Recombinant Human MIP-1α (CCL3L1)

Recombinant Human MIP-1α (CCL3L1) is used in research applications to study immune cell recruitment, inflammation, and HIV-1 infection due to its potent chemotactic and immunomodulatory properties, as well as its high affinity for the CCR5 receptor.

Key scientific reasons to use recombinant CCL3L1 (MIP-1α, LD78β isoform) include:

• Immune Cell Recruitment and Inflammation: CCL3L1 is a chemokine that plays a central role in recruiting immune cells such as monocytes, T cells, B cells, and eosinophils to sites of inflammation, making it essential for studying leukocyte migration and inflammatory responses.
• CCR5 Ligand with High Affinity: The LD78β isoform encoded by CCL3L1 binds CCR5 with much higher affinity than the LD78α isoform (encoded by CCL3), making it particularly relevant for experiments involving CCR5-mediated signaling, immune cell trafficking, and receptor-ligand interactions.
• HIV-1 Research: CCL3L1 is a potent suppressor of HIV-1, as it competes with the virus for binding to the CCR5 co-receptor, thereby inhibiting viral entry into host cells. This makes recombinant CCL3L1 valuable for studying HIV-1 pathogenesis, resistance mechanisms, and potential therapeutic interventions targeting the CCR5 axis.
• Genetic and Functional Studies: Variation in CCL3L1 gene copy number and its interaction with CCR5 genotypes have been linked to differences in susceptibility to HIV/AIDS and other immune-related conditions, supporting its use in genetic association and functional studies.
• Standardization and Reproducibility: Using recombinant protein ensures batch-to-batch consistency, defined activity, and the ability to perform quantitative assays such as chemotaxis, receptor binding, and signaling studies.

Additional applications include:

• Modeling inflammatory diseases (e.g., COPD, autoimmune disorders) where CCL3L1-mediated cell recruitment is implicated.
• Drug screening for CCR5 antagonists or modulators of chemokine signaling.

In summary, recombinant human MIP-1α (CCL3L1) is a critical tool for dissecting chemokine biology, immune cell dynamics, and host-pathogen interactions, especially in the context of CCR5-dependent processes.

Recombinant Human MIP-1α (CCL3L1) can be used as a standard for quantification or calibration in ELISA assays, provided it is highly purified, its concentration is accurately determined, and it matches the isoform recognized by your assay antibodies.

Key considerations and supporting details:

• Recombinant proteins are commonly used as ELISA standards when they are well-characterized and their concentration is precisely known. This allows for the generation of a standard curve, which is essential for quantitative analysis in ELISA assays.

• CCL3L1 (LD78β) is an isoform of MIP-1α that shares 94% sequence homology with CCL3 (LD78α), but there are functional and structural differences between the isoforms. It is critical to confirm that your ELISA antibodies recognize CCL3L1 specifically, as some kits are designed for total MIP-1α (CCL3/CCL3L1), while others may be isoform-specific.

• Commercial ELISA kits often use recombinant MIP-1α as the standard, and results for natural and recombinant proteins are typically parallel, indicating suitability for quantification. However, these kits usually specify which isoform is used as the standard, and the antibodies are validated for that isoform.

• For accurate quantification:

  • Use a recombinant CCL3L1 standard that is highly purified and has a precisely determined concentration (e.g., by amino acid analysis or HPLC).
  • Prepare the standard curve according to best practices, ensuring the range covers the expected sample concentrations.
  • Confirm that the ELISA kit or antibodies you are using are validated for CCL3L1, not just CCL3 (LD78α), as cross-reactivity may vary.
  • If using a custom or in-house assay, validate the parallelism between your recombinant standard and endogenous analyte in your sample matrix.

• If your ELISA kit is designed for CCL3L1 (LD78β), using recombinant human CCL3L1 as a standard is appropriate. If your kit is for total MIP-1α or specifically for CCL3 (LD78α), verify antibody specificity before using CCL3L1 as a standard.

• Always follow the manufacturer’s recommendations for standard preparation, reconstitution, and storage, as these can affect assay accuracy.

In summary, recombinant human MIP-1α (CCL3L1) is suitable as an ELISA standard if it matches the isoform detected by your assay and is properly quantified. Always confirm antibody specificity and validate standard curve performance in your assay system.

