Recombinant Human CD4 (Soluble)

Recombinant Human CD4 (Soluble) is used in research applications primarily for its ability to modulate immune responses and to study or inhibit viral entry, especially in the context of HIV research.

Key scientific applications and rationales include:

• Inhibition of HIV Entry: Soluble CD4 (sCD4) binds to the HIV-1 envelope glycoprotein gp120, blocking the virus from attaching to and entering CD4+ T cells. This property makes sCD4 a valuable tool for studying HIV entry mechanisms and for screening antiviral compounds. In vitro, sCD4 can neutralize HIV infection by triggering premature conformational changes in the viral envelope, preventing productive infection. However, high concentrations are often required to neutralize primary clinical isolates, and its clinical efficacy has been limited by pharmacokinetic and resistance issues.

• Immunomodulation and Inflammation Research: Recent studies demonstrate that sCD4 can suppress excessive inflammatory activation of macrophages by disrupting the MHC II–TLR4 signaling complex, thereby reducing pro-inflammatory cytokine production. This anti-inflammatory effect is independent of TCR or CD40L signaling and may be beneficial in models of sepsis or systemic inflammatory response syndrome (SIRS). sCD4 administration in animal models has been shown to attenuate inflammatory pathology and improve outcomes in severe infections.

• Mechanistic Studies of CD4–MHC II Interactions: sCD4 is used to dissect the molecular interactions between CD4 and MHC class II molecules, which are central to T cell activation and antigen presentation. Recombinant sCD4 allows researchers to study these interactions in a controlled, cell-free system.

• Tool for Antibody and Vaccine Research: sCD4 can be used to expose or stabilize specific conformations of viral envelope proteins, enhancing the efficacy of certain antibodies or cytotoxic immune complexes in neutralization assays.

• Autoimmunity and Inflammatory Disease Models: Fusion proteins or immunoadhesins based on CD4 have been explored for their ability to modulate T cell activation and reduce disease severity in models of autoimmune and inflammatory diseases.

Summary of advantages:

• Enables precise mechanistic studies of immune signaling.
• Serves as a potent inhibitor of HIV entry in vitro.
• Provides a tool for dissecting inflammatory pathways and testing anti-inflammatory strategies.
• Facilitates antibody and vaccine research by stabilizing viral envelope conformations.

Limitations:

• High concentrations may be required for some applications, especially for neutralizing primary HIV isolates.
• Clinical translation has been limited by pharmacokinetics and potential immunogenicity.

In summary, recombinant human soluble CD4 is a versatile reagent for immunology and virology research, with applications ranging from HIV inhibition to the study of immune regulation and inflammation.

Recombinant Human CD4 (Soluble) can be used as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately determined. This approach is common in immunoassays where native protein standards are unavailable or impractical.

Key considerations for using recombinant CD4 as an ELISA standard:

• Purity and Characterization: The recombinant CD4 should be highly purified (typically >90–95% by SDS-PAGE/HPLC). Confirm the protein’s identity and purity using appropriate analytical methods.
• Concentration Determination: Accurately quantify the protein concentration using reliable methods such as UV absorbance at 280 nm, BCA assay, or amino acid analysis.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant CD4 in the same buffer or matrix as your samples to minimize matrix effects. Use these dilutions to generate a standard curve for quantification.
• Epitope Compatibility: Ensure that the recombinant CD4 contains the relevant epitopes recognized by the capture and detection antibodies in your ELISA. Most recombinant soluble CD4 proteins correspond to the extracellular domain and are suitable for assays targeting soluble CD4.
• Assay Validation: Validate the standard curve by comparing recovery and linearity using known concentrations. If possible, compare results with a native CD4 standard or reference material to confirm equivalence.
• Kit-Specific Recommendations: Some commercial ELISA kits are optimized for native CD4 and may not recommend recombinant standards due to potential differences in glycosylation or folding. Always consult the kit’s technical manual for compatibility.

Limitations and Best Practices:

• Recombinant standards may differ from native proteins in post-translational modifications (e.g., glycosylation), which can affect antibody binding and quantification accuracy.
• For regulatory or clinical applications, reference to an international standard (e.g., WHO Reference Material) is preferred if available.
• Always document the source, lot, and characterization data of the recombinant standard used.

Summary Table: Recombinant CD4 as ELISA Standard

In summary: You can use recombinant human CD4 (soluble) as a standard for ELISA quantification, provided it is well-characterized, highly purified, and compatible with your assay’s antibodies and protocol. Always validate its performance in your specific assay system.

