Recombinant Human Interferon-alpha 2b (IFNα 2b)

Recombinant Human Interferon-alpha 2b (IFNα 2b) is widely used in research due to its well-characterized antiviral, immunomodulatory, and antitumor properties, making it a valuable tool for studying immune responses, cancer biology, and infectious diseases.

Key reasons to use IFNα 2b in research applications:

• Potent Immunomodulation: IFNα 2b enhances immune surveillance by activating natural killer (NK) cells, promoting dendritic cell maturation, and stimulating T-cell responses, while inhibiting regulatory T cells and immunosuppressive cytokines. This makes it ideal for dissecting immune mechanisms in both infection and cancer models.

• Antiviral Activity: IFNα 2b induces the expression of antiviral proteins and upregulates endogenous interferons, providing robust protection against a wide range of viruses in vitro and in vivo. It is commonly used as a positive control in antiviral assays and to model innate immune responses.

• Antitumor Effects: IFNα 2b inhibits tumor cell proliferation, induces apoptosis via multiple pathways (mitochondrial, ER stress, death receptor), and suppresses oncogene expression. It is used to study cancer cell biology, immune-oncology, and as a reference in preclinical drug testing.

• Standardization and Reproducibility: As a recombinant protein, IFNα 2b offers high purity, batch-to-batch consistency, and defined biological activity, which are critical for reproducible experimental results.

• Clinical Relevance: IFNα 2b is FDA-approved for several cancers and viral infections, and its mechanisms are well-documented in clinical and preclinical studies. Using IFNα 2b in research provides translational relevance, especially for studies aiming to bridge basic science and therapeutic development.

• Versatility: IFNα 2b can be used in diverse experimental systems, including cell culture, animal models, and ex vivo assays, to investigate immune regulation, viral pathogenesis, tumor immunology, and cytokine signaling.

In summary, recombinant human IFNα 2b is a scientifically validated, versatile cytokine that enables detailed investigation of immune, antiviral, and antitumor mechanisms, with direct relevance to both basic research and translational applications.

Yes, recombinant human Interferon-alpha 2b (IFNα 2b) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly quantified, and compatible with your assay’s antibody specificity and detection system.

Key considerations and supporting details:

• Recombinant IFNα 2b is commonly used as an ELISA standard: Many commercial ELISA kits for human IFN-alpha 2 or IFN-alpha 2b use recombinant proteins as their calibration standards. These standards are typically well-characterized and quantified, ensuring reliable calibration curves.

• Purity and quantification: The recombinant IFNα 2b should be of high purity (typically >95%) and accurately quantified, often by methods such as RP-HPLC or UV absorbance. This ensures that the standard curve reflects the true concentration of IFNα 2b.

• Antibody specificity: Ensure that the antibodies used in your ELISA recognize IFNα 2b. Some ELISAs are subtype-specific (e.g., IFNα 2b only), while others detect multiple IFN-alpha subtypes. If your assay is designed for IFNα 2b, a recombinant IFNα 2b standard is appropriate. If your assay detects multiple subtypes, using a single subtype as a standard may introduce quantification bias, as antibody affinities can differ between subtypes.

• Matrix effects and recovery: When using recombinant standards, it is important to validate recovery and linearity in your sample matrix (e.g., serum, plasma, culture media), as matrix components can affect quantification. Most commercial kits report recovery and linearity data for recombinant IFNα 2b spiked into relevant matrices.

• Documentation and traceability: For publication or regulatory purposes, document the source, lot, and quantification method of your recombinant standard. Some standards are calibrated against international reference preparations, which improves comparability.

• Not all ELISAs are validated for recombinant standards: Some kits are designed to detect only native IFNα 2b and may not recognize recombinant forms if they differ in glycosylation or folding. Always check your assay’s documentation or perform a validation experiment.

Summary Table: Use of Recombinant IFNα 2b as ELISA Standard

In summary: Recombinant human IFNα 2b is widely used as a standard for ELISA quantification, but you must ensure compatibility with your specific assay and validate performance in your sample matrix for accurate results.

Recombinant Human Interferon-alpha 2b (IFNα 2b) has been validated in published research for a broad range of applications, primarily as an antiviral, antitumor, and immunomodulatory agent in both clinical and experimental settings.

