Recombinant Human ICOS

Using Recombinant Human ICOS in research applications enables precise investigation of the ICOS (Inducible T-cell COStimulator) pathway, which is critical for understanding T cell activation, differentiation, and immune regulation in contexts such as cancer, autoimmunity, and transplantation.

Key reasons to use Recombinant Human ICOS include:

• Mechanistic Studies: Recombinant ICOS allows for controlled in vitro and in vivo studies of T cell co-stimulation, enabling detailed analysis of how ICOS engagement affects T cell proliferation, cytokine secretion, and differentiation into subsets such as Th1, Th2, Th17, and T follicular helper (Tfh) cells.
• Protein-Protein Interaction Assays: It is commonly used in binding assays (e.g., SPR, ELISA) to characterize interactions with its ligand (ICOSL) or with therapeutic antibodies, supporting drug discovery and mechanistic immunology research.
• Cancer Immunotherapy Research: ICOS is a promising target for immuno-oncology, as its activation can enhance anti-tumor immune responses, while its blockade may suppress unwanted immune activity in autoimmunity or transplantation. Recombinant ICOS is essential for preclinical evaluation of ICOS-targeted therapies and for dissecting the role of ICOS in the tumor microenvironment.
• Assay Development and Controls: Recombinant ICOS serves as a standard or positive control in functional assays, SDS-PAGE, and apoptosis assays, ensuring reproducibility and reliability in experimental workflows.
• Engineering and Screening: The recombinant format allows for modifications (e.g., Fc fusion, His-tag), facilitating purification, detection, and functional studies, and enabling high-throughput screening of ICOS-targeted molecules.

Additional context:

• Immunological Relevance: ICOS is upregulated upon T cell activation and is involved in germinal center formation, immunoglobulin class switching, and the regulation of both innate and adaptive immune responses.
• Translational Potential: Manipulating ICOS signaling has therapeutic implications for cancer, autoimmune diseases, and transplantation tolerance, making recombinant ICOS a valuable tool for translational and preclinical research.

In summary, Recombinant Human ICOS is a versatile reagent that enables detailed, reproducible, and translationally relevant studies of T cell co-stimulation and immune modulation across a range of biomedical research applications.

Yes, recombinant human ICOS can be used as a standard for quantification or calibration in ELISA assays, provided it is well-characterized and matches the form of ICOS you intend to measure. This is a common practice in quantitative ELISA development and is supported by both published protocols and manufacturer guidelines.

Key considerations and best practices:

• Protein Form and Purity: The recombinant ICOS used as a standard should be highly purified and, ideally, structurally similar to the endogenous ICOS you wish to quantify (e.g., same isoform, post-translational modifications if relevant). If the recombinant protein is a fragment or fusion (e.g., Fc chimera), you must account for differences in molecular weight and epitope presentation.

• Standard Curve Preparation: Prepare a standard curve using serial dilutions of the recombinant ICOS in the same buffer or matrix as your samples to minimize matrix effects. The concentration range should bracket the expected sample concentrations, typically from low pg/mL to ng/mL, depending on assay sensitivity.

• Correction Factors: If your recombinant standard differs in molecular weight or structure from native ICOS (e.g., contains tags or is a chimera), apply a correction factor to ensure accurate quantification relative to endogenous ICOS.

• Validation: Validate the standard by spiking known amounts into representative sample matrices and confirming recovery and linearity. This ensures the standard behaves similarly to endogenous ICOS in your assay system.

• Documentation: Follow any specific instructions provided with your recombinant protein regarding reconstitution, storage, and handling, as these can affect stability and quantification accuracy.

Limitations and caveats:

• Epitope Recognition: If your ELISA antibodies recognize epitopes altered or absent in the recombinant form, quantification may be inaccurate. Confirm antibody compatibility with your recombinant standard.

• Matrix Effects: Differences between the standard diluent and sample matrix (e.g., serum, plasma) can affect assay performance. Use matched matrices or appropriate controls to account for this.

• Regulatory/Diagnostic Use: For clinical or diagnostic applications, additional validation and regulatory compliance are required. For research use, these steps are generally sufficient.

