Recombinant Human IL-28A

Recombinant Human IL-28A is a type III interferon (also known as IFN-λ2) with distinct antiviral, antitumor, and immunomodulatory properties, making it valuable for a range of research applications involving immune response, viral infection, and cancer biology.

Key reasons to use Recombinant Human IL-28A in research:

• Antiviral Activity: IL-28A is upregulated in response to viral infection and plays a critical role in host defense, especially in epithelial tissues. It inhibits viral replication and induces the expression of antiviral genes, similar to type I interferons but with a more restricted receptor distribution, potentially resulting in fewer side effects.

• Immunomodulation: IL-28A promotes Th1 polarization of dendritic cells, enhances IL-12-dependent cytotoxic T lymphocyte (CTL) responses, and upregulates MHC class I antigen presentation. It also inhibits Th2 and Th17-mediated inflammation, which is relevant for studying immune regulation and allergic responses.

• Antitumor Effects: IL-28A has demonstrated antitumor activity in vivo, partly by enhancing CTL responses. It is also involved in modulating tumor cell migration and invasion, making it useful for cancer research, particularly in models of tumor immunity and metastasis.

• Cellular and Tissue Specificity: The activity of IL-28A is largely restricted to cells expressing the IL-28 receptor complex (IL-28RA and IL-10RB), predominantly found on epithelial cells. This specificity allows for targeted studies on mucosal immunity and tissue-specific immune responses.

• Research Applications: Recombinant IL-28A is commonly used in:

  • Bioassays to study antiviral responses and immune cell activation.
  • Cancer cell migration and invasion assays.
  • Immunological studies involving dendritic cell function, T cell polarization, and cytokine signaling.
  • Development of monoclonal antibodies and detection assays for IL-28A.

• Technical Advantages: Recombinant forms expressed in human cells (e.g., HEK293) are often preferred for their native folding, post-translational modifications, and reduced risk of immunogenic contaminants, which is important for both in vitro and in vivo studies.

In summary, Recombinant Human IL-28A is a versatile tool for investigating antiviral immunity, tumor immunology, and cytokine signaling, especially in epithelial and mucosal contexts, due to its unique biological activities and receptor specificity.

Yes, recombinant human IL-28A 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 system. This is a common practice in quantitative ELISA protocols for cytokines such as IL-28A.

Key considerations and supporting details:

• ELISA Standards: Commercial ELISA kits for human IL-28A routinely use recombinant human IL-28A as the standard to generate a calibration curve for quantification. The standard is serially diluted to create a range of known concentrations, which are then used to interpolate the concentration of IL-28A in unknown samples based on their optical density (OD) readings.

• Protein Quality: The recombinant IL-28A used as a standard should be of high purity (typically >95%), accurately quantified, and ideally endotoxin-free, especially if you are measuring in sensitive biological matrices. The protein should be reconstituted and stored according to the manufacturer’s instructions to maintain stability and activity.

• Matrix Compatibility: Ensure that the recombinant standard is compatible with your sample matrix (e.g., serum, plasma, cell culture supernatant). Some ELISA kits are validated for specific matrices, and matrix effects can influence quantification accuracy.

• Carrier Protein: Recombinant IL-28A is often supplied with a carrier protein (such as BSA) to enhance stability and prevent adsorption to plasticware. If your assay is sensitive to carrier proteins, use a carrier-free preparation.

• Assay Validation: If you are developing a custom ELISA or using a recombinant standard not provided with a commercial kit, validate the standard curve for linearity, sensitivity, and reproducibility within your assay system.

• Bioassay vs. ELISA Standard: Some recombinant IL-28A preparations are specifically tested for use as ELISA standards, while others are intended for bioassays. Use a preparation that is validated for ELISA calibration, as bioactivity does not guarantee immunoreactivity in ELISA.

Summary Table: Use of Recombinant IL-28A as ELISA Standard

In summary: Recombinant human IL-28A is suitable as a standard for ELISA quantification if it meets the above criteria and is validated within your assay system.

