Recombinant Human MIP-4 (CCL18)

Recombinant Human MIP-4 (CCL18) is a chemokine widely used in research to study immune cell migration, tumor biology, and fibrotic diseases due to its specific biological activities and human relevance.

Key reasons to use recombinant CCL18 in research applications:

• Chemoattractant Activity: CCL18 acts as a potent chemoattractant for naive T cells, CD4+ T cells, CD8+ T cells, and nonactivated lymphocytes, making it valuable for studying immune cell trafficking, chemotaxis assays, and T cell biology.
• Cancer Research: CCL18 is implicated in promoting breast cancer metastasis and the recruitment of regulatory T cells, which can facilitate tumor progression and immune evasion. It is also being explored as a biomarker for early detection in certain cancers, such as lung cancer.
• Fibrosis and Inflammation: High levels of CCL18 are associated with fibrotic diseases (e.g., idiopathic pulmonary fibrosis), where it induces collagen production in fibroblasts and contributes to pathological matrix deposition. This makes it a useful tool for modeling fibrosis and studying fibroblast activation.
• Receptor Studies: CCL18 interacts with several receptors, including PITPNM3, GPR30, CCR8, and acts as a neutral antagonist for CCR3. Recent studies also suggest binding to CCR6 on fibroblasts, expanding its utility in receptor-ligand interaction assays and signaling pathway research.
• Human-Specific Biology: CCL18 is primarily produced by human innate immune cells and exerts its main effects on the adaptive immune system. There is no murine homolog, so recombinant human CCL18 is essential for in vitro studies using human cells or for translational research relevant to human disease.
• Versatile Applications: Recombinant CCL18 is suitable for a range of applications, including cell migration assays, receptor binding studies, fibrosis models, cancer cell signaling, and biomarker validation.

Additional context:

• CCL18 is highly expressed in the lung, lymph nodes, placenta, and bone marrow.
• It is structurally related to other CC chemokines, such as MIP-1α, but has distinct biological functions.
• Recombinant forms are typically produced in E. coli or mammalian systems and are available as biologically active proteins for in vitro use.

In summary, recombinant human MIP-4 (CCL18) is a critical reagent for dissecting human immune responses, tumor microenvironment interactions, and fibrotic mechanisms, especially where human-specific pathways are involved and murine models are not applicable.

Yes, you can use recombinant human MIP-4 (CCL18) as a standard for quantification or calibration in your ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common practice in ELISA development and quantification protocols.

Key considerations and supporting details:

• Recombinant CCL18 is routinely used as a standard in commercial ELISA kits for quantifying CCL18 in biological samples. For example, validated kits use E. coli-expressed recombinant human CCL18 as the calibrator, and the resulting standard curves are shown to be parallel to those generated with natural CCL18, indicating equivalence for quantification purposes.
• The standard must be highly purified and accurately quantified. The protein should be reconstituted and diluted according to the manufacturer’s or protocol’s instructions to generate a reliable standard curve. Impurities or inaccurate concentration determination can compromise assay accuracy.
• Standard curve preparation: Prepare a serial dilution of the recombinant CCL18 in the same buffer or diluent as your samples to ensure matrix compatibility and minimize matrix effects. The typical standard curve range for CCL18 ELISAs is from low pg/mL to several ng/mL, depending on assay sensitivity.
• Validation: If you are developing your own ELISA or using a non-kit protocol, it is important to validate that your recombinant standard produces a linear and parallel standard curve compared to natural CCL18, especially if you plan to report absolute concentrations.
• Documentation: Record the lot number, source, and quantification method of your recombinant CCL18 standard for reproducibility and transparency in your experimental records.

Limitations:

• If your recombinant CCL18 is not from the same source or expression system as the kit standard, minor differences in glycosylation or folding may affect antibody recognition, though most commercial antibodies are validated for both natural and recombinant forms.
• For regulatory or diagnostic applications, additional validation may be required to demonstrate equivalence between recombinant and native standards.

Summary:
Using recombinant human MIP-4 (CCL18) as a standard is scientifically valid and widely accepted for ELISA quantification, as long as the protein is pure, accurately quantified, and validated for your assay system.

Recombinant Human MIP-4 (CCL18) has been validated for several key applications in published research, primarily in immunological and cancer studies. The most common validated applications include:

• Immunohistochemistry (IHC): Used to detect CCL18 expression in human tissues, such as tonsillitis, heart, and pancreas. Published studies have used IHC to localize CCL18 in tissue sections, particularly in the context of inflammation and cancer.
• Immunofluorescence (IF): Applied to visualize CCL18 in cells and tissues, supporting studies of its cellular localization and expression patterns.
• Enzyme-Linked Immunosorbent Assay (ELISA): Utilized for quantifying CCL18 protein levels in biological samples, such as serum or tissue lysates, often in biomarker studies.
• Functional cell-based assays: Recombinant CCL18 has been used in chemotaxis assays to demonstrate its role as a chemoattractant for naive T cells, CD4+ and CD8+ T cells, and dendritic cells. These assays validate its biological activity in immune cell recruitment.
• Cancer research models: CCL18 has been validated in studies investigating its role in tumor progression, metastasis, and immune modulation, particularly in breast and colorectal cancer. It is used to study recruitment of regulatory T cells, induction of M2-like macrophages, and promotion of cancer cell invasion and migration.
• Structural and biochemical studies: Recombinant CCL18 has been used for crystallography and oligomerization analyses to understand its molecular structure and interactions.

Additional validated research contexts:

• Biomarker studies: CCL18 is measured as a potential biomarker for chronic inflammatory diseases (e.g., pulmonary fibrosis, Gaucher disease) and cancer, using proteomic and immunoassay platforms.
• Receptor binding and signaling assays: Recombinant CCL18 is used to characterize its interaction with receptors such as PITPNM3, GPR30, and CCR8, and to study downstream signaling effects.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed publications and are widely used in immunology, oncology, and translational research.

To reconstitute and prepare Recombinant Human MIP-4 (CCL18) protein for cell culture experiments, follow these steps:

1. Centrifuge the vial briefly before opening to ensure all lyophilized protein is at the bottom.
2. Warm the vial to room temperature before opening if stored cold.
3. Add sterile distilled water or aqueous buffer (optionally containing 0.1% BSA for protein stability) to achieve a final concentration of 0.1–1.0 mg/mL.
4. Do not vortex or mix vigorously; instead, gently pipette the solution down the sides of the vial and allow several minutes for complete dissolution.
5. Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
6. Storage after reconstitution:
   • At 2–8°C (4°C) for up to 1 week.
   • For longer-term storage, keep at –20°C or below, ideally with a carrier protein such as 0.1% BSA to prevent adsorption and loss of activity.

Preparation for cell culture:

• After reconstitution, further dilute the stock solution into cell culture medium or buffer as required for your experiment.
• Ensure all solutions are sterile and endotoxin-free to avoid confounding cellular responses.

Best practices:

• Avoid concentrations above 1 mg/mL during reconstitution to prevent aggregation.
• Do not vortex, as vigorous mixing can denature the protein.
• Use aliquots to minimize freeze-thaw cycles, which can degrade protein activity.

Summary Table: Recombinant Human MIP-4 (CCL18) Reconstitution

These steps ensure optimal solubility, stability, and biological activity of recombinant MIP-4 (CCL18) for cell culture applications.