Recombinant Human IFN-alpha 2a

Recombinant Human IFN-alpha 2a is widely used in research due to its well-characterized antiviral, immunomodulatory, and anti-proliferative properties, making it a prototypic type I interferon for both basic and translational studies.

Key reasons to use recombinant human IFN-alpha 2a in research applications:

• Antiviral Activity: IFN-alpha 2a is secreted by virus-infected cells and induces an antiviral state in neighboring cells, inhibiting viral replication and spread. It is a standard tool for studying innate immune responses to viral infection and for evaluating antiviral compounds in cell-based assays.

• Immunomodulation: IFN-alpha 2a orchestrates the adaptive immune response by acting on dendritic cells, enhancing antigen presentation, and stimulating the activation of cytotoxic T cells and natural killer (NK) cells. This makes it valuable for immunology research, especially in the context of tumor immunology and vaccine development.

• Anti-proliferative and Antitumor Effects: IFN-alpha 2a inhibits cell proliferation and induces apoptosis in various cancer cell types, and has been used clinically as an adjuvant or primary therapy for several malignancies, including leukemias, lymphomas, and solid tumors. In vitro, it is commonly used to study cell cycle regulation, apoptosis, and mechanisms of tumor suppression.

• Standardization and Reproducibility: Recombinant IFN-alpha 2a is highly purified, with defined activity and minimal batch-to-batch variability, ensuring reproducible results in experimental systems. It is the reference subtype for type I interferon research and is often used as a positive control in bioassays.

• Clinical Relevance: IFN-alpha 2a has a long history of clinical use for viral hepatitis, certain cancers, and immunological disorders, making it directly translatable from bench to bedside. Its effects and mechanisms are well documented, facilitating interpretation and comparison of experimental data.

• Versatility: It is suitable for a wide range of applications, including:

  • Induction of interferon-stimulated genes (ISGs) in cell culture
  • Antiviral and anti-proliferative bioassays
  • Immunological studies involving dendritic cells, T cells, and NK cells
  • Preclinical models of infection, cancer, and immune regulation

In summary, recombinant human IFN-alpha 2a is a gold-standard reagent for dissecting type I interferon biology, modeling antiviral and antitumor responses, and developing new therapeutic strategies.

Yes, recombinant human IFN-alpha 2a can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and matched to the assay's requirements.

Supporting details:

• ELISA Standard Use: Recombinant human IFN-α2a is commonly used as a standard in sandwich ELISA protocols for the quantification of IFN-α2a in biological samples. It is suitable for generating a standard curve, which is essential for accurate quantification.

• Assay Compatibility: Most commercial ELISA kits for human IFN-α2a specify that their standards are recombinant proteins, and the assay antibodies are validated to recognize both natural and recombinant forms. This ensures that the recombinant standard will behave similarly to endogenous IFN-α2a in the assay.

• Calibration and Activity: For quantitative calibration, it is important that the recombinant IFN-α2a standard is calibrated against an international reference standard, such as the WHO reference standard, to ensure accurate unit assignment (e.g., IU/mg). Many suppliers provide recombinant IFN-α2a with activity determined by comparison to these standards.

• Best Practices:

  • Confirm the purity and bioactivity of your recombinant IFN-α2a standard, as these can affect assay accuracy.
  • Use carrier-free preparations if required by your assay protocol, as additives like BSA can interfere with some detection systems.
  • Prepare a serial dilution of the recombinant standard to construct a standard curve, plotting absorbance versus concentration, as described in ELISA kit protocols.

• Specificity: ELISA kits designed for IFN-α2a typically do not cross-react with other interferon subtypes or unrelated cytokines, ensuring specificity when using recombinant IFN-α2a as a standard.

Summary Table: Recombinant IFN-α2a as ELISA Standard

Conclusion:
You can use recombinant human IFN-α2a as a standard for ELISA quantification, provided it is properly calibrated and compatible with your assay system. Always verify the standard's specifications and follow best practices for standard curve preparation.

Recombinant Human IFN-alpha 2a has been validated in published research for a broad range of applications, including antiviral assays, immunomodulation studies, cancer research, and as a therapeutic agent in both in vitro and in vivo models.

