Anti-Human IL-2Rα (CD25) (Basiliximab) [Clone Hu107] — PE

Anti-Human IL-2Rα (CD25) (Basiliximab) [Clone Hu107] — PE

Product No.: LT304

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Product No.LT304
Clone
Hu107
Target
CD25
Product Type
Biosimilar Recombinant Human Monoclonal Antibody
Alternate Names
IL-2-RA; IL2-RA; TAC antigen; p55; CD25
Isotype
Human IgG1κ
Applications
FC
,
IF
,
IHC

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Select Product Size
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Antibody Details

Product Details

Reactive Species
Cynomolgus Monkey
Rhesus Monkey
Human
Host Species
Human
Expression Host
HEK-293 Cells
FC Effector Activity
Active
Immunogen
Human CD25
Product Concentration
0.2 mg/ml
Formulation
This R-phycoerythrin (R-PE) 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.
Storage and Handling
This R-phycoerythrin (R-PE) conjugate 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
Excitation Laser
Blue Laser (488 nm) and/or Green Laser (532 nm)/Yellow-Green Laser (561 nm)
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for Basiliximab 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.
Additional Applications Reported In Literature ?
IF
IHC
Additional Reported Applications For Relevant Conjugates ?
CyTOF®
WB
B
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 Basiliximab. Basiliximab recognizes human CD25. This product is for research use only.
Background
CD25, a 55 kD type I transmembrane glycoprotein, has been shown to play roles in lymphocyte differentiation, activation, and proliferation. Many resting memory T cells constitutively express IL2Rα. It functions as the receptor for HTLV-1, resulting in its expression on neoplastic cells in adult T cell lymphoma/leukemia. CD25 (sIL-2R) has been used to track disease progression. Some additional clinical applications include Chagas disease, a disease characterized by a decline of CD25 expression on immune cells, and Multiple sclerosis, in which treatments with mAbs target CD25. Anti-Human IL-2R alpha (CD25) (Basiliximab) utilizes the same variable regions from the therapeutic antibody Basiliximab making it ideal for research projects.
Antigen Distribution
IL-2Rα is expressed on activated mature T and B lymphocytes, during early stages of thymocytes development, pre-B cells, and in activated CD4+ memory T-lymphocytes.
PubMed
NCBI Gene Bank ID
Research Area
Biosimilars

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 Basiliximab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs to accurately quantify Basiliximab concentrations in serum samples.

In the context of a PK bridging ELISA:

  • Biosimilars serve as surrogates for the original (reference) pharmaceutical Basiliximab, addressing issues of cost, availability, and scale encountered when sourcing clinical-grade drug for research use.
  • Calibration standards are prepared using known concentrations of research-grade Basiliximab biosimilar, which are serially diluted to generate a standard curve spanning the assay's dynamic range (e.g., 0–2000 ng/mL).

PK Bridging ELISA workflow with biosimilar calibration standards:

  • Microwells are coated with anti-Basiliximab antibodies (capture antibody).
  • Serum samples and biosimilar calibration standards are added.
  • The Basiliximab in standards and samples binds the capture antibody.
  • HRP-conjugated anti-Basiliximab (detection antibody) is added.
  • After binding and washing, a colorimetric substrate is introduced, with absorbance proportional to Basiliximab quantity.
  • Absorbance readings from biosimilar standards are used to construct a calibration curve, allowing interpolation of drug concentration in test samples by comparison.
  • Biosimilar antibodies are also used as reference controls to verify assay performance, consistency, and equivalency between test and reference materials.

Advantages of using research-grade biosimilars:

  • Highly similar in structure and binding profile to the original Basiliximab, ensuring assay validity and accuracy for PK studies.
  • Enable robust assay development and validation without needing pharmaceutical-grade drug, which may be expensive or restricted.

Additional context:

  • Biosimilar calibration standards are validated to ensure analytical similarity with original reference drugs, focusing on critical quality attributes (e.g., binding kinetics, post-translational modifications).
  • Using research-grade biosimilars also supports regulatory-compliant assay validation and bioequivalence studies during biosimilar drug development.

In summary, research-grade Basiliximab biosimilars act as reliable calibration standards and reference controls in PK bridging ELISAs, enabling accurate measurement of drug levels in serum samples for preclinical and clinical research applications.

Standard flow cytometry protocols for using a conjugated Basiliximab biosimilar (such as PE or APC-labeled) to validate CD25 expression or assess binding capacity typically involve staining of target cells (e.g., PBMCs or transfected cell lines) and quantifying specific antibody binding via fluorescence intensity.

Protocol Overview:

  • Cell Preparation

    • Use target cells expressing human CD25, such as PHA-activated PBMCs or cell lines transfected with CD25/IL-2Rα.
    • Wash cells in appropriate buffer (e.g., PBS + 2% FBS).
  • Antibody Staining

    • Incubate cells with the PE- or APC-conjugated Basiliximab biosimilar at a predetermined optimal concentration (each laboratory should determine the ideal dilution). Typically, 0.5 µg/mL is a common starting point.
    • Include appropriate isotype controls and, if multiplexing, other directly conjugated antibodies (e.g., CD3-PE, CD4-FITC, FoxP3-APC) as needed for cell subset identification.
    • Incubate for 30 minutes at 4°C in the dark.
  • Washing and Acquisition

    • Wash cells thoroughly with buffer to remove unbound antibody.
    • Resuspend cells in buffer containing viability dye if dead cell exclusion is required.
  • Data Acquisition and Analysis

    • Analyze cells on a flow cytometer, collecting data for the relevant fluorescence channels.
    • Assess mean fluorescence intensity (MFI) or percentage of positive cells as a measure of CD25 expression or Basiliximab binding.
    • Comparisons can be made to untreated cells or to those stained with an isotype control.

