Recombinant Viral CMV UL146

Recombinant Viral CMV UL146 is used in research applications primarily to study its role as a viral chemokine (vCXCL1) that interacts with host immune cells, particularly neutrophils, via the CXCR1 and CXCR2 receptors. This protein is valuable for dissecting the mechanisms of cytomegalovirus (CMV) immune modulation, viral dissemination, and host-pathogen interactions.

Key scientific applications and rationales include:

• Chemotaxis and Signal Transduction Studies: Recombinant UL146 induces chemotaxis, calcium mobilization, and degranulation of neutrophils by acting as a potent agonist for CXCR2 and a less potent but efficacious agonist for CXCR1. This allows researchers to model and quantify immune cell recruitment and activation in vitro.

• Viral Pathogenesis Research: CMV uses UL146 to attract neutrophils to infected endothelial cells, potentially facilitating viral spread and persistence in the host. Studying recombinant UL146 helps elucidate how CMV manipulates the immune system to maintain infection reservoirs.

• Comparative Chemokine Biology: UL146 shares sequence similarity with human CXC chemokines and contains conserved motifs important for receptor binding. Recombinant protein enables direct comparison of viral and host chemokine activities, receptor specificity, and downstream signaling.

• Assay Development: Recombinant UL146 is validated for bioactivity assays, including chemotaxis and ELISA, making it a reliable standard for quantifying immune responses or screening inhibitors.

• Vaccine and Therapeutic Research: Understanding UL146’s immunomodulatory functions can inform the design of CMV-based vectors or vaccines, and identify potential targets for antiviral therapies.

Best practices for using recombinant UL146 in research:

• Employ it in dose-dependent chemotaxis assays to measure neutrophil migration or activation.
• Use it as a positive control for CXCR2/CXCR1 signaling studies.
• Investigate its effects on endothelial cell-neutrophil interactions to model CMV dissemination.
• Apply it in neutralization assays to test antibodies or small molecules targeting viral chemokine activity.

In summary, recombinant CMV UL146 is a specialized tool for probing viral manipulation of host immunity, with broad utility in immunology, virology, and therapeutic development.

Yes, Recombinant Viral CMV UL146 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and validated for your specific application.

Key Points Supporting Use as an ELISA Standard:

• Purity and Characterization: Recombinant CMV UL146 proteins (such as those from R&D Systems, Leinco, Creative Diagnostics, and others) are typically >97% pure by SDS-PAGE and have defined amino acid sequences, making them suitable for use as quantitative standards in ELISA.
• Formulation: Many suppliers offer the recombinant protein in lyophilized form, often with stabilizers like BSA, which is recommended for use as an ELISA standard to ensure stability and reproducibility.
• Guidelines: General ELISA best practices recommend using purified or recombinant proteins for standard curves, especially when quantifying specific antigens or antibodies. The standard curve should be prepared using serial dilutions of the recombinant protein to cover the expected concentration range in your samples.
• Validation: The recombinant protein should be validated for lack of cross-reactivity and for its ability to generate a reliable, reproducible signal in your assay format (direct, indirect, or sandwich ELISA).

Practical Considerations:

• Reconstitution: Follow the manufacturer’s instructions for reconstitution, as improper handling can affect protein activity and quantification accuracy.
• Standard Curve Range: The typical ELISA standard curve range for recombinant proteins is 0–1000 pg/mL, but this can be adjusted based on the sensitivity of your assay and the expected analyte concentration.
• Data Analysis: Use appropriate curve-fitting methods (e.g., 4-parameter logistic or 5-parameter logistic regression) for accurate quantification from the standard curve.

References:

• Boster Bio Blog: Guidelines for Preparing ELISA Standards
• R&D Systems: Recombinant Viral CMV UL146/vCXC1 Protein
• Leinco Technologies: Recombinant Viral CMV UL146
• Creative Diagnostics: Recombinant CMV UL146 Protein

In summary, recombinant CMV UL146 is a suitable and commonly used standard for ELISA quantification and calibration, as long as it is handled and validated according to established protocols.

