CCL15 Antibody, FITC conjugated

What is CCL15 and why is it significant in research?

CCL15, also known as HCC-2, MIP-5, or Lkn1, is a chemokine that exists in both full-length (CCL15(1-92)) and N-terminally truncated forms. The truncated forms, particularly CCL15(27-92), demonstrate increased biological activity. CCL15 has been shown to affect hematopoietic progenitor cells by enhancing CXCL12-induced migration and strengthening shear stress-dependent adhesion to vascular cell adhesion molecule-1 (VCAM-1) . Recent studies have also identified CCL15 as a potential biomarker for hepatocellular carcinoma, suggesting its role in tumorigenesis and invasion . The biological significance of CCL15 makes it an important target for immunological detection.

How does FITC conjugation affect CCL15 antibody performance?

FITC (fluorescein isothiocyanate) conjugation provides direct fluorescent visualization of CCL15 expression without requiring secondary antibodies. This conjugation is particularly valuable for flow cytometry, immunofluorescence microscopy, and fluorescence-based immunoassays. When working with FITC-conjugated antibodies, researchers should consider:

• Photobleaching: FITC is susceptible to photobleaching, requiring careful sample handling with minimal light exposure

• pH sensitivity: FITC fluorescence is optimal at pH 8.0 and decreases significantly below pH 7.0

• Autofluorescence: Tissues and cell types with high autofluorescence may interfere with FITC signal detection

For optimal results, researchers should use appropriate mounting media containing anti-fade compounds and store FITC-conjugated antibodies at 2-8°C protected from light.

What structural features should researchers target when selecting CCL15 antibodies?

When selecting CCL15 antibodies, researchers should consider the specific isoform they wish to detect. The commercially available FITC-conjugated antibody targeting amino acids 22-113 would recognize both full-length CCL15(1-92) and the biologically more active truncated forms like CCL15(27-92) . This is critical as research has shown that N-terminally truncated CCL15 forms (LMW-CCL15) have significantly increased potency in inducing adhesion and chemotactic migration compared to the full-length form . Researchers investigating specific physiological contexts, such as G-CSF mobilization, should select antibodies that can differentiate between these forms or complement antibody studies with chromatographic separation techniques.

What are the optimal sample preparation methods for CCL15 detection using FITC-conjugated antibodies?

For effective CCL15 detection using FITC-conjugated antibodies, sample preparation is crucial. Based on experimental protocols in CCL15 research, the following methodology is recommended:

For serum/plasma samples:

• Collect blood in EDTA or heparin tubes

• Centrifuge at 1500g for 10 minutes at 4°C

• Aliquot plasma to avoid freeze-thaw cycles

• For distinguishing between full-length and truncated forms, consider reverse-phase chromatography separation prior to antibody detection

For cell preparations:

• Isolate target cell populations (e.g., HPCs using lineage depletion and Sca1+ selection)

• Perform fixation with 4% paraformaldehyde (10 minutes at room temperature)

• Permeabilize if detecting intracellular CCL15 (0.1% Triton X-100, 5 minutes)

• Block with 5% normal serum from the same species as the secondary antibody

• Incubate with FITC-conjugated anti-CCL15 antibody (typical dilution 1:50-1:200)

• Wash thoroughly to remove unbound antibody

How can researchers differentiate between various truncated forms of CCL15?

Distinguishing between full-length CCL15(1-92) and truncated forms is challenging with antibody detection alone. Research has shown that standard chromatographic methods can effectively separate these forms based on their different hydrophobic properties . A recommended approach combines:

• Reverse-phase HPLC separation using:

  • Column: Source RPC15 or equivalent

  • Binary gradient: 0.1% TFA to 60% acetonitrile/0.1% TFA over 60 minutes

  • Flow rate: 2.5 ml/min

  • Collection of 1-minute fractions starting at minute 20

• Analysis of fractions using:

  • Immunoassays with the FITC-conjugated CCL15 antibody

  • Mass spectrometry for precise identification

Expected retention times based on research findings:

• CCL15(27-92): ~36 minutes

• CCL15(24-92): ~38 minutes

• CCL15(1-92): ~51 minutes

This combined approach allows researchers to accurately differentiate between clinically relevant CCL15 isoforms.

What controls should be implemented when using CCL15 antibodies for functional studies?

When investigating CCL15 function in migration, adhesion, or other cellular assays, proper controls are essential for result validation:

When studying chemotaxis specifically, researchers should include:

• Migration medium-only controls

• CXCL12-only controls when investigating synergistic effects with CCL15

• Both full-length CCL15(1-92) and truncated CCL15(27-92) to compare activity

How does G-CSF treatment affect CCL15 processing and detection?

Granulocyte colony-stimulating factor (G-CSF) treatment significantly impacts CCL15 processing and should be considered when designing experiments. Research has shown that G-CSF mobilization in stem cell donors results in:

• Increased concentrations of N-terminally truncated CCL15 (LMW-CCL15) in plasma (1.1 ± 0.1 ng/ml vs. 0.4 ± 0.1 ng/ml in untreated controls)

• Enhanced neutrophil activation and release of serine proteases (elastase and cathepsin G)

• Proteolytic processing of CCL15 into multiple isoforms including CCL15(22-92), CCL15(24-91), CCL15(25-92), and CCL15(29-92)

Methodological considerations for research involving G-CSF include:

• Timing sample collection relative to G-CSF administration (optimal at 4-5 days post-treatment)

• Including protease inhibitors during sample collection to prevent ex vivo processing

• Using chromatographic separation to distinguish between isoforms

• Employing antibodies that can detect multiple processed forms or specific epitopes

What experimental approaches can detect the interaction between CCL15 and cellular signaling pathways?

