Alcam Antibody

What is ALCAM and why is it significant in research?

ALCAM (Activated Leukocyte Cell Adhesion Molecule), also known as CD166, is a 105 kD cell-surface adhesion molecule belonging to the immunoglobulin superfamily of receptors. It is a type I transmembrane glycoprotein with a characteristic structure consisting of five extracellular immunoglobulin-like domains (VVC2C2C2), a transmembrane domain, and a short cytoplasmic tail. ALCAM's significance stems from its role in mediating both homotypic (ALCAM-ALCAM) interactions and heterotypic interactions with T-cell antigen CD6 . These interactions are critical for various biological processes including T-cell activation, leukocyte migration, and (lymph)angiogenesis, making ALCAM an important target in research relating to immune function, development, and disease pathologies . Additionally, ALCAM expression serves as a valuable marker for the isolation of pluripotent stem cells, further highlighting its research utility .

How are ALCAM antibodies classified and what are the common types available for research?

ALCAM antibodies can be classified based on their molecular format, binding specificity, and functional properties. The primary formats include:

• Monoclonal antibodies (mAbs): These include clones such as eBioALC48 and AZN-L50, which offer high specificity for ALCAM epitopes and are available with various conjugations (PE, APC) for different detection methods .

• Antibody fragments: These include:

  • Single-chain variable fragments (scFv): Smaller fragments like scFv173 and scFv141 that retain antigen-binding capacity while allowing for improved tissue penetration .

  • Stability-optimized fragments: Advanced versions such as the V2D7 clone with enhanced affinity, stability, and solubility properties specifically designed for applications like topical delivery .

Functionally, ALCAM antibodies can be classified as:

• Blocking antibodies (e.g., AZN-L50) that inhibit ALCAM-ALCAM homotypic interactions .

• Non-blocking antibodies that bind to ALCAM without interfering with its functional interactions, used primarily for detection purposes.

The choice between these formats depends on specific research requirements, whether for detection, functional studies, or therapeutic applications .

What are the standard applications for ALCAM antibodies in research?

ALCAM antibodies are versatile research tools with several standard applications:

• Flow Cytometric Analysis: Monoclonal antibodies like eBioALC48 can be used to identify and quantify ALCAM-expressing cells in various samples. Typically used at concentrations of ≤0.25-0.5 μg per test (with a test defined as the amount of antibody to stain cells in a 100 μL volume), these antibodies are effective for analyzing cell populations ranging from 10^5 to 10^8 cells .

• Immunohistochemistry: Particularly on paraffin-embedded tissue sections, ALCAM antibodies help visualize protein expression patterns in tissues .

• Immunofluorescence: Antibodies such as AZN-L50 (at concentrations around 4 μg/ml) can be used on methanol/acetone-fixed cell monolayers grown on glass surfaces to visualize ALCAM localization .

• Immunoprecipitation: ALCAM antibodies coupled to Protein G Sepharose beads can precipitate ALCAM from cell lysates for further analysis .

• Functional Studies: Blocking antibodies are valuable for investigating ALCAM's role in processes such as cell adhesion, migration, and T-cell activation .

Each application requires specific optimization, including appropriate antibody titration, to achieve optimal performance in the assay of interest .

How should ALCAM antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of ALCAM antibodies are crucial for maintaining their activity and specificity:

• Temperature considerations: Most ALCAM antibodies should be stored at 2-8°C for short-term storage (up to 1 month) and at -20°C for long-term storage, avoiding repeated freeze-thaw cycles which can degrade antibody quality.

• Buffer conditions: For stability-optimized fragments like those developed for topical applications, specific buffer formulations have been developed to maintain solubility and stability during storage .

• Quality controls: Before use, verify antibody quality through:

  • Purity assessment: High-quality antibodies typically show >90% purity as determined by SDS-PAGE .

  • Aggregation check: Less than 10% aggregation as determined by HPLC indicates good antibody quality .

  • Filtration status: Properly filtered antibodies (0.2 μm post-manufacturing filtration) help ensure sterility and remove potential aggregates .

