CCL5 Antibody Pair

What is CCL5 and what biological functions does it serve?

CCL5, also known as RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted), is an 8 kDa beta-chemokine that functions as a chemoattractant for blood monocytes, memory T-helper cells, and eosinophils. It causes the release of histamine from basophils and activates eosinophils. At the molecular level, CCL5 may activate several chemokine receptors including CCR1, CCR3, CCR4, and CCR5 .

CCL5 is recognized as one of the major HIV-suppressive factors produced by CD8+ T-cells. Recombinant CCL5 protein induces a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). The processed forms of CCL5, particularly RANTES(3-68), act as natural chemotaxis inhibitors and more potent inhibitors of HIV-1 infection compared to the full-length protein .

Additionally, CCL5 may function as an agonist of the G protein-coupled receptor GPR75, stimulating inositol trisphosphate production and calcium mobilization. Through this pathway, CCL5 may play roles in neuron survival and insulin secretion by islet cells .

What is a CCL5 antibody pair and how does it function in research applications?

A CCL5 antibody pair consists of two different antibodies that recognize distinct epitopes on the CCL5 protein: a capture antibody and a detector antibody. Together, they form the foundation of sandwich ELISA (Enzyme-Linked Immunosorbent Assay) and other immunometric assays for the specific detection and quantification of CCL5 in biological samples .

In a typical sandwich ELISA protocol:

• The capture antibody is immobilized on a solid surface (usually a 96-well plate)

• Sample containing CCL5 is added and binds to the capture antibody

• The detector antibody (often biotinylated or directly conjugated to an enzyme) binds to a different epitope on CCL5

• A detection system (such as streptavidin-HRP for biotinylated antibodies) is added

• Substrate is added, producing a measurable signal proportional to CCL5 concentration

This dual-antibody system ensures high specificity and sensitivity for CCL5 detection, with minimal cross-reactivity to other chemokines like CCL3 or CCL4, which is crucial when analyzing complex biological samples .

How specific are CCL5 antibody pairs across different species?

CCL5 antibody pairs exhibit varying degrees of cross-reactivity across species, which must be considered when designing experiments. Many commercially available antibody pairs are tested and validated for specific species reactivity .

For example, some antibody pairs can detect both human and non-human primate (monkey) CCL5 with high specificity, making them valuable tools for translational research. The antibody pair described in search result is specifically designed for human and monkey CCL5 detection, while showing minimal cross-reactivity with murine CCL5.

When working with feline models, specialized antibody pairs are available with defined cross-reactivity profiles. For instance, the antibody described in search result shows less than 0.02% cross-reactivity with human, mouse, and cotton rat RANTES in sandwich immunoassays. This specificity is critical when interpreting results from animal models or when studying zoonotic diseases .

To ensure valid experimental outcomes, researchers should thoroughly validate antibody specificity for their species of interest before proceeding with full-scale studies, especially when working with less common research animals.

How can CCL5 antibody pairs be utilized in neutralization assays to study cell migration?

CCL5 antibody pairs can be strategically employed in neutralization assays to investigate chemotaxis and cell migration mechanisms. A methodologically sound approach involves:

• Baseline chemotaxis establishment: First, establish a dose-response curve using recombinant CCL5 to induce chemotaxis in appropriate cell models such as the BaF3 mouse pro-B cell line transfected with human CCR5. Migration can be quantified using fluorescent dyes like Resazurin .

• Neutralization gradient setup: Once the optimal chemotactic dose is determined (typically around 0.01-0.08 μg/mL), set up a neutralization assay by pre-incubating this fixed concentration of CCL5 with increasing concentrations of anti-CCL5 neutralizing antibody .

• Quantitative analysis: Calculate the neutralization dose that inhibits 50% of migration (ND50). For human CCL5 neutralization assays, this typically ranges between 0.1-0.4 μg/mL of antibody, while for feline CCL5, it may be 0.5-2.5 μg/mL .

• Controls implementation: Include critical controls such as isotype antibodies to rule out non-specific effects and positive controls using established CCR5 antagonists.

This approach provides mechanistic insights into how CCL5-CCR5 interactions mediate immune cell trafficking in various physiological and pathological contexts, including inflammation and tumor microenvironments .

What role does CCL5 play in cancer research, and how are antibody pairs used to investigate the CCL5-CCR5 axis in tumor microenvironments?

CCL5 has emerged as a significant mediator in cancer biology, with the CCL5-CCR5 axis influencing several aspects of tumor progression. Research applications using CCL5 antibody pairs in cancer studies include:

• Assessment of tumor-associated inflammation: CCL5 antibody pairs enable quantitative measurement of CCL5 levels within the tumor microenvironment, providing insights into inflammatory components that may promote tumor growth. This is particularly relevant as CCL5/CCR5 interactions can accelerate inflammation processes via the NF-κB pathway .

