Anti-Human PD-L1 (Atezolizumab) [RG7446] — DyLight® 488

Anti-Human PD-L1 (Atezolizumab) [RG7446] — DyLight® 488

Product No.: LT1753

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Product No.LT1753
Clone
RG7446
Target
PD-L1
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
Programmed Death Ligand 1, B7-H1, PD-L1, CD274
Isotype
Human IgG
Applications
FC
,
WB

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Antibody Details

Product Details

Reactive Species
Human
Host Species
Human
Expression Host
HEK-293 Cells
Immunogen
Unknown
Product Concentration
0.2 mg/ml
Formulation
This DyLight® 488 conjugate is formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.4, 1% BSA and 0.09% sodium azide as a preservative.
Pathogen Testing
To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s recombinant biosimilar antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Regulatory Status
Research Use Only (RUO). Non-Therapeutic.
Country of Origin
USA
Shipping
Next Day 2-8°C
Excitation Laser
Blue Laser (493 nm)
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for Atezolizumab biosimilar antibody for staining cells in flow cytometry is ≤ 0.25 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application.
Additional Applications Reported In Literature ?
WB
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
This non-therapeutic biosimilar antibody uses the same variable region sequence as the therapeutic antibody Atezolizumab. Atezolizumab recognizes an epitope on mouse PD-L1. This product is for research use only.
Background
PD-1 is a 50-55 kD member of the B7 Ig superfamily. PD-1 is also a member of the extended CD28/CTLA-4 family of T cell regulators and is suspected to play a role in lymphocyte clonal selection and peripheral tolerance. The ligands of PD-1 are PD-L1 and PD-L2, and are also members of the B7 Ig superfamily. PD-1 and its ligands negatively regulate immune responses. PD-L1, or B7-Homolog 1, is a 40 kD type I transmembrane protein that has been reported to costimulate T cell growth and cytokine production. The interaction of PD-1 with its ligand PD-L1 is critical in the inhibition of T cell responses that include T cell proliferation and cytokine production. PD-L1 has increased expression in several cancers. Inhibition of the interaction between PD-1 and PD-L1 can serve as an immune checkpoint blockade by improving T-cell responses In vitro and mediating preclinical antitumor activity. Within the field of checkpoint inhibition, combination therapy using anti-PD1 in conjunction with anti-CTLA4 has significant therapeutic potential for tumor treatments. PD-L2 is a 25 kD type I transmembrane ligand of PD-1. Via PD-1, PD-L2 can serve as a coinhibitor of T cell functions. Regulation of T cell responses, including enhanced T cell proliferation and cytokine production, can result from mAbs that block the PD-L2 and PD-1 interaction.
Antigen Distribution
PD-L1 is present on T cells, B cells, NK cells, dendritic cells, IFN-γ activated endothelial cells, and monocytes.
Ligand/Receptor
PD-1 (PDCD1)
Research Area
Biosimilars
.
Cancer
.
Costimulatory Molecules
.
Immunology

Leinco Antibody Advisor

Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.

Research-grade Atezolizumab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISA by serving as the quantifiable basis for standard curves against which unknown serum concentrations are measured. These biosimilar standards are carefully validated to match the reference (originator) drug, ensuring assay precision, accuracy, and comparability across batches and laboratories.

Key details on their use:

  • Calibration Curve Construction: The ELISA uses a series of known concentrations of the Atezolizumab biosimilar to generate a standard (calibration) curve. These concentrations span the clinically relevant range (e.g., 50–12,800 ng/mL). All unknown serum samples are interpolated from this curve using their measured absorbance.

  • Reference Controls: In addition to calibration standards, quality control (QC) samples containing known concentrations of Atezolizumab biosimilar and/or originator are run with each assay. This ensures assay integrity and monitors performance over time by checking for recovery, linearity, and precision.

  • Biosimilar as Standard: Best practice in PK bridging ELISA for biosimilars is to establish a single PK assay with the biosimilar as the analytical (calibration) standard, allowing both the biosimilar and the reference (innovator) drug concentrations in study samples to be measured against the same curve. This reduces assay variability and facilitates direct pharmacokinetic comparison between products.