• Working dilutions: Prepare fresh working dilutions on the day of the experiment rather than storing diluted antibody for extended periods.

Adherence to these handling guidelines ensures consistent performance across experiments and maximizes the shelf-life of valuable ALCAM antibody reagents.

How can ALCAM antibodies be used to investigate ALCAM-mediated cell adhesion mechanisms?

Investigating ALCAM-mediated adhesion requires specialized methodological approaches:

• Homotypic vs. Heterotypic Binding Studies:

  • For homotypic (ALCAM-ALCAM) interactions: Blocking antibodies like AZN-L50 completely inhibit these interactions and can be used in comparative studies with non-blocking antibodies .

  • For heterotypic (ALCAM-CD6) interactions: Competition ELISAs using various antibody fragments can determine their potency in blocking these interactions .

• Leukocyte Transmigration Assays:

  • Methodology: Use of transwell chambers where ALCAM-expressing cells are seeded and migration of leukocytes is measured in the presence/absence of antibodies.

  • Antibody format considerations: While both mono- and bivalent antibody fragments can block ALCAM-CD6 interactions in competition ELISAs, research shows that only bivalent fragments efficiently inhibit ALCAM-ALCAM interactions in leukocyte transmigration assays .

• Invasion Assays:

  • Protocol: Cells labeled with 3H-thymidine are mixed with antibodies and seeded in invasion chambers. After 48 hours, the ratio of invading to non-invading cells is measured to assess the effect of ALCAM blockade on invasion.

  • Concentration guidance: Anti-ALCAM antibodies are typically used at concentrations of 10 μg per 1×10^5 cells for effective blockade in these assays .

These methodologies allow researchers to dissect the specific contributions of ALCAM to various cellular processes and to validate it as a potential therapeutic target in conditions where dysregulated cell adhesion contributes to pathology.

What methodologies are recommended for developing novel ALCAM-targeting antibodies with enhanced properties?

The development of next-generation ALCAM antibodies involves several sophisticated methodologies:

• Cell-Based Antibody Selection (CBAS):

  • This approach allows for the isolation of antibody candidates from human recombinant antibody libraries that specifically recognize ALCAM on cancer cells .

  • The method involves screening against native ALCAM expressed on cell surfaces rather than purified protein, ensuring recognition of physiologically relevant epitopes.

• Affinity Maturation Process:

  • Phage display technology with biotinylated human ALCAM (hALCAM V1) immobilized on streptavidin-coated plates.

  • Competitive selection protocol: Phages displaying scFv are added to antigen-coated wells in the presence of competing antibodies to select for variants with improved binding characteristics.

  • Stringent washing steps (20-30 washes with 0.1% Tween in PBS followed by 20-30 washes with PBS) ensure selection of high-affinity binders .

• Stability Optimization:

  • Systematic engineering of antibody frameworks to enhance thermal stability and resistance to aggregation.

  • Production in bacterial expression systems like the pHOG21 secretion vector system in E. coli, which allows for scalable production and screening .

• Format Diversification:

  • Development of various antibody fragment formats (scFv, Fab, minibodies) from parent antibodies like IF8.

  • Each format offers different tissue penetration capabilities, particularly important when developing antibodies for topical applications in tissues like the cornea or lungs .

These methodologies have successfully produced antibodies with improved properties, such as the affinity-matured and stability-optimized V2D7 clone, demonstrating the value of systematic antibody engineering approaches in ALCAM research .

How can researchers optimize ALCAM antibody titration for flow cytometry applications?

Optimal titration of ALCAM antibodies for flow cytometry requires a systematic approach:

• Starting Concentration Guidelines:

  • For eBioALC48 antibodies with PE conjugation: Begin with ≤0.25 μg per test .

  • For eBioALC48 antibodies with APC conjugation: Begin with ≤0.5 μg per test .

  • A "test" is defined as the amount of antibody needed to stain a cell sample in 100 μL final volume .

• Titration Protocol:

  • Prepare serial dilutions of the antibody (typically 2-fold dilutions).

