CLEC4M Antibody, Biotin conjugated

What is CLEC4M and what cellular functions does it perform?

CLEC4M (C-type lectin domain family 4 member M), also known as CD299 or L-SIGN, is a type II integral membrane protein that shares 77% identity with CD209 antigen (DC-SIGN). It functions as a pattern recognition receptor that binds to intercellular adhesion molecule 3 (ICAM3) and HIV-1 gp120, enhancing HIV-1 infection of T cells . This protein is primarily expressed in liver sinusoidal endothelial cells and lymph nodes, where it plays crucial roles in pathogen recognition and immune response modulation. Unlike many other pattern recognition receptors, CLEC4M's binding is calcium-dependent and involves specific recognition of high-mannose oligosaccharides on viral envelope proteins and bacterial cell walls .

What are the key applications for CLEC4M antibody in research?

CLEC4M antibody has multiple validated research applications, including:

• Flow cytometry analysis of cell surface expression patterns

• Adhesion blockade experiments to study receptor-ligand interactions

• CyTOF (mass cytometry) for high-dimensional single-cell analysis

• Immunophenotyping of human samples

These applications make the antibody particularly valuable for research in immunology, virology, oncology, and cellular biology fields where receptor-mediated interactions need to be characterized with high specificity and sensitivity.

How should researchers store and handle biotin-conjugated CLEC4M antibody to maintain activity?

Proper storage and handling of biotin-conjugated CLEC4M antibody is essential for maintaining its activity and specificity. The antibody should be stored at 4°C in the dark to prevent photobleaching of the biotin conjugate . For long-term storage beyond experimental timeframes, aliquoting is recommended to minimize freeze-thaw cycles. When handling the antibody, researchers should:

• Avoid extended exposure to room temperature

• Minimize repeated freeze-thaw cycles

• Centrifuge the vial briefly before opening to collect solution at the bottom

• Use sterile technique when handling the antibody

• Consider adding carrier protein if diluting the antibody for extended storage

These practices will help maintain the antibody's binding capacity and specificity over time.

What is the significance of CLEC4M in viral infection research?

CLEC4M has significant implications in viral infection research as it efficiently binds to HIV-1 gp120 and enhances HIV-1 infection of T cells . This protein serves as an attachment factor for various enveloped viruses including HIV, hepatitis C virus, Ebola virus, and SARS-CoV. The biotin-conjugated antibody enables researchers to:

• Track CLEC4M expression levels during viral infection

• Block viral attachment to study infection mechanisms

• Investigate co-receptor dynamics during viral entry

• Evaluate potential antiviral therapeutics targeting this pathway

Understanding CLEC4M's role in viral pathogenesis is critical for developing new antiviral strategies and vaccines against these important human pathogens.

How can researchers optimize CLEC4M antibody concentration for flow cytometry to detect low-expression samples?

Optimizing CLEC4M antibody concentrations for detecting low-expression samples requires a methodical titration approach. For biotin-conjugated antibodies, researchers should:

• Begin with a titration series (typically 0.1-10 μg/mL) using positive control cells (e.g., NIH-3T3 transfected with human DC-SIGNR/CD299)

• Calculate the signal-to-noise ratio for each concentration by comparing median fluorescence intensity (MFI) between positive populations and negative controls

• Select the concentration that provides maximum separation with minimal background

• Consider signal amplification strategies such as:

  • Using premium streptavidin-fluorophore conjugates with optimal fluorophore/protein ratios

  • Implementing sequential multilayer staining protocols

  • Employing tyramide signal amplification for extreme sensitivity requirements

• Include viability dyes to exclude dead cells which often cause non-specific binding

• Use appropriate blocking reagents (Fc block, serum) to reduce background

These optimization steps ensure maximum sensitivity while maintaining specificity in detecting low CLEC4M expression levels in clinical or experimental samples.

What are the critical considerations when designing experiments to study CLEC4M in tumor progression models?