Key validated applications include:

• Antiviral therapy:

  • Approved and widely used for the treatment of chronic hepatitis B and C.
  • Investigated and applied for prophylaxis and treatment of SARS-CoV-2 (COVID-19), including intranasal formulations for infection prevention.
  • Used in the treatment of genital warts (caused by HPV) and validated in combination therapies for cervical HPV infection.

• Anticancer therapy:

  • Approved for and validated in the treatment of several cancers, including:
    • Hairy cell leukemia
    • Chronic myelogenous leukemia (CML)
    • Follicular lymphoma
    • Malignant melanoma
    • AIDS-related Kaposi sarcoma
    • Multiple myeloma
    • Carcinoid tumor
    • Mastocytosis.
  • Used as adjuvant therapy (e.g., in melanoma) and in combination with other chemotherapeutics (e.g., cytarabine, vinblastine, 5-fluorouracil, tamoxifen, interleukin-2).

• Immunomodulation:

  • Validated for enhancing T and NK cell cytotoxicity in cancer immunotherapy, including head and neck squamous cell carcinoma and tongue squamous cell carcinoma.
  • Shown to induce expression of perforin-granzyme B in NK cells and augment suppressed immune functions.

• Bioassays and in vitro research:

  • Used as a standard cytokine in antiviral bioassays (e.g., protection of HeLa cells from viral infection).
  • Applied in studies of JAK-STAT signaling, apoptosis, and cell proliferation inhibition.

• Other clinical and experimental uses:

  • Investigated for Behçet's disease and as an immunomodulatory agent in various inflammatory and infectious diseases.
  • Used off-label in veterinary medicine due to its cross-species activity.

Summary Table of Validated Applications

Note:

• Most applications are supported by both clinical approvals and extensive published research.
• The protein is frequently used as a reference standard in immunological and virological research due to its well-characterized activity profile.
• Combination therapies and novel delivery routes (e.g., intranasal, vaginal suppositories) are active areas of ongoing research and validation.

To reconstitute and prepare Recombinant Human Interferon-alpha 2b (IFNα 2b) for cell culture experiments, dissolve the lyophilized protein in sterile distilled water at a concentration of 0.1 mg/mL (100 µg/mL) or higher, then dilute as needed in cell culture medium or buffer containing a carrier protein such as 0.1% HSA or BSA to prevent adsorption and loss of activity.

Step-by-step protocol:

• Preparation:

  • Before opening the vial, briefly centrifuge it (20–30 seconds in a microcentrifuge) to collect all powder at the bottom.
  • Allow the vial to reach room temperature before opening to avoid condensation.

• Reconstitution:

  • Add sterile distilled water (or sterile 18MΩ-cm H₂O) directly to the lyophilized IFNα 2b to achieve a concentration of ≥100 µg/mL (e.g., for 100 µg protein, add 1 mL water for 0.1 mg/mL).
  • Gently mix by pipetting; do not vortex or shake vigorously to avoid denaturation.
  • If solubility issues arise, incubate the solution at 4°C overnight.

• Dilution for cell culture:

  • For working concentrations, dilute the reconstituted stock in cell culture medium or phosphate-buffered saline (PBS), ideally containing 0.1%–0.3% carrier protein (e.g., BSA, HSA, or bovine casein) to minimize adsorption to plasticware.
  • Initial dilutions (e.g., 1:10, 1:100) should be made in medium containing 5–10% serum or in PBS with carrier protein.

• Sterility:

  • If necessary, filter-sterilize the final working solution using a low-protein-binding 0.22 µm filter.

• Storage:

  • Store reconstituted IFNα 2b at 4°C for up to 2–7 days.
  • For long-term storage, aliquot and freeze at –20°C or below; avoid repeated freeze-thaw cycles to preserve activity.

Additional notes:

• Do not reconstitute at concentrations above 1 mg/mL to avoid solubility problems.
• Confirm protein integrity by SDS-PAGE if needed; a band at ~19–21 kDa should be visible with as little as 10 ng loaded.
• Always consult the specific product datasheet for any unique formulation or tag (e.g., FLAG-tag) that may affect handling.

This protocol ensures optimal solubility, stability, and biological activity of IFNα 2b for cell culture applications.