FAS Monoclonal Antibody

What is FAS and what cellular mechanisms does it regulate?

FAS (CD95/Apo-1/TNFRSF6) is a cell surface glycoprotein that belongs to the tumor necrosis factor receptor superfamily. The canonical human FAS protein consists of 335 amino acids with a molecular weight of approximately 37.7 kDa and exists in both membrane-bound and secreted forms . FAS plays a critical role in programmed cell death when it binds to its cognate ligand (FAS-L), triggering signal transduction involving FADD-mediated recruitment and activation of caspase-8 that results in apoptosis . FAS-mediated apoptosis is fundamental to immune homeostasis and surveillance against virus-infected or transformed cells . The CD95-CD95L pathway is not only essential for T cell death but also contributes to the deletion of autoreactive B cells, B cell somatic hypermutation, cytotoxicity of NK and CD8 T cells, endothelial cell apoptosis, myeloid suppressor cell turnover regulation, and activation of macrophages' functions against infections .

What are the primary applications of FAS monoclonal antibodies in experimental research?

FAS monoclonal antibodies are versatile tools in immunological research with applications including:

• Western Blot (WB): For detecting FAS protein expression in cell lysates with recommended dilutions ranging from 1:500 to 1:5000 .

• Immunoprecipitation (IP): For isolating FAS protein complexes from cellular extracts .

• Immunofluorescence (IF): For visualizing FAS localization within cells and tissues .

• Immunohistochemistry (IHC): For detecting FAS expression in tissue sections, typically used at dilutions of 1:50 to 1:200 .

• Flow Cytometry (FCM): For quantifying FAS expression on cell surfaces .

• ELISA: For quantitative measurement of FAS protein in solution .

These applications enable researchers to study FAS expression, localization, interaction partners, and functional roles in various cellular contexts.

How do researchers select the appropriate FAS antibody clone for their specific experimental needs?

Selection of an optimal FAS antibody clone should consider several factors:

• Epitope specificity: Different clones recognize distinct epitopes on the FAS protein. For instance, the B-10 clone (sc-8009) detects human FAS protein specifically .

• Species reactivity: Verify the antibody's reactivity with your species of interest. Some antibodies are human-specific, while others may cross-react with mouse or rat FAS .

• Application compatibility: Not all antibodies perform equally across different applications. For example, the recombinant FAS antibody from Cusabio (CSB-RA252392A0HU) has been validated for ELISA, WB, and IHC but may not be optimal for other applications .

• Functional properties: Some FAS antibodies act as agonists (activating FAS signaling), while others are antagonists or simply detection antibodies. For instance, the humanized anti-Fas monoclonal antibody h-HFE7A demonstrates selective apoptosis-inducing activities in inflammatory cells .

• Conjugation requirements: Consider whether you need a conjugated form (HRP, FITC, PE, Alexa Fluor conjugates) for direct detection or an unconjugated form for flexibility in secondary detection systems .

What are the optimal protocols for using FAS monoclonal antibodies in Western blot applications?

When conducting Western blot using FAS monoclonal antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Lyse cells in a buffer containing protease inhibitors

  • For membrane proteins like FAS, include detergents such as NP-40 or Triton X-100

  • Denature samples at 95°C for 5 minutes in Laemmli buffer with reducing agent

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation of the 37.7 kDa FAS protein

  • Transfer to PVDF membranes (preferred over nitrocellulose for hydrophobic membrane proteins)

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST for 1 hour

  • Dilute primary FAS antibody according to manufacturer recommendations (typically 1:500-1:5000)

  • Incubate overnight at 4°C with gentle agitation

  • For HRP-conjugated FAS antibodies (e.g., sc-8009 HRP), skip secondary antibody step

• Detection:

  • Use enhanced chemiluminescence (ECL) for standard HRP-conjugated detection

  • For quantitative analysis, consider fluorescently-conjugated antibodies compatible with infrared imaging systems

• Controls:

  • Include positive control (cell line known to express FAS, such as activated lymphocytes)

  • Use β-actin or GAPDH as loading controls

  • Consider using FAS knockout/knockdown samples as negative controls

The expected molecular weight of human FAS is approximately 37.7 kDa, though post-translational modifications such as glycosylation may result in higher apparent molecular weights .

How can researchers optimize FAS antibody use in immunohistochemistry and immunofluorescence?

