TBST, Lane 1: NCI-H1975 (Human non-small cell lung cancer epithelia), Observed MW 40-60 kDa, Exposure time 3 min.

    For recommended Western Blot (WB) protocol for endogenous PD-L1 expression, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Immunohistochemical staining of PD-L1 in formalin fixed, paraffin embedded human non-squamous non-small cell lung cancer (NSQ-NSCLC) using ab205921 at a dilution of 1/400, incubated for an hour at room temperature. Heat mediated antigen retrieval was carried out in low pH buffer and the sample was blocked with peroxidase blocking buffer for 3 minutes.

    This image was courteously provided by Dr. Kai Schmitt from the Institute of Pathology, Saarbrücken-Rastpfuhl.

    See Abreview

  • Immunohistochemical analysis of CHO PD-L1 cells with ab205921 at 2 µg/ml.
    High power view
    A) Rabbit IgG, 5 µg/mL. No staining
    B) Anti PD-L1, 2 µg/mL (ab205921 batches 1)
    C) Anti PD-L1, 2 µg/mL (ab205921 batches 3)
    D) Anti PD-L1, 2 µg/mL (ab205921 batches 4)
    E) Anti PD-L1, 2 µg/mL (ab205921 batches 5)
    F) Anti PD-L1, 2 µg/mL (ab205921 batches 6)
    G) Anti PD-L1, 2 µg/mL (ab205921 batches 7)

     

    All batches/lots (1,3,4,5,6,7) showed consistent results.


    Note strong, moderate, and weak (red, yellow, and white arrows respectively) plasma membrane staining of CHO PD-L1 transfected cells

     

    For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Immunohistochemical analysis of CHO Parental cells with ab205921 at 2 µg/ml.
    High power view
    A) Rabbit IgG, 5 µg/mL. No staining
    B) Anti PD-L1, 2 µg/mL (ab205921 batches 1)
    C) Anti PD-L1, 2 µg/mL (ab205921 batches 3)
    D) Anti PD-L1, 2 µg/mL (ab205921 batches 4)
    E) Anti PD-L1, 2 µg/mL (ab205921 batches 5)
    F) Anti PD-L1, 2 µg/mL (ab205921 batches 6)
    G) Anti PD-L1, 2 µg/mL (ab205921 batches 7)

     

    All batches/lots (1,3,4,5,6,7) showed consistent results.

     

    Note absence of PD-L1 expression in CHO parental cells.

     

    For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Immunohistochemical analysis of Human Lung NSCLC with ab205921 at 2 µg/ml.
    High power view
    A) Rabbit IgG, 5 µg/mL. No staining
    B) Anti PD-L1, 2 µg/mL (ab205921 batches 1)
    C) Anti PD-L1, 2 µg/mL (ab205921 batches 3)
    D) Anti PD-L1, 2 µg/mL (ab205921 batches 4)
    E) Anti PD-L1, 2 µg/mL (ab205921 batches 5)
    F) Anti PD-L1, 2 µg/mL (ab205921 batches 6)

     

    All batches/lots (1,3,4,5,6) showed consistent results.


    Note linear and complete or partial (arrows) PD-L1 staining of tumor cells. Tumor associated immune cells localized over the tumor margin exhibit positive plasma membrane staining (small arrows).

     

    For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human non-small cell lung cancer tissue labelling PD-L1 with ab205921. Staining can be seen in tumor associated macrophages and tumor cells.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling PD-L1 with ab205921 on Ventana Ultra. Tumor cells and immune cells show PD-L1 positive plasma membrane staining.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Paraformaldehyde-fixed, Triton X-100 permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell line) cells stained for PD-L1 (red) using ab205921 at 1/200 dilution in ICC/IF, followed by CF568 Donkey anti-rabbit IgG(H+L) secondary antibody at 1/500 dilution.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human non-small cell lung cancer tissue labelling PD-L1 with ab205921. Tumor cells and immuno cells localized within the stroma show PD-LA plasma membrane staining.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue labelling PD-L1 with ab205921. Tumor cells show weak and partial postive PD-L1 expresseion in the plasma membrane. PD-L1 positive tumor associated immunoe cells are also stained. 

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human tonsil tissue labeling PD-L1 with ab205921 at 2 µg/ml. Counterstained with Hematoxylin.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human head and neck squamous cell carcinoma tissue labeling PD-L1 with ab205921 at 2 µg/ml. Counterstained with Hematoxylin.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded PD-L1 negative Non-small cell lung carcinoma (NSCLC) tissue with ab205921 at 2 µg/ml. Counterstained with Hematoxylin.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Paraformaldehyde-fixed, paraffin-embedded human placenta tissue stained for PD-L1 using ab2205921 at 1/100 dilution in immunohistochemical analysis.