  • Use consistent cell numbers across titration points (cell densities can range from 10^5 to 10^8 cells per test, but should be determined empirically for your specific cell type) .

  • Include appropriate isotype controls at each concentration to account for non-specific binding.

  • Analyze using signal-to-noise ratio rather than just mean fluorescence intensity.

• Cell-Specific Considerations:

  • For mouse splenocytes, which have been validated with eBioALC48 antibodies, standard protocols can be followed .

  • For other cell types, especially those with potentially different ALCAM expression levels, preliminary experiments should determine the optimal cell number range.

• Fluorochrome Selection:

  • Match fluorochrome to available lasers: PE (excitation: 488-561 nm; emission: 578 nm) works with Blue, Green, or Yellow-Green lasers .

  • APC (excitation: 633-647 nm; emission: 660 nm) requires Red laser availability .

  • Consider potential spectral overlap with other fluorochromes in multi-color panels.

Careful optimization through systematic titration not only ensures reliable detection of ALCAM-expressing cells but also maximizes cost-efficiency by determining the minimum effective antibody concentration needed.

What are the common challenges when using ALCAM antibodies in tissue penetration studies and how can they be addressed?

Tissue penetration studies with ALCAM antibodies present several challenges that can be addressed through specific methodological approaches:

• Size-Dependent Penetration Limitations:

  • Challenge: Different antibody formats show variable penetration abilities in tissues like human corneal epithelium.

  • Solution: Select appropriate antibody format based on the target tissue. Research demonstrates a clear size-dependence in penetration ability, with smaller fragments generally achieving better tissue distribution .

• Maintaining Functional Activity After Topical Application:

  • Challenge: Ensuring antibodies retain their functional activity in the target tissue microenvironment.

  • Solution: For applications like intranasal delivery in asthma models, use stability-optimized antibody fragments specifically designed to withstand the conditions of topical application .

• Target Accessibility in Complex Tissues:

  • Challenge: ALCAM expression on various cell types may complicate targeting specificity in heterogeneous tissues.

  • Solution: Use immunohistochemistry or immunofluorescence with antibodies like AZN-L50 (4 μg/ml) on fixed tissue sections to first map ALCAM distribution before functional studies .

• Quantification of Tissue Penetration:

  • Challenge: Accurately measuring the degree of antibody penetration.

  • Solution: Employ fluorescently-labeled antibody fragments and quantitative imaging techniques to assess penetration depth and distribution patterns in target tissues.

• Distinguishing Specific vs. Non-specific Tissue Binding:

  • Challenge: Determining whether tissue retention is due to specific ALCAM binding versus non-specific interactions.

  • Solution: Include appropriate control antibody fragments of similar size but lacking ALCAM specificity to establish baseline non-specific retention levels.

Addressing these challenges requires careful selection of antibody format and application methodology based on the specific tissue and research question under investigation.

How are ALCAM antibodies being utilized in therapeutic development for immune-mediated disorders?

Recent research has demonstrated promising applications of ALCAM antibodies in therapeutic development:

• Topical Applications for Surface-Exposed Tissues:

  • Respiratory disorders: Intranasal delivery of anti-ALCAM fragments has shown efficacy in reducing leukocyte infiltration in mouse models of asthma, confirming ALCAM as a viable target for topical lung treatments .

  • Ocular conditions: Previous research demonstrated that systemic administration of the ALCAM-blocking antibody IF8 can prevent corneal allograft rejection in mice, leading to development of topical formulations for direct ocular application .

• Antibody Engineering Advances:

  • Cross-species reactive antibodies: Development of antibody fragments with reactivity towards mouse, rat, monkey, and human ALCAM facilitates translation from preclinical to clinical studies .

  • Format optimization: Research comparing mono- and bivalent fragments has revealed format-specific effects on different ALCAM interactions (ALCAM-CD6 versus ALCAM-ALCAM), allowing for more targeted therapeutic approaches .

• Safety Considerations:

  • Localized delivery approaches: Given ALCAM's broad expression on many cell types, topical application strategies (e.g., to lungs or cornea) are being developed to circumvent potential systemic side effects that might occur with systemic mAb applications .