Recent research has revealed connections between CLEC4M expression and tumor progression, necessitating careful experimental design considerations:

• Expression analysis validation

  • Use multiple detection methods (flow cytometry, immunohistochemistry, qPCR) to confirm expression patterns

  • Include appropriate controls for antibody specificity (knockout/knockdown controls)

• Model selection

  • Choose models that recapitulate the relevant microenvironment, as CLEC4M function is context-dependent

  • Consider both in vitro and in vivo systems to address different aspects of tumor-CLEC4M interactions

• Functional assessment approaches

  • Implement both gain-of-function and loss-of-function studies

  • Use blocking antibodies at optimized concentrations to specifically inhibit CLEC4M interactions

  • Consider downstream signaling pathway analysis when interpreting results

• Clinical correlation strategies

  • Incorporate analysis of CLEC4M expression in patient-derived samples

  • Correlate with clinical outcomes for prognostic assessment

Evidence from recent studies indicates CLEC4M levels may serve as a potential biomarker in cervical cancer, with elevated levels showing moderate diagnostic potential (71.4% sensitivity, 68.6% specificity) . Furthermore, research suggests CLEC4M may promote metastatic progression in certain cancer types, highlighting the importance of comprehensive experimental designs in this field .

How can researchers troubleshoot inconsistent results when using CLEC4M antibody for CyTOF applications?

CyTOF (Cytometry by Time-of-Flight) applications using metal-tagged antibodies present unique challenges. When troubleshooting inconsistent results with CLEC4M antibody:

• Metal conjugation issues

  • Ensure optimal biotin-streptavidin ratios if using secondary metal labeling

  • Verify metal conjugation efficiency through quality control experiments

  • Test multiple metal isotopes if signal interference is suspected

• Sample preparation factors

  • Standardize fixation protocols, as overfixation can mask epitopes

  • Optimize permeabilization conditions for consistent antibody access

  • Include dead cell removal steps to prevent non-specific binding

  • Standardize cell concentration to ensure consistent staining

• Instrument and acquisition considerations

  • Regularly clean and calibrate the instrument

  • Use EQ calibration beads to normalize signal between runs

  • Implement standard operating procedures for acquisition parameters

• Data analysis approaches

  • Apply appropriate transformation and normalization methods

  • Use dimensionality reduction techniques (tSNE, UMAP) to identify populations

  • Consider batch correction algorithms if combining multiple experiments

• Validation strategies

  • Confirm key findings with orthogonal methods (flow cytometry, immunohistochemistry)

  • Use biological replicates to assess reproducibility

  • Include appropriate positive and negative controls in each experiment

By systematically addressing these factors, researchers can improve consistency and reliability in CyTOF experiments involving CLEC4M antibody detection.

What are the technical considerations for using biotin-conjugated CLEC4M antibody in multiplex immunoassays?

Multiplex immunoassays present unique challenges when incorporating biotin-conjugated CLEC4M antibody:

• Platform-specific optimizations

  • For bead-based assays: optimize antibody coating concentration and binding buffers

  • For planar arrays: determine optimal spotting concentration and surface chemistry

  • For in situ multiplex imaging: test antibody performance after harsh conditions (heat, pH changes)

• Biotin-related considerations

  • Account for endogenous biotin in samples by using appropriate blocking reagents

  • Optimize streptavidin-reporter conjugate concentration to prevent high background

  • Consider sequential staining approaches to minimize cross-reactivity

  • Test for interference with other biotin-containing reagents in the multiplex panel

• Cross-reactivity mitigation

  • Perform extensive cross-reactivity testing across all antibodies in the panel

  • Use appropriate isotype controls to assess non-specific binding

  • Consider cross-adsorption of antibodies if needed

  • Implement computational approaches to correct for spillover between channels

• Validation parameters

  • Establish assay-specific limits of detection and quantification

  • Determine dynamic range for CLEC4M detection in relevant sample types

  • Validate specificity using appropriate biological controls

  • Assess reproducibility through intra- and inter-assay variation measurements

This methodical approach ensures robust integration of biotin-conjugated CLEC4M antibody into complex multiplex immunoassay systems while maintaining specificity and sensitivity.

