Anti-Human IL-6R (Sarilumab) – Fc Muted™ Biotin

Anti-Human IL-6R (Sarilumab) – Fc Muted™ Biotin

Product No.: LT1706

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Product No.LT1706
Clone
Hu137
Target
IL-6
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
Interleukin-6, CDF; HGF; HSF; BSF2; BSF-2; IFNB2; IFN-beta-2
Isotype
Human IgG1κ
Applications
ELISA
,
FA
,
FC

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Antibody Details

Product Details

Reactive Species
Human
Host Species
Human
Expression Host
HEK-293 Cells
FC Effector Activity
Muted
Product Concentration
0.5 mg/ml
Formulation
This Biotinylated antibody 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.
Storage and Handling
This biotinylated antibody is stable when stored at 2-8°C. Do not freeze.
Regulatory Status
Research Use Only (RUO). Non-Therapeutic.
Country of Origin
USA
Shipping
Next Day 2-8°C
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for Alemtuzumab biosimilar antibody for staining cells in flow cytometry is ≤ 1.0 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application.
ELISA
Additional Applications Reported In Literature ?
FA
Additional Reported Applications For Relevant Conjugates ?
B
N
IHC (Paraffin)
IHC (Frozen)
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 antibody uses the same variable region sequence as the therapeutic antibody Sarilumab. Sarilumab binds to the transmembrane and soluble forms of the IL-6 receptor. This product is for research use only.
Background
IL-6 and its signaling pathway play a part in immune response regulation, inflammation, and hematopoiesis.2 Sarilumab is a research-grade recombinant human monoclonal IL-6 receptor antagonist. It specifically binds to both the transmembrane and soluble forms of the IL-6 receptor, thus inhibiting IL-6–mediated cis and trans-signaling in a dose-dependent manner.1 Therapeutic Sarilumab, also known by the trade name Kevzara, is currently used to treat Rheumatoid Arthritis1, however, as of March 2020, The Feinstein Institute of Northwell Health publicized a study on "a human antibody that may prevent the activity" of IL-6 for the treatment of COVID-19.3 Anti-Human IL-6 (Sarilumab) utilizes the same variable regions from the therapeutic antibody Sarilumab making it ideal for research projects.
Antigen Distribution
IL-6R is ubiquitously expressed.
PubMed
NCBI Gene Bank ID
Research Area
Biosimilars
.
Cell Biology
.
Immunology
.
Innate Immunity
.
Neuroscience
.
Other Molecules

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 Sarilumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs to generate standard curves and ensure accurate quantification of drug concentration in serum samples. The underlying principle is to provide a consistent, well-characterized material to calibrate the assay, enabling comparison of biosimilar and reference products during method validation and in analytical runs.

Essential context and process details:

  • Calibration Standards: The biosimilar (or reference Sarilumab) is serially diluted in a relevant biological matrix (typically human serum) to create a standard curve. This curve covers the full expected range of Sarilumab concentrations in study samples, for example: 50 to 12,800 ng/mL.
  • Reference Controls: Quality control (QC) samples made from the biosimilar and/or reference drug at known concentrations are included within each run to monitor assay performance (precision, accuracy, reproducibility).
  • Single Standard Approach: Regulatory and industry best practice is to use a single, well-characterized biosimilar or reference Sarilumab as the analytical calibrator for both biosimilar and reference product quantification. The choice is justified by prior demonstration of bioanalytical equivalence between biosimilar and reference in parallel testing. This minimizes cross-assay variability and confounding factors.
  • Bridging ELISA Format: In a PK bridging ELISA, the immunoassay setup typically involves:
    • Capture: One anti-Sarilumab antibody is coated onto the plate.
    • Detection: Another, often biotinylated, anti-Sarilumab antibody binds the drug, allowing for quantification (often via HRP-streptavidin and colorimetric detection).
    • Structural Principle: Both reference and biosimilar Sarilumab must be equally recognized by the assay antibodies—this equivalence is separately tested during assay validation.

Typical Steps in PK Bridging ELISA Calibration:

  1. Prepare a dilution series of Sarilumab biosimilar (calibrator) in human serum.
  2. Load wells in triplicate with each standard and test sample.
  3. Run detection and readout steps per kit protocol (incubation, washing, HRP/TMB, read plate at 450 nm).
  4. Generate a standard curve from calibration points; calculate unknown sample concentrations by interpolation.

Key Considerations:

  • Assay validation requires that the biosimilar and reference (if both are used) show parallelism and equivalence in response across the dynamic range, with comparability ideally shown within tight predefined limits (e.g., 0.8-1.25 fold range in relative response).
  • Using biosimilar standards helps demonstrate the interchangeability of biosimilar and reference in the test system, ensuring that PK data reflect true pharmacokinetic behavior, not assay bias.

Summary Table: Use of Sarilumab Biosimilars in PK Bridging ELISA

PurposeHow Biosimilar Is UsedSupporting Details
Calibration StandardSerially diluted to form standard curveStandards in human serum, broad concentration range
Reference Control (QC)Spiked at fixed concentrations for quality checksUsed to monitor assay accuracy, precision
Assay Comparability CheckBiosimilar vs. reference analyzed for equivalencyParallelism and equivalence required

This approach ensures rigorous, reproducible measurement of Sarilumab concentrations for both biosimilar and reference products in PK studies.

The standard flow cytometry protocol for validating IL-6 receptor (IL-6R) expression or binding capacity using a conjugated Sarilumab biosimilar antibody (e.g., PE or APC-labeled) follows conventional procedures for surface protein staining, with specific optimizations for antibody titration and receptor quantification.

