Validated using a knockout cell line
Recombinant
RabMAb

Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889)

Overview

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

    Recombinant full length protein within Human PD-L1. The exact sequence is proprietary.
    Database link: Q9NZQ7

  • Positive control
    • Tissue: Human tonsil and head and neck squamous cell carcinoma tissues; L2987 cell line. Cell Lines: Positives: B-CPAP- high, ES-2- medium, HCC70 - low 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

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

    Additional information on positive controls:

    Tissue:
    Tonsil- with hyperreactive changes
    Note: Tonsil Specimens- is recommended to screen several 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
    Note: Tumor Specimens- PD-L1 expression varies by tumor type so screening is recommended to find positive and negative tumor controls. Refer to web link publication below to find some suggested tumor types. Many tumor specimens have some inflammatory macrophages and mononuclear leukocytes. Best to look for specimens with high numbers of these cells

    Cell Lines:
    Positives: B-CPAP- high, ES-2- medium, HCC70 - low

    For primary negative control, isotype control, RabMAb negative control antibody (ab172730) is recommended.

    For negative control sample, cell line COLO205 is recommended.

    For PD-L1 protein, see ab167713

    Recommended protocols:

    For recommended Western Blotting (WB) protocol, please refer to the protocol book (line 3) in the protocol section.

    For recommended Immunohistochemistry (IHC) protocol, please refer to the protocol book (line 2) in the protocol section and/or here (downloadable copy)

    For IHC usage on FFPE tissues, the following antigen solution is recommended with clone 28-8 -  Universal HIER antigen retrieval reagent (ab208572) KO Validated

    For recommended Flow Cytometry (Flow Cyt) protocol, please refer to the protocol book (line 1) in the protocol section and/or here (downloadable copy)

    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. 
    For colormetric detection of PDL1, it is recommended to use Anti-PD-L1 antibody (ab58810)

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    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 ab209889 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 at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB Use at an assay dependent concentration. Detects a band of approximately 33-43 kDa (predicted molecular weight: 33 kDa).

Please check the parent abID, ab205921, for more information on dilutions.

ICC/IF Use at an assay dependent concentration.

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

  • Representative images of PD-L1 expression in human lung adenocarcinoma and squamous cell carcinoma specimens.

    (A) <1.0%, (B) 1.0–4.9%, (C) 5.0–9.9%, (D) 10.0–49.9%, and (E) ≥50.0% PD-L1-positive cells.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205921).

  • This IHC data was generated using the same anti-PDL1 antibody clone, 28-8, in a different buffer formulation (cat# ab205921).

    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.

  • Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889) + NCI-H1975 (human non-small cell lung cancer) whole cell lysate at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

    Predicted band size: 33 kDa
    Observed band size: 40-60 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking buffer and concentration: 5% NFDM/TBST

    Diluting buffer and concentration: 5% NFDM/TBST

  • All lanes : Anti-PD-L1 antibody [28-8] - Low endotoxin, Azide free (ab209889)

    Lane 1 : CHO-S cell lysate
    Lane 2 : Human PD-L1 transfected CHO-S cell lysate

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

    Predicted band size: 33 kDa
    Observed band size: 40-60 kDa why is the actual band size different from the predicted?


    Exposure time: 1 minute


    Blocking buffer and concentration: 5% NFDM/TBST

    Diluting buffer and concentration: 5% NFDM/TBST

     

References

ab209889 has not yet been referenced specifically in any publications.

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