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The immune system employs a series of checkpoints to protect normal, healthy tissue from an immune response. These consist of receptors on the surface of activated T cells and their corresponding ligands on the surface of antigen presenting cells. A key immune checkpoint is triggered when PD-1 (programmed cell death protein 1) engages its ligand PD-L1. As a result of this interaction, T cell activation is attenuated and an active immune response is prevented(1).

This mechanism is often co-opted by tumors. PD-L1 is upregulated in several tumor types and contributes to the malignancy of these cancers by interacting with PD-1 and inhibiting T cell activation. In this way, the tumors avoid detection and destruction by the immune system(1-3). Accordingly, PD-1 and PD-L1 have garnered much attention for their roles in tumor immunology and as immune-based therapeutic targets(4,5).

CST PD-L1 product comparison for IHC-P analysis

Product Name PD-L1 (E1L3N®) XP® Rabbit mAb #13684 PD-L1 (405.9A11) Mouse mAb #29122 PD-L1 (Extracellular Domain Specific) (E1J2J) Rabbit mAb #15165
Clone E1L3N® 405.9A11 E1J2J
Epitope Intracellular Intracellular Extracellular
Antibody host species Rabbit Mouse Rabbit
IHC analysis of paraffin-embedded human placenta IHC Placenta for PD-L1 (E1L3N) IHC Placenta for PD-L1 (405.9A11) IHC Placenta for PD-L1 (Extracellular Domain Specific)
Recommended usage for IHC Preferred clone based on sensitivity Use when multiplexing with rabbit antibodies Use for studying the extracellular domain of PD-L1

PD-L1 (E1L3N®) Antibody - Target Specificity

Recognizes PD-L1 and does not cross-react with other B7 family members

PD-L1 Western Blotting

Western blot analysis of lysates from:

  1. COS cells transfected with PD-L2
  2. COS cells mock transfected
  3. KARPAS-299 cells
  4. SUP-M2 cells

PD-L1 (E1L3N®) Antibody - Flow Validation

Demonstrates consistent, reliable results in immunohistochemistry, flow cytometry, western blotting and immunoprecipitation.

PD-LI Flow Cytometry

Flow cytometric analysis of untreated SUP-M2 cells using PDL1 (E1L3N®) XP® Rabbit mAb #13864 (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

PD-L1 (E1L3N®) Antibody Comparative Data: IHC

KARPAS-299 and PC-3 Cell Staining

  CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 1:200 Company 1 Rabbit mAb 1:8000 Company 2 Mouse mAb 1:50
KARPAS-299 Karpas 299 CST Karpas 299 Company 1 Karpas 299 Company 1
Strong, membrane associated staining Weaker membrane staining than #13684 No membrane staining
PC-3 PC3 CST Company 1 CST Company 2 CST
No staining Diffuse, Non-specific staining Diffuse, Non-specific staining

IHC analysis demonstrates that CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 at 1:200 gives a strong signal in KARPAS-299, which are known to be PD-L1 high expressing cells, and no staining in PC-3 cells. In contrast, Company 1 Rabbit mAb and Company 2 Mouse mAb stain both KARPAS-299 and PC-3 cells meaning that they are not specific for PD-L1.

Lung and Breast Carcinoma

  CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 1:200 Company 1 Rabbit mAb 1:8000
Lung Carcinoma Lung Carcinoma with CST #13684 Lung Carcinoma with Company #2
Predominantly membrane staining in lung carcinoma and no staining in stromal cells. Staining in cytoplasm and membrane in both lung carcinoma and stromal cells.
Breast Carcinoma Breast Carcinoma with CST #13684 Breast Carcinoma with Company #2
No staining in PD-L1 negative breast carcinoma. Non-specific nuclear and cytoplasmic staining in the PD-L1 negative breast carcinoma.

In IHC analysis of human lung and breast carcinoma CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 demonstrates appropriate staining in lung carcinoma with no staining in surrounding stromal cells, and no staining in breast carcinoma. Company 1 Rabbit mAb, however, demonstrates staining in both lung carcinoma and surrounding stromal cells, and inappropriate staining in breast carcinoma. This antibody is detecting a protein that is not PD-L1.

PD-L1 (E1L3N®) Antibody Comparative Data: WB

PD-L1 Western Blotting

Western blot analysis using CST PD-L1 (E1L3N®) XP® Rabbit mAb #13684 results in detection of a band at the appropriate molecular weight for PD-L1 in KARPAS-299 and SUP-M2 cells, which are known to have high PD-L1 expression, and no protein in A549 and PC-3, cells demonstrating that #13684 is specific for PD-L1. At the dilution recommended by the supplier, Company 1 Rabbit mAb produces multiple signals at various molecular weights in A549, PC-3, KARPAS-299, and SUP-M2 cells, demonstrating that this antibody is not specific for PD-L1.