Recombinant Viral CMV UL146 (vCXCL1) has been validated in published research for several key applications, primarily related to its function as a viral chemokine and its role in modulating host immune responses. The main applications include:

1. Chemotaxis Assays:
   Recombinant UL146/vCXCL1 has been used to study its ability to induce chemotaxis of neutrophils by activating the chemokine receptors CXCR1 and CXCR2. This has been demonstrated in both in vitro and in vivo models, confirming its role in attracting neutrophils to sites of infection and facilitating viral dissemination.

2. Calcium Mobilization Assays:
   The protein has been validated for its ability to trigger intracellular calcium release in cells expressing CXCR1 or CXCR2, confirming its agonistic activity on these receptors. This is typically measured in transfected cell lines such as murine pre-B cell lines (L1.2, 300.19) or CHO cells.

3. Binding and Competition Assays:
   Recombinant UL146/vCXCL1 has been used in radioligand binding and competition experiments to determine its affinity and specificity for CXCR1 and CXCR2, showing that it binds selectively to these receptors and not to other chemokine receptors.

4. Structural and Functional Studies:
   The recombinant protein has been employed in studies to elucidate the structural features of vCXCL1, including its C-terminal domains and the impact of genotype variation on protein folding and function. This includes the use of mass spectrometry and chromatographic purification to confirm protein identity and purity.

5. Viral Dissemination and Virulence Studies:
   In animal models, recombinant UL146/vCXCL1 has been used to investigate its role as a virulence factor, particularly in facilitating viral spread by attracting neutrophils that can act as carriers of the virus.

6. ELISA and Immunoassays:
   Recombinant UL146/vCXCL1 is used as a standard in ELISA and other immunoassays to detect and quantify antibodies or other immune responses against the viral chemokine.

These applications highlight the utility of recombinant CMV UL146/vCXCL1 in studying viral immune evasion, host-pathogen interactions, and the development of potential therapeutic strategies targeting chemokine signaling pathways.

To reconstitute and prepare Recombinant Viral CMV UL146 protein for cell culture experiments, dissolve the lyophilized protein at a concentration of 25 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). This carrier protein helps stabilize the recombinant protein and prevents adsorption to surfaces, which is critical for maintaining activity in cell culture applications.

Step-by-step protocol:

• Aseptic technique: Work in a sterile environment to avoid contamination.
• Reconstitution:
  • Add the recommended volume of sterile PBS with 0.1% BSA directly to the lyophilized protein.
  • Gently pipette up and down or swirl to dissolve. Avoid vigorous mixing or vortexing, which may denature the protein.
  • Let the solution stand at room temperature for at least 20 minutes to ensure complete dissolution.
• Storage after reconstitution:
  • Store the reconstituted protein at 2–8°C for up to one month or at –20°C to –70°C for longer-term storage in a manual defrost freezer.
  • Avoid repeated freeze-thaw cycles to preserve protein integrity.
• Dilution for cell culture:
  • Dilute the stock solution to your desired working concentration using cell culture medium or buffer containing carrier protein (e.g., 0.1% BSA, 5% HSA, or 10% FBS) to further stabilize the protein and prevent loss due to adsorption.
  • Do not use water for dilution, as this may cause protein degradation.
• Quality control:
  • Confirm that the solution is clear and free of particulates. If necessary, filter through a 0.2 μm sterile filter.

Additional notes:

• The protein is typically lyophilized from a solution containing acetonitrile and trifluoroacetic acid (TFA); ensure complete removal of these solvents during reconstitution.
• Endotoxin levels are low (<0.1 EU/μg), making the protein suitable for most cell culture applications.
• The UL146 protein is a chemokine and may induce chemotaxis or activation in immune cell assays.

Summary of best practices:

• Use sterile PBS + 0.1% BSA for initial reconstitution.
• Store aliquots at 2–8°C (short-term) or –20°C to –70°C (long-term).
• Avoid freeze-thaw cycles.
• Dilute with carrier-containing buffer or medium for cell culture.

This protocol ensures optimal stability and activity of recombinant CMV UL146 protein for cell-based experiments.