Recombinant Human IL-28A has been validated for several key applications in published research, primarily in bioassays, cell culture functional studies, and animal models investigating its antiviral, immunomodulatory, angiogenic, and anti-tumor properties.

Validated Applications:

• Bioassays: IL-28A is widely used in cell-based bioassays to study its effects on immune cell polarization (e.g., Th1 induction in dendritic cells), antiviral responses, and cytokine production.
• Cell Culture Functional Studies: It has been applied to various human cell lines (e.g., HUVECs, hepatic cancer cells, epithelial cells) to assess proliferation, migration, invasion, tube formation, and gene expression changes, especially in the context of viral infection and immune modulation.
• ELISA Standard: Recombinant IL-28A is used as a standard in ELISA assays for quantifying IL-28A protein levels in biological samples.
• Western Blot and Immunofluorescence: Monoclonal antibodies generated against recombinant IL-28A have been validated for western blot and immunofluorescent staining to detect endogenous IL-28A in tissues and cells.
• Animal Models: IL-28A has been validated in in vivo and ex vivo models, including murine models of viral hepatitis, angiogenesis (e.g., aortic ring assays, Matrigel plug assays, hind-limb ischemia), and tumor studies to assess its biological effects on disease progression and immune responses.
• Immunohistochemistry: Used to localize IL-28A in tissue sections, particularly in studies of macrophage-like cells and epithelial cells in colon and lung tissue.

Key Research Contexts:

• Antiviral Activity: IL-28A is validated for inhibiting replication of viruses such as HCV in hepatic cell lines and for upregulating antiviral genes (e.g., OAS1, MX1).
• Immunomodulation: It promotes Th1 polarization, inhibits Th2/Th17 responses, and modulates cytokine production in immune cells.
• Angiogenesis: IL-28A enhances angiogenic responses in endothelial cells via eNOS/AKT/ERK1/2 signaling, validated by proliferation, migration, tube formation assays, and animal models.
• Anti-tumor Effects: Shown to enhance anti-tumor cytotoxic T lymphocyte responses and modulate tumor cell migration in cancer models.
• Gene Expression Studies: Used to study transcriptional regulation and gene repression in various cell types.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and are considered standard for recombinant human IL-28A in immunology, virology, cancer, and vascular biology research.

To reconstitute and prepare Recombinant Human IL-28A protein for cell culture experiments, follow these steps for optimal protein stability and biological activity:

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Reconstitution buffer: Use sterile 1× PBS (pH 7.4) containing 0.1% endotoxin-free recombinant human serum albumin (HSA) as a carrier protein. This helps stabilize the cytokine and prevents adsorption to plastic surfaces.
3. Concentration: Reconstitute to a final concentration of 0.1–0.2 mg/mL (100–200 μg/mL) unless your protocol specifies otherwise. For example, if you have 10 μg of protein, add 100 μL of buffer to achieve 0.1 mg/mL.
4. Mixing: Gently swirl or tap the vial to dissolve the protein. Avoid vigorous shaking or vortexing, which can denature the protein or cause foaming.
5. Incubation: Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.
6. Aliquoting: Once fully dissolved, aliquot the solution into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
7. Storage:
   • Short-term (≤1 week): Store reconstituted protein at 4°C.
   • Long-term (≤6 months): Store aliquots at –20°C to –80°C.
   • Avoid repeated freeze-thaw cycles to maintain activity.

Additional notes:

• If a carrier-free preparation is required, reconstitute in sterile PBS without HSA or BSA, but use immediately or aliquot and freeze to minimize loss.
• Always consult the specific Certificate of Analysis (CoA) or product datasheet for any lot-specific recommendations.
• For cell culture, ensure all reagents are endotoxin-free to prevent unwanted immune activation.

Summary Table: Recombinant Human IL-28A Reconstitution

These steps will ensure your recombinant IL-28A is properly prepared for reliable and reproducible cell culture experiments.