• Analysis of macrophage polarization: The CCL5-CCR5 axis influences macrophage polarization within tumors. Using neutralizing antibodies against CCL5 in combination with phenotyping markers helps determine how this chemokine shifts the balance between pro-inflammatory M1 and anti-inflammatory M2 phenotypes. After NF-κB pathway activation through cascade phosphorylation of IKK and IκB subunits, the polarization balance tends toward tumor-associated macrophage M1 (TAM-M1) rather than TAM-M2 .

• Evaluation of tumor cell apoptosis: Research has demonstrated that blocking the CCL5-CCR5 axis using anti-CCL5 antibodies or CCR5 inhibitors results in increased apoptosis in certain cancer cell lines, such as B16-F10 melanoma cells .

• Investigation of metastatic potential: CCL5 antibody pairs can be employed in experimental models to evaluate how CCL5 influences cell migration, angiogenesis, and metastasis through both neutralization studies and quantitative analysis of CCL5 expression in different tumor compartments .

Experimental design should incorporate both in vitro cell line models and in vivo tumor models, with careful consideration of the temporal and spatial dynamics of CCL5 expression throughout tumor progression.

How can CCL5 antibody pairs be utilized to investigate the regulatory mechanisms of CCL5 expression?

CCL5 expression is tightly regulated by transcription factors like RUNX/CBFβ complexes, and antibody pairs can be instrumental in elucidating these mechanisms:

• Chromatin immunoprecipitation (ChIP) approaches: Anti-CBFβ antibodies can be used in ChIP-seq experiments to identify genomic regions bound by regulatory complexes. For example, research has identified RUNX bound regions (RBRs) approximately 5 kb upstream from the CCL5 transcription start site that regulate expression .

• Enhancer element investigation: Combining CCL5 detection using antibody pairs with genomic editing of regulatory elements provides a powerful approach to understand transcriptional control. For instance, deletion of the primary enhancer element (Ccl5-PE) using CRISPR/Cas9 technology resulted in loss of homeostatic CCL5 expression in T cells and non-T cells, confirming its regulatory importance .

• Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP): This advanced technique uses epitope-tagged dCas9 (dead Cas9 lacking endonuclease activity) targeted to genomic regions of interest via single-guide RNAs, followed by immunoprecipitation with anti-epitope antibodies. This approach has successfully identified genomic regions that interact with the CCL5 promoter, revealing distal enhancer elements .

• Quantification across cell types: CCL5 antibody pairs enable comparative analysis of expression levels across different immune cell populations (CD8+ cytotoxic T cells, CD4+ T helper cells, NK cells, γδT cells) in wild-type versus genetically modified systems, revealing cell-type specific regulatory mechanisms .

These methodologies have revealed that RUNX-mediated CCL5 repression can be critical for modulating anti-tumor immunity, highlighting potential therapeutic targets .

What are common sources of variability in CCL5 ELISA assays using antibody pairs, and how can they be addressed?

Several factors can introduce variability in CCL5 ELISA assays. Here's a systematic approach to identify and minimize these variables:

Sample preparation inconsistencies:

• Problem: CCL5 levels can be affected by platelet activation during sample collection and processing, as platelets release CCL5 upon activation.

• Solution: Standardize blood collection using anticoagulants like EDTA or citrate, process samples at consistent temperatures (4°C recommended), and centrifuge samples under identical conditions. Consider adding platelet stabilizing agents when appropriate .

Antibody binding efficiency:

• Problem: Binding kinetics can be affected by buffer conditions, temperature fluctuations, and incubation times.

• Solution: Maintain consistent incubation temperatures (typically room temperature or 37°C as specified by the protocol), use calibrated timers for incubation steps, and prepare fresh buffers according to manufacturer specifications .

Cross-reactivity with related chemokines:

• Problem: Some antibody pairs may exhibit cross-reactivity with structurally similar chemokines like CCL3 or CCL4.

• Solution: Select validated antibody pairs with documented specificity profiles. For instance, in sandwich immunoassays, some antibody pairs show less than 0.02% cross-reactivity with related chemokines, ensuring reliable results .

Standard curve preparation:

• Problem: Improper reconstitution or dilution of CCL5 standards leads to inaccurate quantification.

• Solution: Use carrier proteins (BSA) in diluent for low concentrations, prepare fresh standards for each assay, and employ multi-point standard curves covering the physiological range of your samples. Verify standard curve linearity (R² > 0.98) before interpreting results .

Matrix effects:

• Problem: Components in biological samples may interfere with antibody binding.

• Solution: Perform spike-recovery tests with known quantities of recombinant CCL5 added to your sample matrix. Recovery between 80-120% indicates minimal matrix interference. Consider matrix-matched standards when working with complex samples like tissue homogenates or cell lysates.