  • Tissue penetration studies: Size-dependent tissue penetration capabilities of different antibody formats are being leveraged to optimize therapeutic delivery while minimizing off-target effects .

These developments suggest that ALCAM-targeting strategies, particularly using engineered antibody fragments for topical delivery, represent a promising approach for treating immune-mediated disorders affecting surface-exposed tissues.

What role do ALCAM antibodies play in cancer research and potential therapeutic applications?

ALCAM antibodies have emerging applications in cancer research and potential therapeutics:

• Diagnostic and Prognostic Applications:

  • Novel human recombinant single-chain antibodies like scFv173 have been characterized that specifically recognize ALCAM on cancer cells and in tumor tissues .

  • These antibodies can be used to assess ALCAM expression, which may correlate with invasion capacity and metastatic potential in various cancer types.

• Functional Investigations:

  • Anti-ALCAM antibodies have demonstrated capacity to reduce cancer cell invasion in experimental models, suggesting therapeutic potential .

  • Methodology: In invasion assays using 3H-thymidine-labeled cells, anti-ALCAM antibodies (typically used at 10 μg per 1×10^5 cells) can inhibit invasive capacity, allowing researchers to quantify their anti-metastatic potential .

• Development of Novel Therapeutics:

  • Human recombinant antibody libraries are being screened using cell-based antibody selection (CBAS) methods to identify candidates specifically recognizing ALCAM on cancer cells .

  • Expression and purification systems like the pHOG21 secretion vector in E. coli allow for efficient production of promising candidate antibodies for further testing .

• Targeting ALCAM-Mediated Processes:

  • ALCAM's role in homotypic (ALCAM-ALCAM) adhesion is implicated in tumor progression and maintenance of tissue architecture .

  • Blocking antibodies that specifically inhibit these interactions offer potential for disrupting cancer cell cohesion and thereby limiting metastatic spread.

These applications highlight the dual role of ALCAM antibodies in cancer research: as tools for investigating ALCAM's biological functions in tumorigenesis and as potential therapeutic agents targeting ALCAM-mediated processes in cancer progression.

What controls should be included when designing experiments with ALCAM antibodies?

Robust experimental design with ALCAM antibodies requires appropriate controls:

• Antibody-Specific Controls:

  • Isotype controls: Include matched isotype antibodies at equivalent concentrations to assess non-specific binding, particularly critical in flow cytometry and immunohistochemistry applications.

  • Concentration gradients: When evaluating blocking efficacy, include a range of antibody concentrations to establish dose-response relationships.

• Cell and Tissue Controls:

  • Positive control cells: Include known ALCAM-expressing cells such as activated T and B cells, monocyte-derived dendritic cells, or thymic epithelial cells .

  • Negative control cells: Use cell lines with confirmed absence of ALCAM expression.

  • For tissue sections: Include tissues with known ALCAM expression patterns for comparison.

• Functional Assay Controls:

  • For blocking experiments: Include both non-blocking anti-ALCAM antibodies and antibodies against unrelated surface proteins to distinguish specific ALCAM blockade from general effects of antibody binding.

  • For invasion/migration assays: Compare anti-ALCAM antibodies (used at established effective concentrations like 10 μg per 1×10^5 cells) with known inhibitors of cell migration/invasion to contextualize efficacy .

• Technical Validation:

  • Antibody quality verification: Confirm antibody purity (>90% by SDS-PAGE) and low aggregation (<10% by HPLC) before experimental use .

  • For novel antibody formats: Include parent antibodies (e.g., IF8-Fc when testing derived fragments) to benchmark performance .

Incorporating these controls ensures experimental rigor and facilitates accurate interpretation of results when working with ALCAM antibodies across various research applications.

How should researchers approach cross-species reactivity when working with ALCAM antibodies?

Cross-species reactivity is an important consideration when selecting ALCAM antibodies for research:

• Species Reactivity Profiling:

  • Some antibodies, like the stability-optimized fragments derived from the IF8 antibody, demonstrate broad cross-species reactivity towards mouse, rat, monkey, and human ALCAM .