What is the recommended protocol for using CLEC4M antibody in flow cytometry applications?

Recommended Flow Cytometry Protocol for CLEC4M Antibody (Biotin-conjugated):

• Sample preparation

  • Harvest cells (1-5 × 10^6 cells per sample)

  • Wash twice with flow cytometry buffer (PBS + 2% FBS + 0.1% sodium azide)

  • Centrifuge at 400 × g for 5 minutes, discard supernatant

• Blocking step

  • Resuspend cell pellet in 100 μL of flow cytometry buffer

  • Add 5-10 μL of Fc blocking reagent (when working with Fc receptor-expressing cells)

  • Incubate for 10 minutes at room temperature

• Primary antibody incubation

  • Add optimized concentration of biotin-conjugated CLEC4M antibody (typically 1-5 μg/mL)

  • Incubate for 30 minutes at 4°C protected from light

  • Wash twice with 2 mL flow cytometry buffer

• Secondary detection reagent

  • Resuspend cells in 100 μL flow cytometry buffer

  • Add streptavidin-fluorophore conjugate at manufacturer's recommended dilution

  • Incubate for 20 minutes at 4°C protected from light

  • Wash twice with 2 mL flow cytometry buffer

• Optional viability staining

  • Resuspend cells in 100 μL flow cytometry buffer

  • Add viability dye according to manufacturer's instructions

  • Incubate as directed, then wash once with flow cytometry buffer

• Final preparation

  • Resuspend cells in 200-500 μL flow cytometry buffer

  • Filter through 70 μm cell strainer if needed

  • Analyze immediately or fix with 2% paraformaldehyde for later analysis

This protocol has been validated for detecting CLEC4M in human samples, particularly using the 120604 clone biotinylated antibody .

How should researchers design appropriate controls when using CLEC4M antibody in adhesion blockade experiments?

Adhesion blockade experiments using anti-CLEC4M antibodies require rigorous controls to ensure valid interpretations:

• Essential control conditions

  • Isotype control: Use biotinylated mouse IgG2b at the same concentration as test antibody

  • Concentration gradient: Include multiple antibody concentrations (0.1-50 μg/mL) to demonstrate dose-dependence

  • Positive blocking control: Include a well-validated blocking antibody of the same target

  • Alternative blocking approach: Use soluble CLEC4M ligand as complementary approach

  • Negative cell line control: Include cells lacking CLEC4M expression

• Experimental design considerations

  • Pre-test antibody for potential functional effects on cell viability

  • Determine optimal pre-incubation time (typically 30-60 minutes at 37°C)

  • Standardize washing steps to remove unbound antibody

  • Set consistent criteria for quantifying adhesion (e.g., number of adherent cells per field)

• Data analysis approach

  • Calculate percent inhibition relative to untreated control

  • Generate IC50 values when dose-response is observed

  • Use appropriate statistical tests to evaluate significance

  • Consider kinetic measurements to detect temporal effects

This comprehensive control strategy ensures that observed effects are specifically attributable to CLEC4M blockade rather than non-specific antibody interactions or experimental artifacts.

What are the key considerations for using CLEC4M antibody in investigating viral entry mechanisms?

When investigating viral entry mechanisms using CLEC4M antibody, researchers should consider:

• Experimental design fundamentals

  • Cell model selection: Choose appropriate CLEC4M-expressing cell lines or primary cells

  • Viral system: Select authentic virus or pseudotyped particles based on biosafety and research questions

  • Timing of intervention: Apply antibody at different stages (pre-binding, during binding, post-binding)

  • Quantification methods: Use multiple readouts (viral RNA, reporter gene expression, immunostaining)

• Antibody application approaches

  • Blocking studies: Pre-incubate cells with antibody before virus addition

  • Competition assays: Add virus and antibody simultaneously

  • Post-attachment studies: Add antibody after initial virus binding

  • Dose-response analysis: Use a range of antibody concentrations (0.1-50 μg/mL)

• Critical controls

  • Isotype control antibody (mouse IgG2b)

  • Cells lacking CLEC4M expression

  • Alternative entry pathway controls

  • Positive control inhibitors of viral entry

• Advanced methodological considerations

  • Super-resolution microscopy to visualize CLEC4M-virus colocalization

  • Real-time imaging of viral attachment and internalization

  • CRISPR/Cas9-mediated CLEC4M modification for mechanistic studies

  • Co-immunoprecipitation to identify binding partners

What methods can be used to validate CLEC4M antibody specificity for research applications?