For optimal results in IHC and IF applications using FAS antibodies, consider the following methodology:

• Tissue preparation:

  • For IHC: Fix tissues in 10% neutral buffered formalin, embed in paraffin, and section at 4-6 μm

  • For IF: Consider both paraformaldehyde fixation (for structural preservation) and methanol fixation (for epitope accessibility)

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimization may be required as FAS epitope accessibility can vary between antibody clones

• Antibody incubation:

  • For IHC: Dilute FAS antibody at 1:50-1:200 as recommended

  • For IF: Start with similar dilutions but optimize based on signal-to-noise ratio

  • Incubate primary antibody overnight at 4°C in a humidified chamber

• Detection systems:

  • For IHC: Use biotin-streptavidin systems or polymer-based detection kits

  • For IF: Select appropriate fluorescent secondary antibodies or use directly conjugated FAS antibodies (e.g., sc-8009 AF488, sc-8009 AF546)

• Controls and counterstaining:

  • Include positive control tissues (lymphoid tissues expressing FAS)

  • Use isotype control antibodies at matching concentrations

  • For IHC: Counterstain with hematoxylin

  • For IF: Counterstain nuclei with DAPI

• Dual staining considerations:

  • When performing dual staining with other markers, select antibodies raised in different host species

  • If using multiple mouse antibodies, employ sequential immunostaining with blocking steps

What methodologies are recommended for studying FAS-mediated apoptosis in cell culture?

To effectively study FAS-mediated apoptosis in experimental settings, researchers should consider these methodological approaches:

• Induction of FAS-mediated apoptosis:

  • Using agonistic FAS antibodies: Some anti-FAS antibodies require crosslinking with secondary antibodies or Fcγ receptor-positive cells to induce apoptosis efficiently

  • Alternative approach: Use recombinant FAS ligand (FAS-L) to trigger the pathway

• Measuring apoptosis (multiple complementary methods recommended):

  • Annexin V/PI staining: For flow cytometric analysis of early/late apoptotic populations

  • TUNEL assay: For detection of DNA fragmentation

  • Caspase activity assays: Particularly caspase-8 (initiator) and caspase-3 (effector)

  • Western blot for cleaved PARP and cleaved caspases

  • Morphological assessment using fluorescence microscopy

• Cell type considerations:

  • Different cell types show varying sensitivity to FAS-mediated apoptosis

  • Lymphoid cells typically exhibit high sensitivity

  • Some cell types (e.g., certain synoviocytes and chondrocytes) may be resistant

• Experimental controls:

  • Positive control: FAS-sensitive cell line (e.g., Jurkat T cells)

  • Negative control: FAS-resistant cell line or FAS-knockout cells

  • Include pan-caspase inhibitor (e.g., Z-VAD-FMK) to confirm apoptosis is caspase-dependent

• Data analysis:

  • Quantify percentage of apoptotic cells

  • Generate dose-response curves with increasing antibody concentrations

  • Analyze time-course experiments to determine kinetics of apoptosis induction

This methodological approach allows for comprehensive analysis of FAS-mediated apoptosis, distinguishing between different apoptotic stages and confirming pathway specificity.

How do agonistic and antagonistic FAS antibodies differ mechanistically in research applications?

The functional properties of anti-FAS antibodies vary significantly, with distinct mechanisms underpinning their agonistic or antagonistic activities:

Agonistic FAS Antibodies:

• Mechanism of action: Agonistic antibodies mimic FAS ligand by inducing receptor trimerization and subsequent signaling cascade activation . Crystal structure analysis at 1.9 Å resolution has provided insights into how these antibodies bind epitopes that facilitate receptor clustering .

• Crosslinking requirements: Many agonistic antibodies require crosslinking to effectively induce apoptosis. For example, h-HFE7A monoclonal antibody induces apoptosis in human activated lymphocytes only when crosslinked with a secondary antibody or Fcγ receptor-positive cells .

• Affinity-activity paradox: Interestingly, higher-affinity anti-FAS antibodies may demonstrate reduced agonistic activity. Research has shown that affinity-matured versions of agonist antibodies can exhibit significantly diminished signaling capability at the FAS receptor . This counterintuitive relationship suggests that optimal receptor activation involves specific binding kinetics rather than simply maximizing binding strength.

