CLE8 Antibody

What is CLEC4D/CLECSF8 and what cellular functions does it perform?

CLEC4D (also known as MCL and CLECSF8) is a 30 kDa type II transmembrane glycoprotein belonging to the C-type Lectin Receptor family. It is synthesized as a 215 amino acid protein with a 17 aa N-terminal cytoplasmic domain, 21 aa transmembrane segment, and 177 aa C-terminal extracellular region. The extracellular portion contains a carbohydrate recognition domain (CRD) of 118 aa, though its specific carbohydrate ligand remains unidentified .

Functionally, CLEC4D expression is restricted to monocytes and macrophages, where it serves primarily as an endocytic receptor. On the cell surface, it forms homodimers and homotrimers that participate in immune recognition processes . While its complete functional profile continues to be investigated, its structural similarities to other CLRs suggest roles in pathogen recognition and immune signaling.

What are the recommended storage and handling protocols for CLEC4D antibodies?

For optimal antibody performance in research applications, CLEC4D antibodies should be handled according to specific storage protocols:

• Store unopened antibody at -20°C to -70°C for up to 12 months from the date of receipt

• After reconstitution, store at 2-8°C for up to 1 month under sterile conditions

• For longer storage post-reconstitution (up to 6 months), maintain at -20°C to -70°C

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles that can damage antibody structure and function

These conditions help maintain antibody specificity and affinity, ensuring consistent experimental results across multiple studies.

In which experimental systems can CLEC4D antibodies be applied?

CLEC4D antibodies have been validated for several experimental applications, including:

• Flow cytometry: Effective for detecting CLEC4D on monocytes in human whole blood samples, typically using antigen affinity-purified polyclonal antibodies followed by fluorochrome-conjugated secondary antibodies

• Immunophenotyping: Useful for identifying CLEC4D-expressing cell populations in complex samples such as peripheral blood

• Functional studies: Can be employed to study receptor-ligand interactions and downstream signaling

When selecting antibodies for specific applications, researchers should consider the epitope targeted, clonality, and validated applications provided by manufacturers.

What methodological approaches should be employed when validating CLEC4D antibody specificity?

Rigorous validation of antibody specificity is essential for accurate data interpretation. For CLEC4D antibodies, researchers should implement:

• Positive and negative control cell comparisons: Test antibody binding on cells known to express CLEC4D (monocytes) versus non-expressing cell types

• Competitive binding assays: Pre-incubate with recombinant CLEC4D to demonstrate specific blocking

• Knockout/knockdown controls: Compare staining between wild-type and CLEC4D-deficient cells

• Cross-reactivity assessment: Test against related C-type lectin receptors, particularly those with high sequence homology

In flow cytometry applications, controls should include isotype-matched antibodies to establish background staining levels, as demonstrated in validation studies with human monocytes .

How do structural features of C-type lectin receptors influence antibody selection for specific applications?

The unique structural features of C-type lectin receptors like CLEC4D require careful consideration when selecting antibodies:

• Extracellular domain targeting: Antibodies recognizing the carbohydrate recognition domain (CRD) may interfere with ligand binding, making them suitable for functional blocking studies but potentially problematic for ligand-binding assays

• Accessibility in native conformation: Native receptor presentation often involves dimeric or trimeric forms on the cell surface, affecting epitope accessibility

• Post-translational modifications: Glycosylation patterns may impact antibody binding, particularly for antibodies targeting glycosylation-proximal epitopes

When targeting membrane-associated receptors like CLEC4D, researchers should select antibodies that recognize native protein conformations rather than only denatured epitopes, especially for applications like flow cytometry or functional studies.

What are the optimal flow cytometry protocols for CLEC4D detection in primary human samples?

For detecting CLEC4D expression in primary human samples by flow cytometry:

• Sample preparation:

  • Isolate peripheral blood mononuclear cells using density gradient centrifugation

  • For whole blood analysis, use red blood cell lysis buffers that preserve surface epitopes

• Staining protocol:

  • Use 0.25-1.0 μg antibody per 10^6 cells in 100 μL staining volume

  • Include lineage markers (e.g., CD14 for monocytes) to identify specific cell populations

  • Perform staining at 4°C for 30 minutes followed by washing steps

• Controls and analysis:

  • Include isotype-matched control antibodies at equivalent concentrations

  • Set gates based on fluorescence-minus-one (FMO) controls

  • Analyze monocyte population separately from other leukocytes

This approach allows accurate detection of CLEC4D expression on monocytes while minimizing background and non-specific staining .

How can antibody-mediated targeting approaches developed for other C-type lectins inform CLEC4D research?

Recent advances in antibody-targeting strategies for CLEC9A provide valuable insights for CLEC4D research applications:

Studies of CLEC9A antibodies conjugated to tumor antigens (NY-ESO-1) have demonstrated efficient antigen delivery to CD141+ dendritic cells, enhancing cross-presentation and T cell activation . Similar approaches could be explored with CLEC4D antibodies for targeted delivery to monocytes and macrophages.

Key transferable methodologies include:

• Antibody-antigen conjugation strategies that preserve binding specificity

• Validation of receptor-mediated internalization efficiency

• Assessment of antigen processing and presentation following receptor-mediated endocytosis

• Evaluation of downstream immune activation

These approaches could be adapted to investigate CLEC4D's potential as a target for immunomodulatory interventions or vaccine development.