Critical Details and Controls:

  • Blocking/Competition Assays:

    • To specifically validate binding capacity, you can perform blocking experiments: pre-incubate cells with unlabeled Basiliximab (or IL-2) before staining with the conjugated antibody to confirm specificity of the conjugated Basiliximab’s binding.
  • Clone Specificity and Epitope Consideration:

    • Basiliximab binds a unique epitope on CD25. Notably, it can block the binding of certain anti-CD25 clones (e.g., clone 2A3), but not others (e.g., M-A251).
      • This should be considered when designing panels for multicolor flow cytometry or interpreting staining patterns.
      • Preincubation with Basiliximab can decrease detection by other antibodies recognizing overlapping epitopes.
  • Indirect Detection:

    • If only unconjugated Basiliximab biosimilar is available, detection can be performed using a fluorescently labeled anti-human IgG secondary antibody:
      • Primary incubation: Basiliximab biosimilar antibody.
      • Wash and incubate with fluorescent anti-human IgG (e.g., APC-labeled).

Applications:

  • Measurement of CD25 Expression: Quantifying CD25 on activated T cells or Tregs by directly measuring MFI of the fluorophore conjugated to Basiliximab.
  • Binding/Blocking Capacity: Demonstrating that the conjugated Basiliximab specifically blocks IL-2 or other anti-CD25 antibody binding in competition assays.

Sample Protocol (based on manufacturer and literature):

  1. Incubate cells (e.g., 1 x 10^6) with PE- or APC-conjugated Basiliximab biosimilar (e.g., 0.5 µg/mL) for 30 min at 4°C, in the dark.
  2. Wash cells twice with staining buffer.
  3. Optionally: Perform secondary anti-human IgG staining if using unconjugated primary antibody.
  4. Stain with other cell markers as required (e.g., CD3, CD4, FoxP3).
  5. Wash and resuspend for flow cytometric acquisition.
  6. Analyze data, comparing to isotype and/or blocked controls.

To summarize:
Conjugated Basiliximab biosimilars are used in standard flow cytometry protocols for direct detection of CD25, for competition/blocking studies, and to validate the binding capacity to CD25 in both cell lines and primary T cells. Careful consideration of antibody clone and potential epitope overlap with CD25 detection reagents is essential for accurate results.

Biopharma companies employ a comprehensive battery of analytical assays to demonstrate that proposed biosimilars are highly similar to their reference products in both structure and function. These assessments form the foundation of biosimilar development and regulatory approval.

Primary Structure Characterization

The analytical similarity assessment begins with confirming that the biosimilar has an identical primary amino acid sequence to the reference product. Peptide mapping using liquid chromatography-mass spectrometry (LC-MS) serves as the gold standard technique for this analysis. This method fragments the protein into smaller peptides and analyzes each piece to ensure complete sequence identity.

Higher-Order Structure Analysis

Beyond primary structure, companies must demonstrate that the three-dimensional folding and conformation of the biosimilar matches the reference product. Key analytical techniques include:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements like alpha-helices and beta-sheets
  • Fourier-transform infrared (FTIR) spectroscopy for additional conformational analysis
  • Differential scanning calorimetry (DSC) to evaluate thermal stability and protein folding
  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for detailed conformational mapping
  • Nuclear magnetic resonance (NMR) spectroscopy for high-resolution structural characterization

Post-Translational Modifications Assessment

Post-translational modifications significantly impact protein function and immunogenicity. Critical assays include:

  • High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for glycosylation analysis
  • LC-MS techniques to detect oxidation, deamidation, and other chemical modifications
  • These modifications are particularly important as they can affect both efficacy and safety profiles

Purity and Impurity Profiling

The analytical similarity assessment must include rigorous comparison of purity and impurity profiles between the biosimilar and reference product. Key techniques encompass:

  • Size exclusion chromatography (SEC-HPLC) to detect aggregates and fragments
  • Ion exchange chromatography (IEX-HPLC) to identify charge variants
  • Analytical ultracentrifugation sedimentation velocity (AUC-SV) for additional aggregate analysis

Functional Characterization

Demonstrating structural similarity is necessary but insufficient - the biosimilar must also exhibit equivalent biological function. Comprehensive functional testing includes:

Binding Assays:

  • Enzyme-linked immunosorbent assays (ELISA) for target binding assessment
  • Surface plasmon resonance (SPR) for kinetic binding analysis
  • Fc receptor binding assays for antibody products to evaluate immune system interactions

Potency and Activity Assays:

  • Cell-based bioassays to measure biological activity
  • Enzyme kinetics studies for enzymatic products
  • Multiple orthogonal methods to comprehensively assess all known biological activities

Statistical and Comparative Analysis

Manufacturers conduct head-to-head comparisons of the proposed biosimilar's properties against the reference product, with results required to fall within appropriate limits, ranges, or distributions. This extensive characterization uses highly sensitive analytical methods to measure molecular properties across multiple lots of both products.

The analytical studies focus on critical quality attributes (CQAs) that are ranked by their risk of impact on the product's activity, pharmacokinetics, pharmacodynamics, safety, efficacy, or immunogenicity. This risk-based approach ensures that the most important molecular properties receive particular attention during the similarity assessment.

Regarding the Leinco biosimilar, the search results do not contain any specific information about this particular product or its use in analytical studies. The analytical techniques and approaches described above represent the standard industry practices that would typically be applied to any biosimilar development program, but without additional information, I cannot provide details about how Leinco specifically utilizes these methods in their biosimilar studies.

References & Citations

Flow Cytometry
IF
IHC

Certificate of Analysis

Formats Available

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