Summary Table: Use of Recombinant Human ICOS as ELISA Standard

In summary: Recombinant human ICOS is suitable as a standard for ELISA quantification if you ensure structural compatibility, proper standard curve preparation, and thorough validation.

Recombinant Human ICOS has been validated for a range of applications in published research, primarily in immunology and cell biology. The most common validated applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or for screening high-affinity anti-human ICOS monoclonal antibodies, and for competition assays with anti-ICOS-L antibodies.
• Flow Cytometry: Employed to detect binding to ICOS ligand (ICOSL) on cell surfaces and to characterize protein-protein interactions.
• Protein-Protein Interaction Assays: Used in binding assays, such as surface plasmon resonance (SPR) and biolayer interferometry (BLI), to study the interaction between ICOS and ICOSL.
• Functional Cell-Based Assays: Validated for triggering intracellular signaling (e.g., AKT activation), modulating cytokine release, and influencing cell migration and adhesion in immune cells.
• In Vivo Immunomodulation: Recombinant ICOS-Fc has been used as an immunomodulatory agent in animal models, such as sepsis, to modulate cytokine storms, reduce inflammation, and protect against organ damage.
• Apoptosis Assays: Applied to study the role of ICOS in cell death pathways.
• Media Additive: Used to enhance protein or antibody production in cell culture systems.
• SDS-PAGE Control: Utilized as a molecular weight control in protein electrophoresis.

Additional research applications include:

• CAR-T Cell Engineering: ICOS domains have been incorporated into chimeric antigen receptor (CAR) constructs to enhance T cell proliferation, persistence, and antitumor activity in preclinical cancer models.
• Immunological Mechanism Studies: Used to investigate the costimulatory role of ICOS in T cell activation, B cell differentiation, and cytokine secretion.
• Autoimmunity and Inflammation: Soluble ICOS has been evaluated in studies of autoimmune diseases and inflammatory responses.

Summary Table of Validated Applications

These applications are supported by peer-reviewed studies and product validation data, confirming the utility of recombinant human ICOS in both basic and translational immunology research.

To reconstitute and prepare Recombinant Human ICOS protein for cell culture experiments, first consult the product’s Certificate of Analysis (CoA) for any lot-specific instructions. In the absence of a CoA, follow these general best practices:

1. Centrifuge the vial:
Before opening, briefly centrifuge the lyophilized protein vial at 4 °C to ensure all material is at the bottom and not lost when opening.

2. Reconstitution buffer:

• If the protein was lyophilized from PBS with trehalose (common for ICOS), reconstitute with sterile PBS to the recommended concentration (e.g., 200 μg/mL).
• If the datasheet suggests, you may also use sterile water for initial reconstitution, then dilute with PBS or cell culture medium as needed.

3. Reconstitution procedure:

• Add the calculated volume of buffer (e.g., PBS or sterile water) directly to the vial to achieve the desired stock concentration (e.g., 0.2 μg/μL or 200 μg/mL).
• Gently swirl or invert the vial to dissolve. Avoid vigorous shaking or vortexing to prevent protein denaturation and foaming.
• Allow the protein to fully dissolve at room temperature for 15–30 minutes with gentle agitation.

4. Dilution for cell culture:

• For working concentrations, dilute the reconstituted stock in cell culture medium immediately before use.
• To minimize protein loss from adsorption, dilute using medium containing a carrier protein such as 0.1% BSA or 10% FBS unless your experiment requires serum-free conditions.
• For serum-free or in vivo applications, avoid animal-derived carriers and consider using trehalose as a stabilizer.

5. Storage:

• After reconstitution, aliquot the stock solution to avoid repeated freeze-thaw cycles.
• Store aliquots at –20 °C to –80 °C for long-term stability. For short-term use (up to one week), store at 2–8 °C.
• If storing at low concentrations, add carrier protein or glycerol (5–50%) to improve stability.

6. Usage:

• Thaw aliquots on ice before use.
• Add the required amount directly to your cell culture medium.

Summary Table: Recombinant Human ICOS Protein Reconstitution

Always verify the specific requirements for your recombinant ICOS protein batch, as formulations and recommended procedures may vary between preparations and manufacturers.