Anti-Human PD-L1 (CD274) [Clone 29E.2A3] — Purified in vivo GOLD™ Functional Grade

Clone 29E.2A3 is a mouse monoclonal antibody that targets human PD-L1 (CD274, B7-H1) and is widely used in in vivo mouse studies to block human PD-L1 signaling and to investigate mechanisms of immune checkpoint blockade, particularly in tumor immunology.

Common in vivo applications in mice include:

• Blocking PD-L1 signaling to study immune checkpoint inhibition and the functional consequences of disrupting the PD-1/PD-L1 interaction.
• Assessment of anti-tumor immune responses: The antibody is used in humanized mouse models (e.g., xenograft models with human immune and/or tumor cells) to test the enhancement of T cell-mediated tumor rejection in the context of immunotherapy research.
• Therapeutic studies of immune modulation: Investigations include combination therapies with other checkpoint inhibitors to model and optimize clinical immunotherapy regimens.
• Functional cellular assays in vivo: Beyond cancer, studies may evaluate acute and chronic infection models or autoimmune responses by modulating human PD-L1 in reconstituted mouse systems.

Key details:

• Clone 29E.2A3 is specific to human PD-L1 and does not cross-react with murine PD-L1; thus, it is effective mainly in mice engrafted with human immune system components or human tumor cells.
• The main mechanism is blocking the interaction between PD-L1 and PD-1, leading to increased T cell proliferation and cytokine production, thereby reversing immune suppression.
• It is frequently used in preclinical evaluation of PD-L1 checkpoint blockade therapies and as a tool for dissecting the biology of human PD-L1 signaling in vivo.

It is not suitable for Western blotting or detecting mouse PD-L1; its use is directed at functional in vivo studies involving human PD-L1 only.

The 29E.2A3 antibody is a monoclonal antibody specific for human PD-L1 (CD274, B7-H1) and is widely used in immunology and tumor immunology research. In the literature, it is commonly used alongside antibodies targeting related proteins involved in immune checkpoint pathways and immune cell characterization.

Frequently co-used antibodies or proteins include:

• PD-1 (CD279): As PD-L1 is a ligand for PD-1, functional studies and detection panels often pair anti-PD-L1 (29E.2A3) with anti-PD-1 antibodies to assess checkpoint signaling in T cells and tumor immune evasion.
• CD3, CD4, CD8: These are core T-cell markers. In flow cytometry, researchers frequently analyze PD-L1 expression on tumor cells or immune infiltrates in tandem with T cell markers to study immune contexture.
• CTLA-4: Another major immune checkpoint receptor, CTLA-4 (CD152) is often studied with PD-L1 and PD-1 to compare or combine checkpoint blockade strategies.
• CD28: As a co-stimulatory T cell receptor, CD28 is sometimes included to explore the broader regulation of T cell activation states alongside inhibitory checkpoints.
• Isotype controls & secondary detection reagents: Goat anti-mouse secondary antibodies are commonly paired with 29E.2A3 for detection in flow cytometry or immunohistochemistry panels.
• Tumor or immune markers (e.g., HLA-DR, CD45): In tumor microenvironment studies, researchers often combine PD-L1 detection with lineage or activation markers to characterize different cell populations expressing PD-L1.

Published studies using 29E.2A3 commonly employ multicolor flow cytometry panels where PD-L1 detection is combined with checkpoint, lineage, and activation markers for immunophenotyping or tumor-immune profiling.

Summary Table of Commonly Used Antibodies/Proteins with 29E.2A3:

Specific references and reagent providers such as BioLegend, ThermoFisher, and others confirm these co-usage practices in flow cytometry and immunohistochemistry.

The key findings from scientific literature regarding clone 29E.2A3 center on its specificity, functional applications, and a notable mode of antibody-fluorochrome interaction:

• Specificity and Epitope Recognition: Clone 29E.2A3 is a monoclonal antibody that specifically recognizes human PD-L1 (CD274, B7-H1), binding an epitope within the PD-L1–CD80 binding region.

• Applications:

  • Widely used for flow cytometry, immunohistochemistry, and blocking assays in immunological studies.
  • It is suitable for detecting PD-L1 expression on various immune cell types and tumor cells with surface and intracellular staining.
  • Recommended for functional assays due to low endotoxin and azide-free preparations.

• Blocking Function: Clone 29E.2A3 can block the interaction between PD-L1 and PD-1, making it useful for studying immune checkpoint pathways.

• Immunofluorescence Cross-Reactivity: A critical finding is that clone 29E.2A3 can directly bind to certain fluorochromes, notably Alexa Fluor® 700 (AF700), when used in multi-color flow cytometry. This cross-reactivity can produce false-positive PD-L1 detection and can disrupt fluorescence intensity, a problem not present with some other anti-PD-L1 clones. Sequential antibody staining or selecting alternative clones is recommended to avoid artifacts in multi-color panels.

• Expression Profile: PD-L1 detected by 29E.2A3 is abundant on many human cancers and upregulated by interferon gamma stimulation.

  • PD-L1 expression assessed with 29E.2A3 is used in cancer immunotherapy and immune regulation research.

• Product Reference: Clone 29E.2A3 is used extensively as a reference standard for PD-L1 detection in comparative studies.

Summary Table of Core Findings

The most critical insights pertain to both its epitope specificity and blocking capacity and the need for rigorous antibody validation to avoid cross-reactivity-induced artifacts in immunofluorescence experiments.

The dosing regimens of clone 29E.2A3 (anti-human PD-L1 antibody) in mouse models are not standardized and appear to vary significantly depending on the experimental context, mouse model used, and the target expression (human vs. mouse PD-L1). This is in contrast to well-established anti-mouse PD-L1 antibody clones (such as 10F.9G2), which have clearly defined in vivo dosing protocols in mouse models.

Key details:

• Clone 29E.2A3 is specific for human PD-L1 and is commonly used in flow cytometry, blocking assays, and immunohistochemistry for detection of human PD-L1. Its use in vivo in mice typically requires models expressing human PD-L1, such as humanized mice or xenograft systems with human tumors.
• Dosing guidance is limited: There is no universally established in vivo dose or schedule for clone 29E.2A3 in published protocols. This is due both to the human specificity and the variety of humanized mouse models (characterized by different engraftment and PD-L1 expression profiles).
• Examples from suppliers and literature:
  • Leinco Technologies explicitly notes that “dosing regimens for clone 29E.2A3 are not standardized across mouse models, as its primary applications are in vitro assays and not for routine in vivo use in mice,” highlighting the lack of dosing consensus for in vivo experiments.
  • Most published in vivo mouse studies of PD-L1 blockade use antibodies specific for mouse PD-L1 (such as clone 10F.9G2), typically at 100–250 μg per mouse intraperitoneally, 2–3 times per week. These regimens cannot be directly applied to clone 29E.2A3 unless validated for the specific humanized system in use.
• In vivo use of 29E.2A3 in humanized mice is possible but requires model-specific titration and validation to establish both efficacy and lack of off-target effects. Published protocols typically do not report detailed dosing for this clone.

Summary Table: Mouse anti–human PD-L1 antibody dosing (clone 29E.2A3)

For in vivo studies in humanized mice, dosing should be empirically optimized, often starting with amounts similar to other in vivo antibodies (e.g., 100–250 μg per mouse per dose, intraperitoneally), but adjusted according to model response and pilot experiments. Always reference the experimental literature for your specific model and consult with the antibody supplier for additional guidance.