• Cell type selectivity: Some agonistic antibodies show selective apoptosis-inducing activities. The humanized anti-Fas mAb h-HFE7A selectively induces apoptosis in inflammatory cells while sparing synoviocytes and chondrocytes, making it potentially valuable for treating conditions like rheumatoid arthritis .

Antagonistic FAS Antibodies:

• Mechanism of action: These antibodies bind FAS but block conformational changes or prevent FAS-L binding, inhibiting apoptotic signal transduction.

• Applications: Antagonistic antibodies are valuable for studying FAS biology without triggering cell death, and for potentially blocking pathological FAS-mediated apoptosis in disease models.

• Epitope specificity: Antagonistic properties often correlate with binding to specific regions of FAS that do not promote receptor clustering.

This mechanistic distinction has profound implications for experimental design. Researchers must carefully select antibodies based on their functional properties and validate their activity in the specific experimental system being used.

What are the methodological approaches for studying FAS antibody efficacy in animal models of autoimmune disease?

When investigating FAS antibody efficacy in autoimmune disease models, researchers should implement the following methodological framework:

• Model selection and validation:

  • SCID-HuRAg mice: These immunodeficient mice implanted with human rheumatoid arthritis tissue provide a valuable model for testing human-specific FAS antibodies

  • Collagen-induced arthritis models: For testing species-specific antibodies in immunocompetent animals

  • Validation of model through histological and immunological characterization before intervention

• Antibody administration protocol:

  • Dose optimization: Typically starting with 1-10 mg/kg body weight

  • Administration route: Intravenous, intraperitoneal, or subcutaneous depending on pharmacokinetics

  • Treatment schedule: Determine appropriate frequency and duration based on antibody half-life and disease progression

• Safety assessment:

  • Liver function monitoring: Critical due to potential hepatotoxicity of FAS activation

  • Histological examination of liver, cartilage, and other potentially sensitive tissues

  • Complete blood count analysis to monitor for hematological abnormalities

• Efficacy evaluation (multi-parameter approach):

  • Clinical scoring: Joint swelling, mobility, grip strength

  • Histopathology: Assessing inflammation, pannus formation, and cartilage/bone destruction

  • Immunohistochemistry: Quantifying inflammatory cell infiltration and apoptosis

  • Cell-specific effects: As demonstrated with h-HFE7A, which significantly decreased inflammatory cells in implanted tissue while sparing synoviocytes and chondrocytes

• Mechanistic investigations:

  • Flow cytometry of isolated cells from affected tissues to assess FAS expression

  • TUNEL assays on tissue sections to quantify apoptosis

  • Analysis of apoptotic pathway activation through detection of cleaved caspases in tissue lysates

  • Cytokine/chemokine profiling to assess inflammatory mediators

• Comparison with standard-of-care treatments:

  • Include control groups receiving current standard treatments

  • Consider combination therapies to assess additive or synergistic effects

This comprehensive methodological approach allows for rigorous evaluation of FAS antibody efficacy while addressing safety concerns that have historically limited clinical development of FAS-targeting therapeutics.

How can researchers investigate the relationship between FAS antibody affinity and agonistic activity?

The counterintuitive relationship between antibody affinity and agonistic activity represents an intriguing area of FAS biology. To systematically investigate this phenomenon, researchers should consider the following methodological approach:

• Generation of affinity variants:

  • Create an affinity maturation library through techniques such as phage display or yeast display

  • Perform directed evolution with decreasing concentrations of antigen to select higher-affinity variants

  • Alternatively, introduce specific mutations in the complementarity-determining regions (CDRs)

• Binding kinetics characterization:

  • Measure association (kon) and dissociation (koff) rate constants using surface plasmon resonance (SPR)

  • Determine equilibrium dissociation constants (KD) for each variant

  • Create a panel of antibodies with a range of affinities spanning at least 2-3 orders of magnitude

• Structural analysis:

  • Crystallography of antibody-FAS complexes at high resolution (comparable to the 1.9 Å resolution achieved for previous anti-FAS antibodies)

  • Epitope mapping to confirm binding to the same region of FAS

  • Molecular dynamics simulations to assess binding conformational dynamics

• Functional activity assessment:

  • Quantify apoptosis induction using multiple complementary assays (Annexin V/PI staining, caspase activation, PARP cleavage)

  • Determine EC50 values for each antibody variant

  • Plot correlation between affinity (KD) and biological activity (EC50)

