Recombinant Human GITR Ligand

Recombinant Human GITR Ligand is used in research applications to modulate immune responses, particularly by providing co-stimulatory signals to T cells and natural killer (NK) cells, enhancing anti-tumor immunity, and investigating immune regulation mechanisms.

Key scientific reasons to use Recombinant Human GITR Ligand include:

• T Cell Activation and Proliferation: GITR Ligand binding to its receptor (GITR) delivers a potent co-stimulatory signal to activated CD4⁺ and CD8⁺ T cells and NK cells, promoting their activation, proliferation, and survival.
• Modulation of Regulatory T Cells (Tregs): Engagement of GITR by its ligand inhibits the suppressive function of Tregs, thereby enhancing effector T cell responses and potentially improving anti-tumor immunity.
• Macrophage Activation and Cytokine Production: GITR Ligand can induce macrophage activation and modulate cytokine expression, which is relevant for studying inflammation and autoimmune disease mechanisms.
• Anti-Tumor Immunity: Preclinical studies show that GITR Ligand, either alone or in combination with other immunotherapies (e.g., vaccines, checkpoint inhibitors), can increase resistance to tumors by expanding antigen-specific CD8⁺ T cells, depleting intratumoral Tregs, and inducing long-lasting immunologic memory.
• Adjuvant for Vaccines: Multimeric forms of GITR Ligand have been used as adjuvants to enhance the efficacy of DNA vaccines by boosting antigen-specific immune responses.
• Reverse Signaling in Dendritic Cells: GITR Ligand can induce reverse signaling in dendritic cells, affecting their activation and potentially contributing to immune tolerance or suppression studies.
• Autoimmune and Inflammatory Disease Research: The GITR/GITRL pathway is implicated in the pathogenesis of autoimmune diseases, making recombinant ligand a valuable tool for dissecting these mechanisms.

Typical applications include:

• In vitro bioassays to study T cell activation, proliferation, and cytokine production.
• In vivo models to assess anti-tumor efficacy and immunologic memory.
• ELISA and other immunoassays to quantify ligand-receptor interactions or downstream effects.
• Mechanistic studies of immune regulation, tolerance, and inflammation.

Best practices: Use recombinant human GITR Ligand at concentrations validated for your specific cell type and assay. Include appropriate controls (e.g., isotype, untreated) and consider combination with other immune modulators for synergy studies. Always confirm species cross-reactivity if using in non-human models.

In summary, Recombinant Human GITR Ligand is a versatile tool for immunology, oncology, and inflammation research, enabling precise modulation and analysis of immune cell functions and therapeutic mechanisms.

Yes, recombinant Human GITR Ligand can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose in your specific assay system. Several ELISA kits and protocols explicitly use recombinant Human GITR Ligand as a standard for generating calibration curves in quantitative assays.

Key considerations and supporting details:

• ELISA Standard Use: Multiple sources confirm that recombinant Human GITR Ligand (TNFSF18) is suitable as a standard in ELISA assays designed to quantify GITR Ligand in biological samples such as serum, plasma, and cell culture supernatants. These kits typically include lyophilized recombinant protein for standard curve preparation, allowing quantification of unknown samples by comparison to the standard curve.

• Validation and Compatibility: It is essential to ensure that the recombinant protein is compatible with the antibodies used in your ELISA. Commercial ELISA kits for GITR Ligand use recombinant protein as the standard and report good recovery and linearity in relevant sample types. If you are developing your own assay, confirm that your capture and detection antibodies recognize the recombinant form equivalently to the native protein.

• Standard Preparation: Follow manufacturer or published protocols for reconstitution and dilution of the recombinant standard. Avoid harsh mixing (such as vortexing) to maintain protein stability. Prepare serial dilutions to cover the expected concentration range in your samples.

• Assay Sensitivity and Range: Typical ELISA kits using recombinant Human GITR Ligand as a standard report sensitivity down to 25 pg/mL and standard curve ranges from approximately 40–10,000 pg/mL.

• Sample Types: Validated sample types include serum, plasma, cell culture supernatants, and urine, depending on the kit and protocol.

• Research Use Only: Note that these standards and assays are for research use only and not for diagnostic purposes.