Validating antibody specificity is critical for reliable research outcomes. For CLEC4M antibody, recommended validation methods include:

• Genetic validation approaches

  • CRISPR/Cas9 knockout cells as negative controls

  • siRNA or shRNA knockdown with quantified reduction

  • Overexpression systems (e.g., NIH-3T3 cells transfected with human CLEC4M)

• Peptide competition assays

  • Pre-incubate antibody with immunizing peptide

  • Observe elimination of specific signal

  • Include irrelevant peptide as negative control

• Cross-platform validation

  • Compare results across multiple techniques (Western blot, flow cytometry, immunohistochemistry)

  • Verify concordance of expression patterns

  • Use different antibody clones targeting distinct epitopes

• Orthogonal method comparison

  • Correlate protein detection with mRNA expression (qPCR, RNA-seq)

  • Compare with mass spectrometry-based protein identification

  • Validate functional readouts with genetic modulation

• Species cross-reactivity assessment

  • Test reactivity on samples from multiple species

  • Align epitope sequences across species to predict reactivity

  • Validate experimentally when using in non-human samples

Implementing multiple validation strategies from this comprehensive approach provides robust evidence for antibody specificity, ensuring reliable research outcomes when working with CLEC4M.

How is CLEC4M antibody being used in oncology research, and what are the recent findings?

Recent oncology research utilizing CLEC4M antibody has revealed significant findings:

• Diagnostic biomarker potential

  • A 2025 study demonstrated significantly elevated serum CLEC4M levels in cervical cancer patients compared to healthy controls

  • ROC curve analysis showed moderate diagnostic potential with 71.4% sensitivity and 68.6% specificity

  • This suggests potential utility as part of a biomarker panel for cervical cancer screening or monitoring

• Cancer progression mechanisms

  • Multiple studies have established associations between CLEC4M and tumor growth

  • Research indicates CLEC4M may promote metastatic progression in certain cancer types

  • The specific molecular mechanisms remain under active investigation

• Tumor microenvironment interactions

  • CLEC4M expression in liver sinusoidal endothelial cells may influence hepatocellular carcinoma development

  • Research suggests complex roles in modulating immune responses within the tumor microenvironment

  • Antibody-based studies are clarifying cell-specific expression patterns

• Therapeutic targeting investigations

  • Blocking antibodies are being used to assess CLEC4M as a potential therapeutic target

  • Preliminary studies suggest inhibiting CLEC4M-mediated interactions may impact tumor progression

  • Combined approaches with immune checkpoint inhibitors are under exploration

These findings highlight the growing importance of CLEC4M as a research target in oncology, with potential diagnostic and therapeutic implications.

What role does CLEC4M play in infectious disease research beyond HIV studies?

CLEC4M's significance extends beyond HIV research to multiple infectious disease areas:

• Viral pathogen interactions

  • Hepatitis C virus: CLEC4M serves as an attachment factor for HCV, with antibody blocking studies revealing entry mechanisms

  • SARS coronaviruses: Recognized as a binding receptor for SARS-CoV spike protein

  • Ebola and Marburg viruses: Involved in initial attachment and enhancement of infection

  • Influenza viruses: Interactions with highly glycosylated hemagglutinin proteins being characterized

• Bacterial pathogen studies

  • Mycobacterium tuberculosis: CLEC4M binds mannosylated lipoarabinomannan on bacterial surface

  • Streptococcus pneumoniae: Recognition of capsular polysaccharides influences immune response