Recombinant Human MIP-1α (CCL3L1), also known as LD78β, has been validated in published research for several key applications, primarily in immunology and infectious disease studies.

Key validated applications include:

• Chemoattractant assays: CCL3L1 is widely used to study chemotaxis, particularly for its ability to attract monocytes, lymphocytes, and dendritic cells via CCR1 and CCR5 receptors. It is notably more potent than the CCL3 (LD78α) isoform in inducing chemotaxis of human monocytes and lymphocytes.
• HIV-1 inhibition studies: CCL3L1 has been validated as a highly potent natural ligand and antagonist for CCR5, the co-receptor used by HIV-1 for cell entry. It is used in assays to block HIV-1 infection and to study mechanisms of viral entry inhibition, showing superior antiviral activity compared to other chemokines such as CCL3 and CCL5.
• Antigen-presenting cell (APC) targeting in vaccine research: CCL3L1 has been used as a targeting unit in DNA vaccine constructs to enhance antigen uptake and presentation by APCs, thereby boosting both humoral and T cell responses in immunization models.
• Genotype-phenotype association studies: Recombinant CCL3L1 is used in studies examining the impact of CCL3L1 gene copy number variation on immune function, susceptibility to infectious diseases (notably HIV/AIDS), and autoimmune conditions.
• Functional studies of chemokine receptor signaling: CCL3L1 is employed to dissect signaling pathways downstream of CCR1 and CCR5, including calcium flux and cell migration assays.

Additional details:

• CCL3L1 is often compared to its paralog CCL3 (LD78α) for potency and receptor specificity, with CCL3L1 showing higher affinity and efficacy for CCR5-mediated responses.
• Truncated forms of CCL3L1, generated in vivo, have also been studied for their altered receptor binding and functional properties.

In summary, recombinant human CCL3L1 is a validated tool for:

• Chemotaxis and immune cell migration assays
• HIV-1 entry and inhibition studies
• Vaccine targeting and immunogenicity enhancement
• Genetic association and functional genomics research
• Chemokine receptor signaling analyses

These applications are well-supported by published research in immunology, virology, and vaccine development.

To reconstitute and prepare Recombinant Human MIP-1α (CCL3L1) protein for cell culture experiments, follow these best-practice steps:

1. Equilibrate the vial: Allow the lyophilized protein to reach room temperature before opening to minimize condensation.

2. Centrifuge the vial: Briefly spin down the vial to collect all lyophilized material at the bottom before opening.

3. Reconstitution:

   • Add sterile water to achieve a concentration of 0.1 mg/mL (100 μg/mL), unless your protocol specifies otherwise.
   • Alternatively, some protocols recommend reconstituting in sterile PBS (phosphate-buffered saline) containing at least 0.1% BSA or HSA (bovine or human serum albumin) to stabilize the protein, especially for storage or sensitive applications.
   • Gently pipette the solution down the sides of the vial to dissolve the protein. Do not vortex; allow several minutes for complete dissolution.

4. Aliquoting and Storage:

   • Once fully dissolved, aliquot the solution to avoid repeated freeze-thaw cycles.
   • For short-term storage (up to 1 month), keep at 2–8 °C.
   • For long-term storage, freeze aliquots at –20 °C to –70 °C.
   • If storing for more than a few days, include 0.1% BSA or HSA as a carrier protein to prevent adsorption and loss of activity.

5. Preparation for Cell Culture:

   • Before use, dilute the reconstituted stock to the desired working concentration in cell culture medium or appropriate buffer.
   • Typical working concentrations for bioassays range from 1–100 ng/mL, but optimize based on your specific cell type and assay requirements.

Summary Table: Key Steps and Recommendations

Additional Notes:

• Always use sterile technique to prevent contamination.
• If your application is sensitive to carrier proteins, use only sterile water or PBS for reconstitution and minimize storage time.
• Avoid repeated freeze-thaw cycles, as this can degrade the protein and reduce activity.

These guidelines are based on standard protocols for recombinant chemokines and are suitable for most cell culture applications. Adjustments may be necessary depending on your specific experimental requirements.