What are the current technical challenges in studying C-type lectin receptor interactions with antibodies?

Researchers face several technical challenges when investigating C-type lectin receptor interactions:

• Receptor redundancy: Functional overlap between multiple C-type lectin receptors complicates interpretation of blocking studies

• Heterogeneous expression: Variable expression levels across cell types and activation states require careful sample handling and analysis

• Conformational dependencies: Antibody binding may be affected by receptor oligomerization and ligand-induced conformational changes

• Species differences: Human CLEC4D shares only 63% sequence identity with mouse in the extracellular region, limiting translational research applications

A comprehensive approach involves employing multiple antibody clones targeting different epitopes, combined with genetic approaches to receptor modification, to fully characterize receptor function and interactions.

How can structural insights from antibody-receptor complexes inform development of CLEC4D-targeting reagents?

Structural studies of antibody-receptor complexes provide valuable insights for developing CLEC4D-targeting reagents:

Research on CCR8-antibody complexes has revealed that effective antagonist antibodies can target specific extracellular loops, forming extensive interaction interfaces dominated by electrostatic interactions . Similar structural approaches could inform the development of function-blocking CLEC4D antibodies.

Key considerations drawn from structural studies include:

• Targeting critical interaction interfaces (epitope mapping)

• Understanding the role of post-translational modifications in antibody recognition

• Identifying antibody binding modes that distinguish between receptor conformational states

• Developing antibodies that specifically block ligand binding without affecting receptor expression

These structural insights can guide the rational design of next-generation antibodies with enhanced specificity and functional properties for CLEC4D research.

What criteria should researchers use when selecting between different anti-CLEC4D antibody clones?

Selecting the appropriate anti-CLEC4D antibody requires systematic evaluation of several criteria:

For flow cytometry applications, antibodies validated on human whole blood monocytes have demonstrated reliable performance when paired with appropriate secondary antibodies .

How do methodological approaches for studying CLEC4D differ from those for other C-type lectin receptors?

While general principles of antibody-based research apply across C-type lectin receptors, CLEC4D research requires specific methodological considerations:

• Cell type specificity: CLEC4D expression is restricted to monocytes/macrophages, unlike some other CLRs that have broader expression patterns across dendritic cell subsets

• Functional assays: While CLEC9A targeting can be evaluated through cross-presentation assays and T cell activation , CLEC4D functional studies focus on receptor-mediated endocytosis and innate immune signaling

• Structural considerations: CLEC4D forms homodimers and homotrimers on the cell surface , which may affect antibody binding and functional blocking efficiency compared to monomeric receptors

• Species differences: The 63% sequence identity between human and mouse CLEC4D extracellular regions necessitates careful validation of cross-reactivity for translational studies

These differences highlight the importance of receptor-specific optimization of experimental protocols rather than direct transfer of methods from other C-type lectin receptor systems.

What novel applications are emerging for antibodies targeting C-type lectin receptors in immunotherapy research?

Recent advances suggest several promising applications for antibodies targeting C-type lectin receptors:

• Antigen delivery vehicles: Antibody-antigen conjugates can deliver specific antigens to target cells for enhanced processing and presentation, as demonstrated with CLEC9A-NY-ESO-1 conjugates

• Immune response modulation: Targeting specific C-type lectin receptors can enhance or suppress immune responses in different contexts

• Cell type-specific targeting: The restricted expression pattern of many CLRs enables selective targeting of specific immune cell subsets

• Combination therapies: CLR-targeting antibodies may complement checkpoint inhibitors or other immunotherapeutic approaches

While most advanced work has focused on CLEC9A and dendritic cell targeting , similar principles could be applied to CLEC4D for monocyte/macrophage-directed therapies, opening new avenues for immunomodulatory interventions.

What are common pitfalls in flow cytometry experiments with CLEC4D antibodies and how can they be addressed?

Researchers frequently encounter several challenges when using CLEC4D antibodies in flow cytometry:

• High background staining: Can be addressed by optimizing antibody concentration, including proper blocking steps, and using appropriate isotype controls

• Low signal intensity: May require signal amplification through sequential primary-secondary antibody staining rather than direct conjugates

• Sample preparation artifacts: Red blood cell lysis protocols can affect surface epitopes; optimize lysis conditions or use density gradient separation

• Receptor internalization: Sample processing can trigger receptor endocytosis; minimize processing time and keep samples cold (4°C)

• Antibody lot variability: Validate each new lot against previously used lots to ensure consistent staining patterns

Proper experimental design includes parallel staining with isotype controls at equivalent concentrations and including known positive cell populations (monocytes) as internal controls .

How can researchers distinguish between technical artifacts and genuine biological variability in CLEC4D expression studies?

Distinguishing technical artifacts from biological variability requires systematic controls:

• Within-sample controls: Include well-characterized cell populations with known CLEC4D expression levels as internal references

• Technical replicates: Perform multiple staining reactions per sample to assess staining consistency

• Biological validation: Confirm unusual expression patterns using alternative detection methods or antibody clones

• Correlation with biological parameters: Assess whether expression changes correlate with other cellular activation markers or functional readouts

• Standardization approach: Use quantitative beads to convert fluorescence intensity to antibody binding capacity (ABC) units for more objective comparisons across experiments and instruments

By implementing these approaches, researchers can distinguish genuine biological variation in CLEC4D expression from technical artifacts that might otherwise confound interpretation of experimental results.