  • Helicobacter pylori: Emerging evidence for CLEC4M-mediated recognition

• Parasitic infection research

  • Leishmania species: Initial recognition studies showing CLEC4M binding to parasite glycoconjugates

  • Schistosoma mansoni: Interactions with egg antigens influence immunopathology

• Immunological consequences

  • Modulation of pattern recognition responses

  • Influence on adaptive immune polarization

  • Potential impact on vaccine-induced immunity

These diverse roles make CLEC4M antibodies valuable tools across multiple infectious disease research domains, with applications in binding studies, cellular localization, and functional blocking experiments.

How can researchers integrate CLEC4M antibody into multiparametric analysis workflows?

Integrating CLEC4M antibody into multiparametric analysis requires strategic planning:

• Panel design considerations

  • Spectral compatibility: The biotin-conjugated format allows flexible pairing with various streptavidin-fluorophores

  • Expression level assessment: Allocate brightest fluorophores if CLEC4M has low expression

  • Co-expression analysis: Plan markers based on biological questions (e.g., combining with DC-SIGN, viral receptors)

  • Functional correlation: Include activation/functional markers relevant to CLEC4M biology

• Optimization protocols

  • Titration matrix: Perform antibody titrations in the context of the full panel

  • Fluorescence minus one (FMO) controls: Essential for setting boundaries in high-parameter analysis

  • Compensation: Critical when using streptavidin-conjugated fluorophores to detect biotinylated antibodies

  • Fixation compatibility: Test performance after various fixation/permeabilization protocols

• Advanced analytical approaches

  • Dimensionality reduction: Apply tSNE, UMAP, or PhenoGraph algorithms to identify novel populations

  • Trajectory analysis: Pseudotime algorithms to map developmental or activation sequences

  • Clustering approaches: FlowSOM, PhenoGraph for automated population identification

  • Correlation analysis: SPADE or Scaffold maps to visualize marker relationships

• Specialized applications

  • Mass cytometry integration: Use biotin-conjugated primary with metal-tagged streptavidin

  • Imaging mass cytometry: For spatial context of CLEC4M expression

  • Spectral flow cytometry: Leveraging full emission spectra for increased parameters

This comprehensive integration strategy enables researchers to position CLEC4M analysis within complex cellular phenotyping workflows, maximizing biological insights from each experiment.

What are the emerging therapeutic applications being investigated using CLEC4M antibodies?

Emerging therapeutic applications being investigated with CLEC4M antibodies include:

• Antiviral therapeutic approaches

  • Blocking antibodies: Development of therapeutic antibodies that prevent viral attachment to CLEC4M

  • Antibody-drug conjugates: Targeting CLEC4M-expressing cells that may harbor viral reservoirs

  • Combination approaches: Using CLEC4M antibodies alongside conventional antivirals for synergistic effects

• Cancer immunotherapy applications

  • Diagnostic companion tools: Using CLEC4M antibodies to identify patients likely to respond to immunotherapy

  • Immunomodulatory approaches: Blocking CLEC4M to potentially enhance anti-tumor immune responses

  • Targeted drug delivery: Exploiting CLEC4M expression for selective delivery to liver endothelial cells

• Inflammatory disease interventions

  • Pathway modulation: Using antibodies to modify CLEC4M-mediated immune signaling

  • Cell-specific targeting: Delivering therapeutics to CLEC4M-expressing cells involved in inflammatory processes

  • Biomarker applications: Monitoring treatment response in diseases with altered CLEC4M expression

• Vaccine development applications

  • Adjuvant targeting: Directing vaccine components to CLEC4M-expressing antigen-presenting cells

  • Glycan modification: Optimizing glycosylation patterns for enhanced CLEC4M recognition

  • Vector targeting: Improving vaccine vector uptake through CLEC4M-mediated pathways

These therapeutic applications remain in early research stages, with most current work focusing on proof-of-concept studies using biotinylated antibodies like the 120604 clone for target validation before development of therapeutic-grade antibodies.