Recombinant Mouse CD80

Recombinant Mouse CD80 is widely used in research applications to study immune co-stimulation, T cell activation, and anti-tumor immunity due to its critical role as a co-stimulatory molecule in the immune response.

Key scientific reasons to use recombinant Mouse CD80:

• T Cell Co-stimulation: CD80 (also known as B7-1) is expressed on antigen-presenting cells (APCs) and binds to CD28 on T cells, providing a necessary co-stimulatory signal for full T cell activation, proliferation, and cytokine production. This is essential for dissecting mechanisms of immune activation in vitro and in vivo.

• Checkpoint Inhibition Studies: Recombinant CD80 can block the PD-1/PD-L1 pathway by binding to PD-L1, thereby overcoming immune suppression and restoring T cell function. This makes it valuable for research on immune checkpoint blockade and cancer immunotherapy.

• Anti-tumor Immunity: Soluble or Fc-fusion forms of recombinant CD80 have demonstrated significant anti-tumor effects in mouse models, both by directly activating T cells and by enhancing immune infiltration into tumors. These properties are useful for preclinical studies of novel immunotherapeutic strategies.

• Functional Assays: Recombinant CD80 is used in cell-based assays to evaluate T cell responses, screen for immunomodulatory drugs, and study interactions with other immune checkpoint molecules.

• Versatility in Experimental Design: Recombinant proteins allow for controlled, reproducible experiments, including dose-response studies, mechanistic investigations, and combination therapies with other immune modulators.

Additional context:

• Recombinant CD80 can be engineered as soluble proteins, Fc-fusion proteins, or tagged constructs, enabling diverse applications such as flow cytometry, ELISA, and in vivo functional studies.
• Using recombinant proteins ensures batch-to-batch consistency and eliminates variability associated with primary cell isolation or endogenous protein expression.

In summary, recombinant Mouse CD80 is a powerful tool for immunological research, particularly in studies of T cell activation, immune checkpoint regulation, and cancer immunotherapy.

Yes, recombinant mouse CD80 can be used as a standard for quantification and calibration in ELISA assays, but with important considerations regarding its application and validation.

Suitability for ELISA Standardization

Recombinant mouse CD80 proteins are specifically designed and validated for use as ELISA standards. These recombinant proteins are expressed in mammalian systems (such as HEK 293 cells) and are formulated with appropriate storage buffers to maintain stability and biological relevance. The recombinant CD80 standard is guaranteed to work with validated capture and detector antibody pairs in sandwich ELISA formats.

Critical Calibration Considerations

However, there is a fundamental distinction you must understand: recombinant proteins purchased separately from ELISA kit components are not pre-calibrated to the same master calibrator standards used in commercial ELISA kits. This creates a significant technical challenge. When using a recombinant protein as a quality control or standard, you should expect approximately ±25% recovery variation between the mass values calculated by ELISA versus those reported on the vial label.

The primary source of this discrepancy is the large dilution factor required to place the recombinant protein within the ELISA detection range—typically diluting from micrograms per milliliter to picograms per milliliter. Each dilution step introduces potential inaccuracy, and larger dilution factors compound this error.

Recommended Approach

For optimal results, use recombinant CD80 standards that are specifically included with or validated for your particular ELISA kit system. If you must use a separately purchased recombinant protein standard, ensure it has been validated against your specific antibody pair and calibrated to an appropriate master calibrator. Additionally, account for the expected ±25% variation in your data analysis and validation protocols.

Applications of Recombinant Mouse CD80 in Published Research

Recombinant mouse CD80 has been validated for several important applications in immunological and oncological research:

T Cell Activation and Costimulation

Recombinant CD80 proteins, particularly in Fc-fusion formats, have been extensively validated for enhancing T cell activation. The CD80-Fc fusion protein effectively activates T cells through CD28 costimulation, which is a fundamental mechanism in adaptive immunity. This application leverages CD80's natural role as a ligand for CD28 on T cells, resulting in enhanced cell proliferation and cytokine production.

Tumor Immunotherapy

CD80-based recombinant proteins have demonstrated significant anti-tumor activity in preclinical models. In murine tumor models, CD80-Fc fusion proteins have shown the ability to inhibit tumor growth in vivo, even in tumors with poor response to immune checkpoint inhibitors. The therapeutic mechanism involves both direct T cell activation through CD28 engagement and inhibition of PD-1-mediated suppression. Engineered CD80 variants with enhanced PD-L1 affinity have been validated to provide superior anti-tumor efficacy compared to checkpoint blockade alone in mouse tumor models.

Immunoassay Applications

Recombinant mouse CD80 proteins have been validated for use in enzyme-linked immunosorbent assays (ELISA) and flow cytometry applications. These formats are commonly employed for detecting CD80 expression and measuring immune responses in research settings.

Combination Immunotherapy Studies

CD80-Fc fusion proteins have been validated in combination approaches, demonstrating that co-administration with other therapeutic agents—such as discoidin domain receptor inhibitors—can significantly promote T cell infiltration in the tumor microenvironment and enhance tumor growth inhibition.

To reconstitute and prepare Recombinant Mouse CD80 protein for cell culture experiments, use sterile technique and follow these general steps, which are consistent with best practices for recombinant protein handling and supported by multiple technical sources:

1. Check the Product Datasheet or Certificate of Analysis (CoA):

   • Always refer to the lot-specific datasheet or CoA for precise instructions, as formulation and recommended diluent may vary by manufacturer and protein tag.

2. Reconstitution:

   • Most lyophilized recombinant proteins, including CD80, are reconstituted in sterile, deionized water or sterile PBS (phosphate-buffered saline) at a concentration recommended by the supplier (commonly 100 μg/mL, but verify for your specific product).
   • Add the appropriate volume of diluent directly to the vial. Do not use buffers containing calcium, magnesium, or preservatives unless specified.
   • Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing to prevent protein denaturation or foaming.
   • Allow the vial to stand at room temperature for 15–30 minutes with gentle agitation until fully dissolved.

3. Aliquoting and Storage:

   • Once reconstituted, aliquot the protein into single-use volumes to avoid repeated freeze-thaw cycles, which can degrade protein activity.
   • Store aliquots at 2–8°C for up to one month, or at –20°C to –70°C for longer-term storage.
   • Avoid storing at –20°C in frost-free freezers due to temperature cycling.

4. Preparation for Cell Culture:

   • Before adding to cell cultures, dilute the reconstituted protein to the desired working concentration using sterile cell culture medium or buffer compatible with your assay.
   • Filter-sterilize the final working solution if sterility is a concern and the protein is not already sterile-filtered.

5. General Notes:

   • The typical working concentration for functional assays (e.g., T cell activation) is in the range of 1–10 μg/mL, but this should be optimized for your specific application.
   • If the protein is fused to Fc or His tags, confirm compatibility with your downstream assay.

Summary Table: Recombinant Mouse CD80 Reconstitution

Key Reminders:

• Always use aseptic technique.
• Refer to the lot-specific datasheet for any unique instructions.
• Avoid repeated freeze-thaw cycles to maintain protein integrity.

These steps will ensure that your recombinant Mouse CD80 protein is properly prepared for reliable use in cell culture experiments.