By systematically addressing these variables, researchers can achieve reproducible and reliable quantification of CCL5 across experiments and laboratories.

How should researchers interpret discrepancies between CCL5 protein levels measured by antibody pairs and mRNA expression data?

Discrepancies between CCL5 protein levels and mRNA expression are common and can provide valuable biological insights when properly interpreted:

• Post-transcriptional regulation: CCL5 undergoes significant post-transcriptional regulation. While mRNA may be detected, translation might be suppressed by microRNAs or RNA-binding proteins. Concurrent analysis of both protein (via antibody pairs) and mRNA provides a more complete picture of regulatory mechanisms .

• Protein processing considerations: CCL5 exists in multiple processed forms with different biological activities. The full-length CCL5(1-68) and processed forms like CCL5(3-68) and CCL5(4-68) have varying functions in chemotaxis and HIV suppression. Depending on antibody epitope specificity, certain antibody pairs may preferentially detect specific forms, potentially explaining discrepancies with total mRNA measurements .

• Cell-type specific expression patterns: RUNX/CBFβ complexes regulate CCL5 expression differently across cell types. For example, homeostatic CCL5 expression patterns differ significantly between CD8+ T cells, CD4+ T cells, NK cells, and γδT cells. When analyzing heterogeneous samples, protein measurements represent the net expression across all cell types, which may not correlate with mRNA from bulk analysis .

• Technical approach to reconciliation: When encountering discrepancies:

  • Verify antibody specificity for all CCL5 isoforms

  • Consider cell sorting before analysis to assess cell-type specific expression

  • Implement pulsed protein labeling to distinguish newly synthesized from stored CCL5

  • Evaluate protein stability through cycloheximide chase experiments

This integrated approach helps distinguish between transcriptional, post-transcriptional, and post-translational regulatory mechanisms affecting CCL5 levels in your experimental system.

What controls should be included when using CCL5 antibody pairs in neutralization studies?

A robust neutralization study using CCL5 antibody pairs requires carefully selected controls to ensure valid interpretation:

Essential experimental controls:

• Isotype control antibodies: Include matched isotype control antibodies at equivalent concentrations to the anti-CCL5 antibody. This controls for non-specific effects mediated by the antibody's Fc portion or other antibody characteristics unrelated to CCL5 binding .

• Dose-response validation: Establish complete dose-response curves for CCL5-induced effects (e.g., chemotaxis) before attempting neutralization. This confirms that the observed biological response is specific to CCL5 and occurs within the dynamic range of the assay .

• Positive control inhibitors: Include established CCR5 antagonists (such as maraviroc) as positive controls. These provide a reference point for complete inhibition through a different mechanism (receptor blockade versus ligand neutralization) .

• Recombinant protein controls: Test neutralization against both recombinant and native CCL5 sources, as their conformation and activity may differ. For instance, comparing neutralization efficiency between recombinant E. coli-derived CCL5 and natural CCL5 from activated T cell supernatants can reveal important differences .

• Cross-reactivity assessment: Include related chemokines (CCL3, CCL4) in parallel experiments to confirm antibody specificity. This is crucial since these chemokines can act through some of the same receptors as CCL5 .

Data analysis controls:

• ND50 determination: Calculate the neutralization dose that inhibits 50% of the biological activity (typically 0.1-0.4 μg/mL for human CCL5, or 0.5-2.5 μg/mL for feline CCL5) to enable quantitative comparisons between experiments .

• Statistical validation: Apply appropriate statistical tests to determine if neutralization effects are significant compared to control conditions.

Implementing these controls ensures that observed effects can be confidently attributed to specific neutralization of CCL5 rather than experimental artifacts or non-specific interactions.

How are CCL5 antibody pairs being utilized to investigate the role of CCL5 in COVID-19 pathogenesis?

The COVID-19 pandemic has expanded research interest in the CCL5-CCR5 axis, with antibody pairs providing critical tools for investigation:

CCL5 has been implicated in the immunopathology of COVID-19 through several mechanisms. The CCL5-CCR5 axis participates in various aspects of SARS-CoV-2 infection and the resulting inflammatory responses. Researchers are utilizing CCL5 antibody pairs to:

• Quantify CCL5 levels in COVID-19 patients: Antibody pairs in ELISA formats allow precise measurement of CCL5 in serum, plasma, and bronchoalveolar lavage fluid from COVID-19 patients, enabling correlation with disease severity, progression, and outcomes. This approach has helped identify CCL5 as a potential biomarker for disease stratification .

