Recombinant Human IFNγ

Recombinant Human IFNγ (Interferon-gamma) is a highly valuable tool for research applications due to its central role in regulating immune responses and its broad biological effects. Here are key reasons why you should consider using Recombinant Human IFNγ in your research:

1. Immune System Modulation

• IFNγ is a key cytokine that regulates both innate and adaptive immunity.
• It activates macrophages, dendritic cells, and natural killer (NK) cells, enhancing their antimicrobial and antitumor activities.
• It promotes antigen presentation by upregulating MHC class I and II molecules, which is crucial for T cell activation and immune surveillance.

2. Antiviral, Antiproliferative, and Apoptotic Effects

• IFNγ exhibits direct antiviral activity, making it useful for studying host defense mechanisms against viral infections.
• It inhibits cell proliferation and induces apoptosis in certain cell types, which is relevant for cancer research and studies on cell cycle regulation.

3. Inflammation and Autoimmunity Studies

• IFNγ has dual roles in inflammation: it can act as a pro-inflammatory mediator but also as an anti-inflammatory agent by promoting regulatory T cell development and inhibiting Th17 differentiation.
• This makes it an important molecule for studying autoimmune diseases, chronic inflammation, and immune tolerance.

4. Standardization and Reproducibility

• Recombinant IFNγ provides a consistent and well-characterized source of the cytokine, ensuring reproducible experimental results across different studies and laboratories.

5. Therapeutic and Drug Development Applications

• IFNγ is used in screening assays for antibodies or small molecules that modulate IFNγ signaling (e.g., inhibitors or agonists).
• It is relevant for developing and testing new immunotherapies, including cancer immunotherapy and treatments for infectious diseases.

6. Versatility Across Cell Types and Models

• IFNγ can be applied to a wide range of cell types (e.g., immune cells, epithelial cells, neurons) and experimental models (e.g., organoids, whole cells, bioassays), making it suitable for diverse research areas.

7. Well-Established Detection and Quantification Methods

• Recombinant IFNγ serves as a standard in assays designed to detect or quantify IFNγ levels, such as ELISA, bioassays, and functional readouts.

8. Relevance to Disease Mechanisms

• IFNγ plays a role in the pathogenesis of various diseases, including infections, cancer, autoimmune disorders, and inflammatory conditions. Using recombinant IFNγ allows researchers to dissect its contribution to these processes.

In summary, Recombinant Human IFNγ is essential for studies involving immune activation, host defense, inflammation, cancer biology, and therapeutic development. Its well-defined biological activities and availability as a purified protein make it a reliable and versatile reagent for a wide range of research applications.

Yes, recombinant human IFNγ is commonly used as a standard for quantification or calibration in ELISA assays for human IFNγ. This is a standard practice in immunoassays designed to measure IFNγ concentrations in biological samples.

Key points and best practices:

• Recombinant IFNγ as Standard: Most commercial ELISA kits for human IFNγ include a recombinant human IFNγ protein as the calibration standard. This standard is used to generate a standard curve, allowing quantification of IFNγ in unknown samples by comparison to known concentrations.

• Source and Expression System: Recombinant IFNγ used as a standard is typically expressed in E. coli or mammalian cells (e.g., HEK293). The expression system can affect post-translational modifications (such as glycosylation), but for most immunoassays, both natural and recombinant forms are recognized equivalently by the antibodies used.

• Parallelism and Calibration: ELISA kits are validated to ensure that the standard curve generated with recombinant IFNγ is parallel to the response obtained with natural IFNγ in samples, supporting accurate quantification. Some kits provide conversion factors to relate results to international standards (e.g., NIBSC/WHO 82/587).

• Preparation and Dilution: Follow the specific instructions provided with your ELISA kit or recombinant protein for reconstitution and serial dilution to prepare the standard curve. Use the recommended assay diluent to match the matrix of your samples and minimize matrix effects.

• Compatibility: Ensure that the recombinant IFNγ standard you use is compatible with the antibodies and detection reagents in your ELISA system. Most sandwich ELISAs for human IFNγ are designed to detect both recombinant and natural forms.