Recombinant Human CD4 (Soluble) has been validated for several key applications in published research, primarily in the fields of HIV research, immunology, and drug screening.

Validated Applications:

• HIV-1 Neutralization and Entry Inhibition:
  Soluble CD4 (sCD4) binds to the HIV-1 envelope glycoprotein gp120, blocking the virus from attaching to and infecting CD4+ T cells. This property has been used to study HIV entry mechanisms, screen for HIV inhibitors, and develop neutralization assays.

• Drug Screening:
  sCD4 is used in high-throughput screening assays to identify compounds that block HIV entry or disrupt the gp120-CD4 interaction.

• T-Cell Research and Immunology:
  sCD4 serves as a tool to dissect T-cell receptor signaling, study CD4-MHC class II interactions, and investigate T-cell activation pathways.

• Cell Adhesion Assays:
  Recombinant sCD4 has been validated for supporting the adhesion of certain cell lines (e.g., HeLa cells), which is relevant for studying cell-cell interactions mediated by CD4.

• Vaccine and Antibody Characterization:
  sCD4 is used in binding and competition assays to map the CD4-binding site on HIV Env proteins and to characterize neutralizing antibodies targeting this site.

• Viral Entry Studies Beyond HIV:
  sCD4 has been shown to inhibit entry of other viruses, such as Ebola virus, by interfering with viral entry pathways, demonstrating its broader utility in virology research.

Supporting Details:

• sCD4 is typically produced as a recombinant protein corresponding to the extracellular domain of human CD4, often expressed in mammalian or bacterial systems for functional studies.
• In flow cytometry and ELISA-based assays, sCD4 is used as a competitor or probe to study CD4-dependent interactions.
• sCD4 has also been used to select for viral variants with altered gp120 affinity, aiding in the study of viral evolution and immune escape.

Summary Table of Validated Applications

These applications are well-supported in the literature and demonstrate the versatility of recombinant human soluble CD4 as a research tool.

To reconstitute and prepare Recombinant Human CD4 (Soluble) protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
• Warm the vial to room temperature before opening to minimize condensation.

Reconstitution

• Buffer selection: Use sterile PBS (phosphate-buffered saline, pH 7.2–7.4) or sterile distilled water for reconstitution, as recommended by most protocols.
• Concentration: Common reconstitution concentrations are 0.25 µg/µL (250 µg/mL) in sterile water, 100 µg/mL in PBS, or 0.2 mg/mL (200 µg/mL) in sterile water. Adjust the volume to achieve your desired working concentration.
• Dissolving: Add the buffer gently along the vial wall. Do not vortex; instead, gently pipette up and down or swirl to dissolve the protein completely.
• Incubation: Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation to ensure full dissolution.

Storage After Reconstitution

• Short-term storage: Store at 2–8°C for up to one month.
• Long-term storage: Aliquot and store at –20°C to –80°C to avoid repeated freeze-thaw cycles.
• Stabilizers: For long-term storage, consider adding an equal volume of glycerol (final 50%) if compatible with your downstream application. Alternatively, add a carrier protein such as 0.1% BSA to prevent adsorption and loss of activity.

Preparation for Cell Culture

• Dilution: Before adding to cell culture, dilute the reconstituted protein to the desired working concentration using cell culture medium or PBS. If using serum-free conditions, ensure the protein is fully soluble and compatible with your medium.
• Sterility: Always use sterile technique. If sterility is a concern, filter the final solution through a 0.2 µm filter before use.
• Endotoxin: Confirm the endotoxin level is suitable for cell culture (typically <1 EU/µg for sensitive cells).

Additional Notes

• Do not exceed 1 mg/mL during reconstitution to avoid solubility issues.
• Avoid vigorous mixing (no vortexing) to prevent protein denaturation.
• If solubility issues occur, incubate the solution overnight at 4°C.

Summary protocol example:

1. Centrifuge vial, warm to room temperature.
2. Add sterile PBS or water to achieve 100 µg/mL.
3. Gently pipette to dissolve, incubate at room temperature for 15–30 min.
4. Aliquot and store at –20°C or –80°C with 50% glycerol or 0.1% BSA if needed.
5. Dilute to working concentration in cell culture medium before use.

These steps ensure optimal solubility, stability, and bioactivity of recombinant human CD4 (soluble) protein for cell culture applications.