Key validated applications include:

• Antiviral assays: IFN-alpha 2a is widely used to assess antiviral activity against viruses such as HIV, hepatitis B and C, and SARS-CoV-2 in cell culture systems. It is employed in bioassays measuring viral replication, viral antigen expression, and plaque reduction.

• Immunomodulation studies: The protein is used to study induction of interferon-stimulated genes (ISGs), modulation of immune cell function (e.g., dendritic cells, T cells, NK cells), and regulation of cytokine responses.

• Cancer research: IFN-alpha 2a has been validated for its antiproliferative and pro-apoptotic effects on various cancer cell lines, including studies on renal cell carcinoma, melanoma, Kaposi sarcoma, and hematological malignancies.

• Cell signaling and gene expression: Researchers use IFN-alpha 2a to induce and analyze signaling pathways such as JAK/STAT, and to study transcriptional regulation of genes like hypoxia-inducible factor-1α (HIF-1α) and PD-L1.

• Western blot and ELISA: IFN-alpha 2a is validated as a positive control or stimulant in Western blot and ELISA assays for detection of interferon-responsive proteins and cytokines.

• Cell culture stimulation: It is routinely used to stimulate primary cells and cell lines to model interferon responses, including upregulation of ACE2 in respiratory epithelia and activation of the kynurenine pathway in trisomy 21 models.

• Therapeutic validation: Clinically, recombinant IFN-alpha 2a has been approved and validated for treatment of chronic viral hepatitis B and C, chronic myeloid leukemia, Kaposi sarcoma, follicular lymphoma, renal cell carcinoma, melanoma, and T cell lymphoma.

• Biophysical and aggregation studies: IFN-alpha 2a has been used as a model protein for studying oligomerization, aggregation, and protein stability using techniques like small-angle X-ray scattering and analytical ultracentrifugation.

These applications are supported by numerous peer-reviewed publications, demonstrating reproducible results in both basic research and translational studies. The protein is considered a prototypic type I interferon for experimental modeling of innate immune responses and antiviral defense mechanisms.

To reconstitute and prepare Recombinant Human IFN-alpha 2a for cell culture experiments, follow these best-practice steps:

1. Equilibrate and Centrifuge the Vial

   • Allow the lyophilized protein vial to reach room temperature before opening to prevent condensation.
   • Briefly centrifuge the vial (20–30 seconds in a microcentrifuge) to collect all material at the bottom.

2. Reconstitution Buffer

   • Use sterile water or sterile PBS (pH 7.4) as the solvent.
   • For improved stability and to minimize adsorption, add 0.1% endotoxin-free recombinant human serum albumin (HSA) or 0.1% BSA to the buffer.
   • Avoid using buffers with high ionic strength or extreme pH unless specified by your protocol.

3. Reconstitution Concentration

   • Common reconstitution concentrations are 0.1 mg/mL (100 μg/mL) or 0.2 mg/mL (200 μg/mL).
   • Add the appropriate volume of buffer to achieve the desired concentration (consult the product datasheet for exact mass and volume).

4. Dissolving the Protein

   • Gently swirl or tap the vial to mix. Do not vortex or shake vigorously, as this can denature the protein.
   • If solubility is an issue, allow the solution to incubate at 4°C overnight.

5. Aliquot and Storage

   • After reconstitution, aliquot the solution into working volumes to avoid repeated freeze-thaw cycles.
   • Store aliquots at -20°C to -80°C for long-term storage (up to 6 months), or at 4°C for short-term use (up to 1 week).
   • Always avoid repeated freeze-thaw cycles, as this can degrade the protein.

6. Preparation for Cell Culture

   • Before use, dilute the reconstituted stock to the desired working concentration in cell culture medium.
   • Ensure all solutions are sterile and endotoxin-free to prevent confounding cellular responses.

Summary Table: Key Steps and Recommendations

Additional Notes:

• Confirm protein integrity by SDS-PAGE if needed.
• Always consult the specific product datasheet for any manufacturer-specific recommendations, as minor formulation differences may exist.
• For functional assays, titrate IFN-alpha 2a to determine the optimal concentration for your cell type and experimental endpoint.

These guidelines ensure maximal activity and reproducibility of recombinant IFN-alpha 2a in cell culture experiments.