CCL15 activates multiple signaling pathways, particularly through its receptors CCR1 and CCR3. To investigate these interactions, researchers can employ:

• Calcium mobilization assays:

  • Load cells with calcium-sensitive dyes (Fluo-4 AM)

  • Measure fluorescence changes upon CCL15 stimulation

  • Include pertussis toxin to confirm Gαi-coupling

• GTPase activation assays:

  • Pull-down assays for activated Rac

  • Western blot detection of phosphorylated downstream effectors

  • Include pathway-specific inhibitors like NSC23766

• Adhesion under shear stress:

  • Pre-coat laminar flow chambers with VCAM-1 (2 μg/ml)

  • Subject cells to increasing shear stress (0.35-15 dyn/cm²)

  • Quantify adherent cells under different conditions

• Chemotaxis cooperation assays:

  • Test CCL15 alone and in combination with CXCL12

  • Use 5 μm pore transwell systems

  • Incubate for 6 hours at 37°C

  • Quantify migration using DNA-binding fluorescent dyes

How can CCL15 antibodies be utilized in hematopoietic stem cell research?

CCL15 antibodies can provide valuable insights into hematopoietic stem cell (HSC) biology, particularly in transplantation and regenerative medicine contexts. Research strategies include:

• Monitoring CCL15 forms during HSC mobilization:

  • Measure plasma concentrations of different CCL15 isoforms using chromatography and FITC-antibody detection

  • Correlate with mobilization efficacy and neutrophil activation

• Investigating CCL15 effects on HSC function:

  • Colony formation assays (CFU-A) with different concentrations of CCL15 isoforms

  • Competitive repopulation assays to assess engraftment potential

  • Analysis of receptor expression on different HSC subpopulations

• Evaluating adhesion/migration mechanisms:

  • Combined CXCL12/CCL15 stimulation assays

  • Integrin activation studies using flow cytometry

  • Intravital microscopy with fluorescently labeled cells

Research has demonstrated that CCL15(27-92) significantly enhanced CXCL12-induced migration of Lin-/Sca1+ HPCs and strengthened shear stress-dependent adhesion to VCAM-1. Additionally, pretreatment of bone marrow with CCL15(27-92) significantly increased competitive repopulation in murine models . These findings suggest that CCL15 modulates HPC adhesion and migration with potential applications in improving stem cell transplantation outcomes.

How can researchers validate CCL15 as a biomarker in hepatocellular carcinoma?

Research has identified CCL15 as a potential biomarker for hepatocellular carcinoma (HCC). SELDI-TOF-MS revealed a specific 7777 M/Z band in HCC patient serum samples that was absent in healthy controls and benign disease patients . To validate CCL15 as an HCC biomarker, researchers should implement:

• Multi-cohort validation studies:

  • Include diverse patient populations

  • Compare HCC samples with cirrhosis, hepatitis, and healthy controls

  • Correlate with established HCC markers (AFP, DCP)

• Quantitative analysis methods:

  • ELISA with sensitivity verification

  • Immunohistochemistry of tumor versus adjacent tissues

  • Flow cytometry of circulating tumor cells

• Functional validation:

  • Migration/invasion assays of HCC cell lines with CCL15 stimulation

  • Receptor expression analysis

  • Pathway inhibition studies

• Clinical correlation:

  • Stage-specific expression patterns

  • Survival and recurrence analysis

  • Treatment response prediction

These approaches would help establish the clinical utility of CCL15 as an HCC biomarker, potentially offering new diagnostic and therapeutic opportunities.

What methodological considerations are important when analyzing CCL15 in patient samples?

Clinical sample analysis presents unique challenges for CCL15 research. Based on published protocols, researchers should consider:

• Pre-analytical variables:

  • Sample collection timing (diurnal variations)

  • Processing delays can affect proteolytic activation

  • Storage conditions (-80°C recommended with minimal freeze-thaw cycles)

• Analytical variables:

  • For mass spectrometry detection: Protein depletion strategies to remove abundant proteins

  • For immunoassays: Potential interference from other chemokines and heterophilic antibodies

  • Standardization using recombinant CCL15 isoforms

• Population variables:

  • Age and sex-dependent reference ranges

  • Concomitant inflammatory conditions affecting CCL15 levels

  • Medication effects, particularly G-CSF and other immune modulators

• Isoform-specific analysis:

  • Chromatographic separation prior to detection

  • Use of isoform-specific antibodies when available

  • Mass spectrometry confirmation of specific truncated forms

Implementing these methodological considerations will improve the reliability and clinical relevance of CCL15 measurements in patient samples.

How might novel CCL15 antibody technologies advance our understanding of chemokine biology?

Emerging antibody technologies offer new opportunities for CCL15 research:

• Bispecific antibodies targeting CCL15 and its receptors could provide insights into:

  • Receptor-specific signaling outcomes

  • Synergistic effects with other chemokines like CXCL12

  • Tissue-specific chemokine networks

• Antibody engineering approaches:

  • Single-domain antibodies for improved tissue penetration

  • pH-sensitive fluorophore conjugates to track internalization

  • Photoactivatable antibodies for spatiotemporal studies

• Single-cell analysis applications:

  • Combining CCL15 detection with single-cell transcriptomics

  • Spatial mapping of CCL15 gradients in tissues

  • Real-time imaging of chemokine-receptor interactions

These advanced technologies could reveal new aspects of CCL15 biology in stem cell mobilization, cancer progression, and inflammatory responses, potentially leading to novel therapeutic strategies.