    See Abreview

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded L2987 (Human lung adenocarcinoma cell line with endogenous PD-L1 expression) cells labeling PD-L1 with ab205921 at 2 µg/ml. Counterstained with Hematoxylin.

    For antigen retrival buffer, Universal HIER antigen retrieval reagent (ab208572) was used.
    For IHC detection kit, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) is recommended.

  • Ab205921 specificity testing by Flow Cytometry (KO testing): Loss of detection on KO Cells
    Strong detection with anti-PD-L1 (ab205921, clone 28-8) TALEN constructs targeting exon4 of human PD-L1, transcript variant 1 (NM_014143.3) and complete knock out (K.O) confirmed by deep sequencing in clone L2-14. Cell surface staining is almost completely eliminated in the L2987 L2-14 K.O. cell line.

     

    For recommended Flow Cytometry (Flow Cyt) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).



  • Predicted band size : 33 kDa

    (A and B) Western blots of recombinant PD-L1 protein (Lane 1), cell lysates of CHO-PD-L1 (Lane 3), CHO (Lane 4), ES-2 (Lane 5) and Colo205 (Lane 6) cell lines. In B, anti-PD-L1 (ab205921, clone 28-8) was pre-incubated with purified recombinant PDL1 protein overnight at 4°C.

    Blank/no sample (Lane2). Lane 2 is blank on purpose.

    For recommended Western Blot (WB) protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

Protocols

References

This product has been referenced in:
  • Miyazaki T  et al. Assessment of PD-1 positive cells on initial and secondary resected tumor specimens of newly diagnosed glioblastoma and its implications on patient outcome. J Neurooncol N/A:N/A (2017). IHC ; Human . Read more (PubMed: 28447277) »
  • Gambichler T  et al. Expression of PD-L1 in keratoacanthoma and different stages of progression in cutaneous squamous cell carcinoma. Cancer Immunol Immunother N/A:N/A (2017). Read more (PubMed: 28501937) »

See all 39 Publications for this product

Customer reviews and Q&As

Filter by Application

Filter by Species

Filter by Ratings

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (U87MG glioblastoma)
Permeabilization
Yes - 0.1% v/v Triton X-100 pH 7.4, 5 min at RT
Specification
U87MG glioblastoma
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde
Username

Dr. Dimitra Kalamida

Verified customer

Submitted Sep 12 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (lymph node)
Antigen retrieval step
Heat mediated
Permeabilization
No
Specification
lymph node
Blocking step
ultratek kit, ScyTek Laboratories, Logan, Utah, USA as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 20°C
Fixative
Formaldehyde
Username

Sara Belluco

Verified customer

Submitted Sep 12 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rabbit Tissue sections (Colon/Tonsil)
Antigen retrieval step
Enzymatic - Buffer/Enzyme Used: pH9 20 mins 100oC
Specification
Colon/Tonsil
Blocking step
No blocking step used for 30 minute(s) · Concentration: 3%
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted May 30 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Ovarian Tumor)
Permeabilization
No
Specification
Ovarian Tumor
Blocking step
Peroxidase Blocking Solution + UV block as blocking agent for 10 minute(s) · Concentration: 100% · Temperature: 25°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted May 25 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (NSQ-NSCLC)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EnVision FLEX TRS, Low pH
Permeabilization
No
Specification
NSQ-NSCLC
Blocking step
EnV FLEX Peroxidase-Blocking Reagent as blocking agent for 3 minute(s) · Concentration: 100%
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted May 11 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Tumor generated by a carcinogen (DMBA) or PANC02 m)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer, pH6
Permeabilization
No
Specification
Tumor generated by a carcinogen (DMBA) or PANC02 m
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 15 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Placenta)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: TRIS-EDTA Buffer pH 9,0
Permeabilization
Yes - Commercial Wash Buffer with Tween
Specification
Placenta
Fixative
Paraformaldehyde
Username

Mr. Rudolf Jung

Verified customer

Submitted Feb 04 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Placenta)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EDTA pH8.5
Permeabilization
No
Specification
Placenta
Blocking step
Ventana blocking agent as blocking agent for 4 minute(s) · Concentration: 100% · Temperature: 36°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 04 2015

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