  • Assay Qualification and Validation: Before use, the biosimilar standard and the originator are compared in PK bridging studies to confirm bioanalytical equivalence. This involves rigorous evaluation of precision, accuracy, recovery, matrix effects, and linearity in human serum/plasma. Regulators expect such assays to be validated to EMA/FDA standards for use in clinical PK studies.

  • Calibration Source and Traceability: For Atezolizumab, standards in commercial ELISA kits are typically calibrated against TecentriqTM (originator drug) or against international standards (NIBSC/WHO) if available, often using research-grade biosimilar as the practical working standard.

  • Application in PK Bridging: In comparative PK studies (bridging studies), this setup enables objective quantification and statistical assessment of exposure equivalence between biosimilar and reference drug, since both are measured using the same qualified curve.

In summary, a research-grade Atezolizumab biosimilar is used as the quantifiable reference in the ELISA calibration curve, QC controls, and method validation process, enabling accurate, equivalent measurement of both biosimilar and reference drug concentrations in serum for regulatory pharmacokinetic bridging studies.

Standard flow cytometry protocols using a conjugated Atezolizumab biosimilar (e.g., PE- or APC-labeled) to validate PD-L1 expression or binding capacity generally involve direct staining of cells expressing PD-L1, titration of the antibody, and analysis of fluorescence intensity to quantify surface PD-L1 levels or binding affinity. This approach is suitable for both cell line validation and, in some contexts, patient or preclinical samples.

Essential Protocol Steps and Key Points

  • Antibody and Conjugate: Use directly labeled (e.g., APC- or PE-conjugated Atezolizumab biosimilar) that specifically binds human PD-L1 for direct flow cytometry staining.
  • Cell Preparation: Prepare target cells (e.g., tumor cell lines, PBMCs, or engineered cells expressing PD-L1) at appropriate densities (typically 0.5–1 x 10^6 per sample for standard tubes, fewer for plates).
  • Blocking (Optional): Some protocols use Fc receptor blockers or isotype controls to reduce nonspecific binding and permit background correction.
  • Antibody Titration: Serially dilute the conjugated antibody, often across a broad range (e.g., 0.98–1000 ng/ml, two-fold dilutions) to generate a binding curve and subsequently determine EC50 (half-maximal effective concentration) if needed.
  • Staining: Incubate cells with diluted conjugated antibody (either alone or in combination with experimental blocking competitors) for 30–60 minutes at 2–8°C in the dark.
  • Washing: Wash cells (typically ×2–3) with buffer such as PBS+1% BSA to remove unbound antibody and minimize background signal.
  • Fixing (Optional): For endpoint fixation, resuspend cells in buffer with 1–2% paraformaldehyde (if necessary).
  • Detection and Analysis: Acquire data by flow cytometry using appropriate lasers and filter sets (e.g., red laser for APC). Analyze the geometric mean fluorescence intensity (gMFI) or percentage of positive cells to quantify binding or expression.
  • Data Interpretation: Compare fluorescence intensity with isotype controls, or (if performing blocking or competition assays) measure the reduction in signal to confirm specificity and calculate binding parameters such as EC50.

Example – Use Case and Reporting

A study evaluating the binding of Atezolizumab to PD-L1-expressing cells by flow cytometry serially titrated the antibody, stained cells, and detected the bound antibody using an APC-conjugated anti-human IgG secondary (in the absence of a directly labeled antibody, but this is analogous to the use of direct conjugates). The mean fluorescence intensity correlates with available surface PD-L1, and binding curves are used to calculate affinity parameters such as EC50.

Commercial sources provide APC-conjugated Atezolizumab biosimilar for research specifically validated for flow cytometry and recommend titration to optimize staining for the precise sample type.