• Research Use Only: Note that these standards and assays are generally for research use only and not for diagnostic or therapeutic applications.

In summary: You can use recombinant human IFNγ as a standard for quantification or calibration in your ELISA assays, provided it is compatible with your assay system and you follow the recommended protocols for preparation and dilution.

Recombinant human IFN-γ has been validated across a diverse range of research applications in published studies, reflecting its importance as a tool for immunological, oncological, and infectious disease research.

Immunological Applications

Recombinant human IFN-γ has been extensively used to study immune cell activation and function. The protein stimulates antimicrobial and anti-tumor activity in macrophages, natural killer cells, and neutrophils. It has been validated for examining the upregulation of major histocompatibility complex (MHC) class I and II molecules, which are critical for priming antigen-specific cytotoxic T lymphocytes and promoting cancer cell death. Additionally, the protein has been used to investigate IFN-γ-stimulated anti-parasitic defenses in both murine and human neurons against intracellular pathogens such as Toxoplasma gondii.

Assay Development and Standardization

Recombinant human IFN-γ serves as a standard for IFN-γ detection and quantification assays. It has been validated in multiple assay formats including binding assays, bioassays, ELISA (both capture and standard applications), cell culture differentiation studies, and Western blot controls. The protein has demonstrated biological activity in cell-based bioassays, including protection of A549 cell lines and anti-viral activity in HeLa human cervical epithelial carcinoma cells infected with encephalomyocarditis virus.

Therapeutic Development and Screening

The protein has been validated for screening and release assays for antibodies that block IFN-γ signaling, as well as for screening engineered IFN-γ variants. Recombinant human IFN-γ can be used in combination with specialized cell lines to evaluate inhibitory molecules targeting IFN-γ and its receptor interactions.

Cancer Research

Recombinant human IFN-γ has been validated for dose-dependent inhibition of cancer cell proliferation, as demonstrated in studies using human colorectal adenocarcinoma cell lines. The protein has also been applied to investigate tumor microenvironment modulation and immune checkpoint regulation in cancer research.

Viral and Infectious Disease Research

The protein has been validated for studying anti-viral mechanisms, including investigations of membrane-tethered mucin 1 stimulation by interferon in response to virus infection across multiple cell types, with specific applications to influenza A virus infection in human airway epithelium.

For cell culture experiments, recombinant human IFNγ protein should be reconstituted and prepared according to the following best practices, based on manufacturer guidelines and scientific protocols:

Reconstitution:

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein in sterile, endotoxin-free water or a neutral buffer (such as PBS, pH 7.4). Most manufacturers recommend:
   • 0.2 mg/mL in sterile PBS (pH 7.4) or sterile deionized water.
   • Some protocols suggest adding 0.1% endotoxin-free recombinant human albumin (HSA) or 1–4% BSA to stabilize the protein and prevent adsorption to surfaces.
3. Gently swirl or tap the vial to mix; do not vortex or pipette vigorously to avoid denaturation.

Preparation for Cell Culture:

1. Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
2. Store aliquots at –20°C to –80°C for long-term storage. For short-term use (within a week), store at 2–8°C under sterile conditions.
3. Dilute the protein in cell culture medium or a neutral buffer containing carrier protein (e.g., 1–4% BSA or 5–10% FBS) before adding to cells.
4. Typical working concentrations for in vitro cell culture experiments range from 0.1 to 10 ng/mL, depending on the cell type and experimental design.

Additional Tips:

• Warm the lyophilized powder to room temperature before opening the vial.
• Avoid repeated freeze-thaw cycles to maintain protein activity.
• Confirm protein concentration and integrity if possible (e.g., by SDS-PAGE or spectrophotometry).

Example Protocol:

• Reconstitute 10 µg of lyophilized IFNγ in 50 µL sterile PBS (pH 7.4) to achieve 0.2 mg/mL.
• Add 0.1% HSA or 1% BSA if desired.
• Aliquot and store at –80°C.
• For cell culture, dilute to the desired concentration (e.g., 10 ng/mL) in culture medium containing 1% BSA or 5% FBS.

Always refer to the specific product datasheet for any manufacturer-specific instructions.