Recombinant Human GITR

Recombinant Human GITR is used in research applications to study and modulate immune responses, particularly in the context of cancer immunotherapy, autoimmune diseases, and inflammation. Its primary value lies in its ability to enhance T cell activation, reduce regulatory T cell (Treg) suppression, and facilitate the investigation of immune checkpoint pathways.

Key scientific applications and rationale:

• Cancer Immunotherapy:
  GITR acts as a co-stimulatory receptor on T cells. Engaging GITR with its ligand or agonists (including recombinant proteins) increases proliferation, survival, and cytokine production of effector T cells (CD4⁺ and CD8⁺), thereby strengthening anti-tumor immune responses. It also reduces the suppressive function of Tregs, which often inhibit effective anti-tumor immunity. Recombinant Human GITR can be used to model these interactions in vitro, screen for agonists, or as a control in functional assays.

• Checkpoint Modulation Studies:
  GITR is overexpressed on exhausted and tumor-reactive TILs (tumor-infiltrating lymphocytes) in certain cancers, such as mismatch repair-proficient colorectal carcinoma. Agonistic targeting of GITR (using recombinant proteins or ligands) has been shown to reinvigorate TIL responses and enhance the efficacy of anti-PD-1 therapies. Recombinant Human GITR enables mechanistic studies of these pathways and supports the development of combination immunotherapies.

• Autoimmunity and Inflammation Research:
  GITR signaling modulates immune responses in autoimmune and inflammatory disease models. Recombinant Human GITR can be used to investigate its role in T cell receptor-mediated cell death, resistance to apoptosis, and the balance between effector and regulatory T cell populations. This is relevant for understanding disease mechanisms and screening potential therapeutics.

• Assay Development and Controls:
  Recombinant Human GITR is essential for developing bioassays, ELISAs, and cell-based assays to quantify ligand binding, receptor activation, and downstream signaling events. It serves as a standardized reagent for validating antibody specificity, screening agonists/antagonists, and calibrating experimental systems.

Best practices for use:

• Employ recombinant GITR in in vitro assays to study T cell activation, cytokine production, and apoptosis resistance.
• Use as a control or standard in ligand-binding assays, functional screening, and immunophenotyping.
• Combine with other immune checkpoint modulators (e.g., PD-1/PD-L1 inhibitors) to assess synergistic effects on immune cell function.
• Validate recombinant protein activity in relevant cell lines or primary immune cells before use in complex models.

Summary of scientific value:
Using Recombinant Human GITR in research provides a controlled tool to dissect immune regulatory mechanisms, optimize immunotherapeutic strategies, and develop robust assays for immune modulation. Its application is supported by extensive preclinical and translational research highlighting its role in enhancing effector T cell function, overcoming immune suppression, and improving therapeutic outcomes in cancer and autoimmune diseases.

You can use recombinant human GITR as a standard for quantification or calibration in your ELISA assays, provided it is properly validated and matches the analyte detected by your assay system.

Key considerations and supporting details:

• Recombinant protein as standard: Most commercial ELISA kits for human GITR (also known as TNFRSF18) use recombinant human GITR as the standard for generating the calibration curve. This is standard practice, as recombinant proteins provide a defined concentration and purity, enabling accurate quantification.

• Validation: The recombinant GITR used as a standard should be of high purity and well-characterized, ideally matching the native form (e.g., correct isoform, post-translational modifications if relevant for antibody recognition). The standard must be reconstituted and stored according to manufacturer or protocol recommendations to maintain stability and activity.

• Assay compatibility: Ensure that the recombinant GITR standard is compatible with the antibodies used in your ELISA. The antibodies should recognize the same epitope(s) present on the recombinant standard and the endogenous protein in your samples. If your ELISA is designed for GITR ligand (TNFSF18) rather than GITR receptor (TNFRSF18), the standard must match the target analyte.

• Standard curve preparation: Prepare the standard curve by serially diluting the recombinant GITR in the same matrix as your samples (e.g., assay buffer, serum, plasma) to minimize matrix effects and ensure accurate quantification. Follow the recommended concentration range for your specific ELISA kit or protocol.

• Documentation: Refer to your ELISA kit’s instructions or published protocols for details on standard preparation, storage, and use. If you are developing a custom assay, validate the recombinant standard by assessing recovery, linearity, and parallelism with endogenous samples.

• Limitations: If the recombinant GITR differs significantly from the endogenous protein in terms of structure or modifications, quantification may be less accurate. Always confirm that the standard and sample analytes are immunologically equivalent in your assay system.

In summary, recombinant human GITR is suitable as a standard for ELISA quantification if it is validated for your assay and matches the analyte detected by your antibodies. Always follow best practices for standard preparation and assay validation to ensure reliable results.