Additional Considerations

  • Blocking Epitope Consideration: If other anti-PD-L1 antibodies are used in combination (for example, in multiplex panels), Atezolizumab may block detection by other anti-PD-L1 antibodies targeting overlapping epitopes.
  • Alternative Detection: For indirect approaches, an unconjugated biosimilar can be used, detected by a secondary anti-human IgG, but direct conjugates (APC or PE) are preferred for single-step protocols.
  • Validation: Proper controls, including PD-L1-negative cells and isotype controls, should always be included to interpret specificity and baseline signal.

In summary: The standard protocol is: label cells with titrated direct Atezolizumab-conjugate, incubate, wash, and analyze by flow cytometry, using controls to establish background and specificity. This method robustly validates PD-L1 expression levels and antibody binding capacity on various cell types.

Biopharma companies typically perform a comprehensive set of analytical assays to confirm the structural and functional similarity of a proposed biosimilar to the originator drug, using validated and highly sensitive techniques to examine both structural features and biological activity.

Core Analytical Assays for Biosimilar Characterization

  • Structural Characterization

    • Primary Structure: Peptide mapping and mass spectrometry to confirm amino acid sequence.
    • Higher-Order Structure: Techniques such as circular dichroism (CD), nuclear magnetic resonance (NMR), and X-ray crystallography to assess secondary, tertiary, and quaternary structure.
    • Physicochemical Properties: Assessments of isoelectric point, molecular weight, and charge variants using high-performance liquid chromatography (HPLC), capillary electrophoresis, and related methods.
    • Post-Translational Modifications: Glycosylation profiling and analysis of disulfide bonds.
    • Impurity and Aggregate Profiling: Quantification of process- and product-related impurities and aggregates, typically using size-exclusion chromatography, electrophoresis, and mass spectrometry.
  • Functional Characterization

    • Biological Activity Assays: Cell-based or reporter assays that measure the drug's intended biological effect.
    • Binding Assays: Assessment of target binding (e.g., ELISA, surface plasmon resonance) and receptor binding properties to establish equivalence in mechanism of action.
    • Enzyme Kinetics and Potency Assays: Functional potency measurements to directly compare the biosimilar’s activity to the reference.
  • Comparative Assessment Approach

    • These analyses are always done head-to-head with both the biosimilar and multiple lots of the originator (reference) drug to ensure high similarity across critical quality attributes (CQAs).
    • Multiple orthogonal methods (different, independent techniques probing the same attribute) are used to increase sensitivity for detecting differences.

Regulatory and Scientific Context

  • Regulatory agencies like the FDA and EMA require that critical quality attributes (CQAs) with possible impact on safety or efficacy are identified and matched as closely as possible.
  • Analytical similarity is a prerequisite before moving to animal studies or clinical trials, and robust data here reduces the required burden for additional clinical studies.

About Leinco Biosimilars

While the search results do not explicitly mention Leinco’s specific use in biosimilar similarity studies, Leinco Technologies is known in the life science industry for providing reference standards and well-characterized biosimilar antibodies that serve as benchmarks in analytical method development and comparability exercises. In biosimilar analytical studies, a “Leinco biosimilar” could be used as either:

  • A well-characterized reference standard (for performance qualification of assay systems or as a comparison), or
  • A model biosimilar used in case studies to validate analytical platforms, processes, and to confirm assay sensitivity and robustness during similarity studies.

In summary, biopharma companies rely on a battery of structural and functional assays, including peptide mapping, higher-order structure analyses, impurity profiling, binding, and potency assays, conducted in a comparative, head-to-head manner—often making use of high-quality standards or biosimilars like those provided by Leinco for reference or assay development purposes.

References & Citations

1. Ardolino, M. et al. (2018) J Clin Invest. 128(10):4654-4668. PubMed
2. Schreiber, RD. et al. (2017) Cancer Immunol Res. 5(2):106-117.
3. Freeman, G. et al. (2000) J. Exp. Med. 192:1027.
Flow Cytometry
General Western Blot Protocol

Certificate of Analysis

Formats Available

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Disclaimer AlertProducts are for research use only. Not for use in diagnostic or therapeutic procedures.