• Mechanism investigation:

  • Analyze receptor clustering efficiency using techniques such as FRET or proximity ligation assay

  • Assess binding valency effects using Fab fragments versus whole IgG

  • Investigate the role of FcγR interaction through experiments with F(ab')2 fragments and Fc-engineered variants

• Mathematical modeling:

  • Develop computational models incorporating binding kinetics and receptor clustering dynamics

  • Test hypotheses regarding optimal dwell time for receptor activation

  • Validate model predictions with experimental data

Based on previous findings, researchers might expect to observe that extremely high-affinity antibodies demonstrate reduced agonistic activity compared to moderate-affinity variants . This phenomenon may be explained by a model where optimal receptor activation requires a specific "hit-and-run" binding dynamic rather than extremely stable complex formation, which might limit the conformational changes or receptor clustering necessary for signal transduction.

What are the common challenges when using FAS antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with FAS antibodies. Here are evidence-based solutions for addressing these issues:

How should researchers validate the specificity and functionality of newly acquired FAS antibodies?

Rigorous validation is crucial before incorporating a new FAS antibody into experimental workflows. A comprehensive validation protocol should include:

• Specificity validation:

  • Western blot analysis comparing FAS-expressing and FAS-deficient cells

  • Peptide competition assay using neutralizing peptides (e.g., sc-8009 P)

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Testing multiple applications to ensure consistent recognition patterns

• Functional validation for agonistic antibodies:

  • Apoptosis assays using FAS-sensitive cell lines (e.g., activated lymphocytes)

  • Assessment of crosslinking requirements with secondary antibodies or Fcγ receptor-positive cells

  • Dose-response studies to determine EC50 values

  • Time-course analysis to characterize kinetics of response

• Epitope characterization:

  • Comparison with established antibody clones

  • Competition assays with antibodies of known epitope specificity

  • Testing binding to truncated or mutant FAS variants

• Application-specific validation:

  • For WB: Confirm expected molecular weight and band pattern

  • For IHC/IF: Verify expected tissue/cellular distribution pattern

  • For FCM: Compare with established antibody clones on the same samples

  • For functional studies: Confirm biological activity matches published observations

• Documentation and reproducibility:

  • Maintain detailed records of validation experiments

  • Test multiple antibody lots if planning long-term studies

  • Consider independent validation by different laboratory members

This systematic validation approach ensures experimental reliability and facilitates troubleshooting if unexpected results arise during subsequent experiments.

What quality control measures are essential when working with FAS antibodies in longitudinal studies?

Maintaining consistency in longitudinal studies requires robust quality control measures:

• Antibody storage and handling:

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

  • Store according to manufacturer recommendations (typically -20°C or -80°C)

  • Track lot numbers and expiration dates

  • Consider stability testing for critical applications

• Standardized positive controls:

  • Maintain frozen aliquots of reference cell lysates or tissues

  • Use calibrated positive control samples in each experimental run

  • Consider recombinant FAS protein standards for quantitative applications

• Protocol standardization:

  • Develop detailed standard operating procedures (SOPs)

  • Use automated systems where possible to minimize operator variability

  • Maintain consistent reagent sources throughout the study

• Regular verification testing:

  • Periodically re-validate antibody performance against reference standards

  • Include internal controls in every experiment

  • Consider antibody titration experiments to ensure optimal working concentration

• Data normalization strategies:

  • Use reference genes/proteins for normalization in expression studies

  • Include calibration curves for quantitative applications

  • Apply consistent analysis parameters across all time points

• Documentation system:

  • Maintain detailed electronic records of all experimental conditions

  • Document any deviations from established protocols

  • Record instrument settings and calibration status

Implementation of these quality control measures minimizes experimental variability and enhances the reliability of longitudinal data, particularly important when studying subtle changes in FAS expression or signaling over time.

How are FAS monoclonal antibodies being utilized in cancer immunotherapy research?