Recombinant Human GITR has been validated for several key applications in published research, primarily in the context of immunology, cancer, and autoimmune disease studies.

Validated Applications in Published Research:

• Bioassays:
  Recombinant Human GITR has been widely used in in vitro bioassays to study its role in T cell activation, proliferation, and costimulatory signaling. These assays often assess the functional consequences of GITR engagement on effector T cells and regulatory T cells (Tregs), including proliferation, cytokine production, and suppression assays.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  It has been used as a standard or capture reagent in ELISA to quantify GITR ligand or to study GITR–GITRL interactions. ELISA-based approaches have also been used to measure soluble GITR or GITRL in biological samples and to validate binding specificity.

• Cell-based Functional Assays:
  Recombinant GITR has been used to stimulate or block GITR signaling in cultured immune cells, allowing researchers to dissect downstream effects such as apoptosis resistance, modulation of Treg suppressive function, and effector T cell survival.

• Preclinical In Vivo Models:
  While not a direct application of the recombinant protein itself, studies have used recombinant GITR or GITR-targeting antibodies in mouse models to evaluate therapeutic effects, particularly in cancer immunotherapy and autoimmune disease models. These studies often use recombinant GITR to validate antibody binding, agonist activity, or to model human GITR function in humanized mice.

• Protein–Protein Interaction Studies:
  Recombinant GITR has been used in binding assays to characterize its interaction with GITR ligand (GITRL), including affinity measurements and structural studies.

Representative Published Research Examples:

• Cancer Immunotherapy:
  Recombinant GITR has been used in bioassays to study its costimulatory effects on T cells and its potential to enhance anti-tumor immunity, both alone and in combination with checkpoint inhibitors.

• Autoimmune and Inflammatory Disease:
  Studies have used recombinant GITR to investigate its role in modulating immune responses in models of rheumatoid arthritis, inflammatory bowel disease, and other autoimmune conditions.

• ELISA Standardization:
  Recombinant GITR is used as a standard in ELISA for quantifying GITR or GITRL in cell culture supernatants or serum samples.

Summary Table: Applications of Recombinant Human GITR in Research

Additional Notes:

• Recombinant Human GITR is typically used in in vitro settings, but its validation in in vivo models is often indirect, supporting the development and testing of GITR-targeting therapeutics.
• Applications are primarily research-focused and not for diagnostic or therapeutic use in humans.

If you require protocols or more detailed application notes for a specific assay type, please specify the context or experimental system.

To reconstitute and prepare Recombinant Human GITR protein for cell culture experiments, follow these best-practice steps based on protein type and general recombinant protein handling protocols:

1. Review Product Documentation

• Always check the product’s Certificate of Analysis (CoA) or data sheet for specific instructions, as formulations and recommended diluents may vary between preparations.

2. Centrifuge the Vial

• Briefly spin down the lyophilized protein vial to ensure all powder is at the bottom before opening.

3. Reconstitution Buffer

• For most recombinant human GITR proteins (including Fc chimeras and His-tagged forms), reconstitute in sterile PBS (phosphate-buffered saline).
• If the protein contains carrier protein (e.g., BSA), or if recommended by the manufacturer, use sterile PBS containing at least 0.1% human or bovine serum albumin to stabilize the protein and prevent adsorption to surfaces.

4. Reconstitution Concentration

• A typical reconstitution concentration is 100 μg/mL. Adjust the volume of buffer added to the vial to achieve this concentration.

5. Gentle Mixing

• Gently swirl or invert the vial to dissolve the protein. Do not vortex, as vigorous agitation can denature or aggregate the protein.
• Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.

6. Aliquoting and Storage

• Once fully dissolved, aliquot the protein into single-use portions to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at –20°C or –70°C for long-term storage. For short-term use (up to 1 month), storage at 2–8°C may be acceptable.

7. Preparation for Cell Culture

• Before adding to cell culture, dilute the reconstituted stock to the desired working concentration using sterile cell culture medium or PBS.
• If using in functional assays, ensure the final buffer is compatible with your cells (e.g., serum-free or serum-containing medium as appropriate).

Summary Table: Key Steps for Recombinant Human GITR Protein Reconstitution

Additional Notes:

• Avoid repeated freeze-thaw cycles to maintain protein integrity.
• If the protein is to be used in sensitive assays, consider adding carrier protein (e.g., BSA) to minimize loss due to adsorption.
• Always use sterile technique to prevent contamination.

These steps are consistent with best practices for recombinant protein handling and specifically tailored for GITR protein preparations as described in the cited protocols.