• Evaluate therapeutic targeting potential: Using neutralizing antibodies against CCL5 in ex vivo models of SARS-CoV-2 infection helps assess whether interrupting the CCL5-CCR5 signaling pathway could modulate the hyperinflammatory response characteristic of severe COVID-19. This builds on previous knowledge of the CCL5-CCR5 axis in other inflammatory conditions .

• Investigate cellular sources of CCL5 during infection: Combining flow cytometry with CCL5 antibody-based detection allows identification of which immune cell populations (T cells, macrophages, etc.) are primary producers of CCL5 during SARS-CoV-2 infection, providing insights into disease pathogenesis.

• Study mechanism of viral entry modulation: Some research suggests CCR5 may influence SARS-CoV-2 infection processes, making antibody-based CCL5 detection crucial for understanding potential interactions between chemokine signaling and viral entry mechanisms .

This emerging research direction highlights how established immunological tools like CCL5 antibody pairs can be rapidly applied to new pathological contexts, potentially uncovering therapeutic targets.

What new methodologies are being developed to integrate CCL5 antibody-based detection with other analytical techniques?

Innovative methodologies are expanding the research applications of CCL5 antibody pairs through integration with complementary analytical techniques:

• Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP): This cutting-edge technique combines CRISPR/Cas9 technology with antibody-based precipitation approaches. By targeting an epitope-tagged dead Cas9 (dCas9) to genomic regions of interest via single-guide RNAs, followed by ChIP with an anti-epitope antibody, researchers can identify genomic interactions with the CCL5 promoter. This has successfully identified enhancer elements regulating CCL5 expression that were previously unknown .

• Multiplex cytokine profiling systems: New platforms incorporate anti-CCL5 antibody pairs with antibodies against multiple other cytokines and chemokines, enabling simultaneous detection of complex immune signatures. This approach provides a more comprehensive understanding of how CCL5 functions within broader inflammatory networks, particularly in diseases characterized by cytokine dysregulation.

• Single-cell secretion assays: Advanced microfluidic platforms equipped with antibody-coated capture surfaces allow detection of CCL5 secretion at the single-cell level, revealing heterogeneity within seemingly homogeneous immune cell populations. This technology has revealed previously unappreciated functional diversity in T cell responses.

• In vivo imaging with labeled antibodies: Near-infrared labeled anti-CCL5 antibodies are being developed for non-invasive tracking of CCL5 expression dynamics in animal models, offering real-time visualization of chemokine production during disease progression.

• Proximity ligation assays: These assays combine antibody recognition with DNA amplification, dramatically increasing sensitivity for detecting CCL5-receptor interactions in tissue sections. This technique enables visualization of CCL5-CCR5 binding events in situ, providing spatial context that traditional methods lack.

These methodological innovations are expanding the research applications of CCL5 antibody pairs beyond traditional ELISA and Western blotting, offering unprecedented insights into CCL5 biology.

How can researchers apply CCL5 antibody pairs to study the role of CCL5 in transcriptional regulation of immune responses?

CCL5 antibody pairs can be strategically implemented to uncover the complex transcriptional regulatory mechanisms governing CCL5 expression in immune responses:

• Chromatin reorganization analysis: By combining chromatin accessibility assays (ATAC-seq) with CCL5 protein quantification using antibody pairs, researchers can correlate changes in chromatin structure with resulting protein expression. This approach has revealed that RUNX/CBFβ complexes bind to specific genomic regions (RBRs) approximately 5 kb upstream from the CCL5 transcription start site, influencing its expression in different immune cell populations .

• Enhancer element functional validation: After identifying potential enhancer elements through computational or ChIP-seq approaches, researchers can validate their function by:

  • Deleting candidate enhancers using CRISPR/Cas9 (as demonstrated with Ccl5-PE)

  • Measuring the resulting changes in CCL5 protein using antibody pairs

  • Correlating these changes with altered immune functions

  This methodology confirmed that Ccl5-PE is essential for homeostatic CCL5 expression in both T cells and non-T cells .

• Cell-type specific regulatory mechanisms: CCL5 antibody pairs enable precise quantification of CCL5 across diverse immune cell populations, revealing distinct regulatory patterns:

  • In NK and γδT cells: Ccl5-PE controls homeostatic CCL5 expression

  • In CD8+ T cells: Activation-induced CCL5 depends on different enhancer elements

  • In CD4+ Th cells: RUNX/CBFβ suppresses inappropriate CCL5 expression

• Temporal regulation assessment: By measuring CCL5 protein at different time points following immune cell activation, researchers can map the kinetics of enhancer activity and transcription factor binding. This approach revealed that RUNX/CBFβ not only regulates the magnitude but also the cell-type specificity and temporal dynamics of CCL5 expression .

These applications demonstrate how antibody-based detection of CCL5 protein complements genomic and transcriptomic approaches, providing crucial validation of regulatory mechanisms identified through other methodologies.