FAS monoclonal antibodies are being investigated in cancer immunotherapy through several innovative approaches:

• Selective targeting of tumor cells:

  • Some tumors overexpress FAS, making them potentially susceptible to FAS agonist therapy

  • Research focuses on developing antibodies with tumor-selective apoptosis induction

  • Similar to the selective effect observed with h-HFE7A in inflammatory cells versus synoviocytes

• Combination therapy strategies:

  • Pre-clinical studies examining synergistic effects of FAS antibodies with conventional chemotherapeutics

  • Potential to overcome chemoresistance through complementary cell death pathways

  • Investigation of sequence-dependent effects (e.g., sensitization with chemotherapy followed by FAS targeting)

• Immune checkpoint modulation:

  • FAS/FAS-L interactions represent an immune checkpoint pathway

  • Antagonistic FAS antibodies may prevent activation-induced cell death of tumor-infiltrating lymphocytes

  • Combination approaches with established checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

• Addressing hepatotoxicity challenges:

  • Development of tumor-targeted delivery systems to minimize systemic exposure

  • Engineering of antibodies with restricted tissue distribution

  • Exploration of administration routes that limit hepatic exposure

• Biomarker development:

  • Identification of predictive biomarkers for response to FAS-directed therapies

  • Correlation of FAS expression patterns with sensitivity to agonistic antibodies

  • Development of companion diagnostics for patient selection

This research area represents a promising frontier in cancer immunotherapy, with the potential to overcome the limitations that have historically restricted clinical application of FAS-targeting approaches.

What methodological approaches are being developed to overcome hepatotoxicity of FAS-targeting therapeutics?

Hepatotoxicity has been the primary limitation in clinical development of FAS-targeting therapeutics. Innovative methodological approaches to overcome this challenge include:

• Antibody engineering strategies:

  • Development of bispecific antibodies that simultaneously target FAS and tumor-specific antigens

  • Engineering antibodies with modified Fc regions that activate FAS only in the presence of a second signal

  • Creation of prodrug-like antibodies that require tumor-associated protease activation

• Selective delivery systems:

  • Antibody-drug conjugate (ADC) approaches where anti-FAS antibodies are coupled to tumor-targeting antibodies

  • Nanoparticle encapsulation with tumor-homing peptides or antibodies

  • Local delivery approaches for accessible tumors (intratumoral injection, implantable devices)

• Cell type-selective agonism:

  • Further development of antibodies like h-HFE7A that demonstrate cell type selectivity

  • Mechanistic investigation of differential sensitivity between cell types

  • Identification of co-receptors or signaling components that determine cell-specific responses

• Alternative signaling pathway exploitation:

  • Investigation of non-apoptotic FAS signaling pathways that may be selectively activated

  • Development of antibodies that trigger specific signaling branches

  • Combination with pathway-specific inhibitors to direct signaling outcomes

• Safety and monitoring protocols:

  • Development of biomarkers for early detection of hepatotoxicity

  • Dose-escalation strategies with comprehensive liver monitoring

  • Combination with hepatoprotective agents

These methodological approaches represent the current frontier in translating the promising biology of FAS-mediated apoptosis into clinically viable therapeutic strategies while mitigating the risk of off-target hepatotoxicity.

How can researchers integrate FAS antibody research with other emerging immunological techniques?

Integration of FAS antibody research with cutting-edge immunological techniques offers powerful new research opportunities:

• Single-cell technologies:

  • Combining FAS antibody treatments with single-cell RNA sequencing to characterize cellular responses

  • Single-cell proteomics to map FAS signaling networks in heterogeneous populations

  • Integration with cellular indexing of transcriptomes and epitopes (CITE-seq) to correlate FAS expression with transcriptional states

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize FAS receptor clustering in real-time

  • Intravital imaging to track FAS-mediated apoptosis in vivo

  • Mass cytometry imaging to simultaneously examine multiple signaling pathways activated by FAS

• CRISPR-based functional genomics:

  • Genome-wide CRISPR screens to identify modulators of FAS sensitivity

  • CRISPR activation/inhibition libraries to map regulatory networks

  • Base editing approaches to introduce specific FAS mutations and assess functional consequences

• Organoid and microphysiological systems:

  • Testing FAS antibodies on patient-derived organoids for personalized approaches

  • Development of liver-on-chip platforms to predict hepatotoxicity

  • Multi-tissue microphysiological systems to assess systemic effects

• Computational and systems biology:

  • Network analysis of FAS signaling in different cellular contexts

  • Machine learning approaches to predict cellular responses to FAS antibodies

  • Multi-scale modeling to link molecular events to tissue-level outcomes

By integrating these advanced technologies with traditional FAS antibody research, investigators can develop a more comprehensive understanding of FAS biology and accelerate the development of safer, more effective therapeutic approaches.