Key steps and considerations include:

  • Antibody Dilution: Use ≤1.0 μg per 10⁶ cells in 100 μl staining buffer as a starting concentration for directly conjugated Sarilumab biosimilars (e.g., APC or PE). Titration is recommended to optimize for signal and minimize background.

  • Sample Preparation: Prepare single-cell suspensions, typically from PBMCs or cell lines expressing human IL-6R. Wash cells in PBS with 1% BSA or similar buffer to minimize non-specific binding.

  • Blocking: To reduce Fc receptor-mediated non-specific staining, include an Fc block (e.g., human IgG or Fc receptor-blocking reagent) during incubation, especially for primary human cells.

  • Staining:

    • Incubate cells with the conjugated Sarilumab biosimilar antibody for 30 minutes at 4°C (or room temperature, depending on antibody stability and manufacturer instructions).
    • For unconjugated formats, follow with a fluorochrome-labeled secondary antibody (e.g., APC-anti-human IgG), ensuring appropriate controls.
  • Washing: Wash cells at least twice in staining buffer after antibody incubation to remove unbound antibody and reduce background.

  • Data Acquisition: Use the appropriate laser (e.g., red laser for APC detection), observing compensation controls if multiplexing with other markers.

  • Quantification: To quantify receptor density or binding capacity, utilize fluorescence quantitation beads, such as QuantiBRITE PE beads, and translate mean fluorescence intensity (MFI) to number of molecules per cell based on the antibody-to-fluorochrome ratio.

Controls and Validation:

  • Include isotype-matched controls to assess non-specific binding.
  • Use irrelevant antibody controls, as well as cell lines with known expression (positive/negative for IL-6R), to validate specificity.

Example Application:

  • HEK293 cells transfected with human IL-6R and eGFP can be stained with APC-Sarilumab biosimilar, then analyzed for co-expression and specific binding; irrelevant protein-transfected cells serve as negative controls.
  • Endogenous IL-6R-expressing cell lines (e.g., U937) can be similarly stained to assess natural receptor presence and biosimilar binding.

Additional Notes:

  • Incubation times and temperatures, as well as blocking and washing steps, should be optimized per experiment to maximize specificity and reproducibility.
  • Protocols for membrane-associated proteins apply directly to IL-6R detection using Sarilumab biosimilar reagents.

In summary, conjugated Sarilumab biosimilars are used in flow cytometry by standard surface staining protocols with titrated antibody concentrations and suitable controls, and quantitation is possible using standardized fluorescence beads to assess IL-6R expression or binding.

Biopharmaceutical companies confirm the structural and functional similarity of a proposed biosimilar to the originator drug primarily through an extensive battery of analytical assays targeting both structure and function, followed by direct head-to-head comparisons between biosimilar and reference product.

Common analytical assays include:

  • Primary structure analysis: Peptide mapping and mass spectrometry to confirm identical amino acid sequences.
  • Higher order structure: Circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and differential scanning calorimetry to compare secondary and tertiary structures.
  • Post-translational modification analysis: Examination of glycosylation patterns and other modifications by mass spectrometry or chromatography.
  • Purity and impurity profiling: Size-exclusion chromatography and capillary electrophoresis to detect and quantify aggregates and fragments.
  • Functional assays:
    • Binding assays: Measurement of the biosimilar’s binding affinity to its target (e.g., receptor or antigen), often using surface plasmon resonance or ELISA methods.
    • Biological (cell-based) assays: Assessment of the in vitro biological activity, such as cell killing, proliferation inhibition, or activation of effector functions, to verify that clinical mechanism of action is preserved.
    • Fc receptor binding (for antibodies): Comparison of interactions with Fc receptors to assess potential immune effector functions.
  • Stability studies: Accelerated stability and forced degradation tests to assess biosimilar robustness under various stress conditions.

These assays are often executed using orthogonal approaches—that is, employing multiple, independent analytical platforms to confirm findings and mitigate method-specific limitations.

Analytical Similarity Assessment:

  • All critical quality attributes (CQAs) relevant to clinical outcomes—such as identity, potency, purity, and safety—must be identified and assessed using validated assays, guided by regulatory requirements.
  • Biosimilar manufacturers conduct side-by-side comparisons across multiple lots to ensure detected variation is within a predefined, non-clinically meaningful range.

Role of Leinco Biosimilars:

Leinco Technologies supplies research-grade biosimilars which are predominantly used as reference standards and test comparators in these analytical studies (not as therapeutics themselves).

  • Leinco biosimilars are typically used to:
    • Benchmark analytical and functional assays.
    • Serve as positive controls in method development.
    • Validate the performance and sensitivity of critical assays such as ELISA, cell-based bioassays, or Fc-binding assessments.
    • Support assay validation and system suitability testing before clinical-grade comparators are introduced.

In summary, biopharma companies utilize a combination of sophisticated structural and functional analytical assays—employing standards such as Leinco biosimilars for method development and benchmarking—to rigorously establish the similarity of proposed biosimilars to originator drugs, as required by regulatory agencies.

References & Citations

1. Kevzara (sarilumab) injection [prescribing information]. Tarrytown, NY: Regeneron Pharmaceuticals; Bridgewater, NJ: sanofi-aventis U.S.; May 2017.
2. Yoshida Y, Tanaka T. Interleukin 6 and rheumatoid arthritis. Biomed Res Int. 2014;2014:698313.
3. "Northwell Health Initiates Clinical Trials of 2 COVID-19 Drugs". 21 March 2020.
Indirect Elisa Protocol
FA
Flow Cytometry

Certificate of Analysis

Formats Available

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