Best Practices:

• Always verify that the recombinant standard is from a reliable source and is of high purity.
• Validate the standard curve in your specific assay matrix to ensure accurate quantification.
• Store and handle the recombinant protein according to recommended conditions to preserve activity and stability.

Summary Table: Recombinant Human GITR Ligand as ELISA Standard

In conclusion, recombinant Human GITR Ligand is widely used and validated as a standard for ELISA quantification, provided assay-specific compatibility and validation are confirmed.

Recombinant Human GITR Ligand (TNFSF18) has been validated in several key applications in published research, primarily in the context of immunology and cancer biology.

Validated Applications in Published Research:

• Bioassays:
  Used to study the costimulatory effects on activated CD4⁺ and CD8⁺ T cells and NK cells, as well as to investigate reverse signaling in dendritic cells and its immunomodulatory effects. Bioassays have also been used to assess the ligand’s ability to modulate immune responses in cancer and autoimmune disease models.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Employed as a standard in ELISA to quantify GITR ligand levels or to measure binding interactions, including functional ELISA for receptor-ligand binding studies.

• Structural and Biophysical Studies:
  Utilized in structural biology to resolve the crystal structure of the human GITR–GITRL complex, validating the receptor–ligand interface and receptor clustering mechanisms.

• In Vivo Functional Studies:
  Applied in mouse tumor models (e.g., CT26, EMT6, JC) to evaluate anti-tumor immune responses, T cell activation, and immunological memory following GITR ligand administration or GITR-L fusion protein treatment. These studies often include tumor growth inhibition, pharmacodynamic analyses, and survival assessments.

• Cellular Activation and Cytokine Production Assays:
  Used in human PBMC costimulation assays to measure T cell proliferation and cytokine production (e.g., IFN-γ, IL-2, TNF-β) upon GITR ligation.

• Autoimmune and Inflammatory Disease Models:
  Investigated for its role in modulating macrophage stimulation, cellular adhesion, and cytokine expression in diseases such as rheumatoid arthritis and ocular inflammation.

Summary Table of Validated Applications

Additional Notes:

• The ligand is commonly used with whole cells or in cell-based assays, and as a standard in ELISA for quantifying or validating GITR–GITRL interactions.
• Applications are supported by both in vitro and in vivo studies, including human and mouse systems.
• Research also includes the use of GITR ligand fusion proteins and bispecific constructs for enhanced immunomodulatory effects in preclinical models.

If you require protocols or more specific details for any of these applications, please specify the intended experimental context.

To reconstitute and prepare Recombinant Human GITR Ligand (TNFSF18) protein for cell culture experiments, follow these general best practices based on manufacturer protocols and scientific recommendations:

Reconstitution

1. Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
2. Reconstitute the protein in sterile PBS (pH 7.2–7.4) at a concentration of 50–100 µg/mL.
3. Add carrier protein such as 0.1–1% human or bovine serum albumin (BSA or HSA) to stabilize the protein and prevent adsorption to surfaces.
   • Example: For 100 µg of protein, add 1 mL of sterile PBS containing 0.1–1 mg/mL BSA.
4. Gently mix by swirling or slow pipetting; avoid vigorous shaking to prevent foaming and protein denaturation.
5. Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation to fully dissolve.

Preparation for Cell Culture

• Filter sterilize the reconstituted solution through a 0.22 µm filter if not already sterile.
• Aliquot the protein into small volumes to minimize freeze-thaw cycles.
• Use immediately or store aliquots at –20°C or –70°C for long-term storage.

Storage After Reconstitution

• Short-term (≤1 week): Store at 2–8°C under sterile conditions.
• Long-term: Store at –20°C or –70°C. Avoid repeated freeze-thaw cycles.

Notes for Cell Culture Use

• Always use sterile technique to prevent contamination.
• Confirm the endotoxin level is low (<1.0 EU/µg) if using for sensitive cell-based assays.
• For functional assays, titrate the protein to determine the optimal working concentration for your specific cell type and experimental setup.

References

• Manufacturer datasheets (R&D Systems, BioLegend, Abcam, etc.)
• General recombinant protein handling guidelines (Thermo Fisher, R&D Systems)

Following these steps will help ensure the protein remains active and suitable for cell culture experiments.