  • This cross-reactivity should be experimentally verified rather than assumed, particularly when working with non-human models.

• Translational Research Considerations:

  • For studies intended to progress from animal models to human applications, prioritize antibodies with verified cross-species reactivity to maintain consistency across research phases.

  • When testing in multiple model organisms, use the same antibody clone where possible to minimize variables when comparing results across species.

• Epitope Conservation Analysis:

  • ALCAM's structure includes five extracellular immunoglobulin-like domains (VVC2C2C2) , with varying degrees of conservation across species.

  • Antibodies targeting highly conserved epitopes offer better cross-species applicability than those binding to more variable regions.

• Validation Methodologies:

  • Flow cytometry: Test antibody binding to equivalent cell types from different species.

  • Western blotting: Verify recognition of ALCAM protein from various species, noting potential differences in glycosylation patterns that might affect apparent molecular weight.

  • Functional assays: Confirm that blocking antibodies like AZN-L50 maintain their ability to inhibit homotypic ALCAM-ALCAM interactions across species .

• Application-Specific Considerations:

  • For therapeutic development: Cross-species reactive antibodies facilitate translation from preclinical to clinical studies by allowing consistent targeting across model systems .

  • For mechanistic studies: Species-specific antibodies may be preferred when investigating unique aspects of ALCAM biology in particular organisms.

Understanding and properly accounting for cross-species reactivity ensures experimental reproducibility and facilitates translational applications of ALCAM antibody research.

How should researchers interpret variations in ALCAM detection across different antibody clones?

Variations in ALCAM detection between antibody clones require careful interpretation:

• Epitope Specificity Considerations:

  • Different antibody clones (e.g., eBioALC48, AZN-L50) bind to distinct epitopes on the ALCAM molecule, which may be differentially accessible depending on protein conformation, interaction state, or post-translational modifications.

  • Epitope masking can occur when ALCAM engages in protein-protein interactions, potentially affecting antibody binding and leading to apparent expression differences.

• Isoform Recognition:

  • ALCAM exists in both membrane-bound (105 kD) and soluble (30 kD) forms . Different antibodies may preferentially detect specific forms.

  • When interpreting results, consider whether the antibody recognizes all isoforms or specifically targets the membrane-bound or soluble variant.

• Methodological Approach to Discrepancies:

  • When significant variations are observed between clones, confirm ALCAM expression using multiple detection methods (e.g., flow cytometry, Western blotting, qPCR).

  • Consider using antibody panels targeting different ALCAM epitopes to generate a more complete picture of expression.

• Technical Factors Affecting Detection:

  • Signal strength variations: Different conjugates (PE vs. APC) have distinct fluorescence intensities and spectral properties .

  • Staining protocols: Fixation methods can affect epitope accessibility—some epitopes may be preserved in methanol/acetone fixation but masked in paraformaldehyde fixation .

• Quantitative Analysis Approaches:

  • For flow cytometry: Compare median fluorescence intensity ratios (sample to isotype control) rather than absolute values when comparing data across different antibody clones.

  • For imaging: Use relative measures of staining intensity normalized to appropriate controls.

By systematically addressing these factors, researchers can distinguish genuine biological variations in ALCAM expression from technical artifacts related to antibody properties.

How are ALCAM antibodies being utilized in stem cell research and regenerative medicine?

ALCAM antibodies have significant applications in stem cell biology and regenerative medicine:

• Marker for Pluripotent Stem Cell Isolation:

  • ALCAM expression serves as an important marker for the identification and isolation of pluripotent stem cells .

  • Flow cytometric protocols using antibodies like eBioALC48 (at ≤0.25-0.5 μg per test) facilitate the purification of stem cell populations based on ALCAM expression .

• Mesenchymal Stem Cell Characterization:

  • ALCAM functions as a marker for pluripotent mesenchymal stem cells, which have applications in various regenerative therapies .

  • Antibodies targeting ALCAM contribute to the standardized characterization of these therapeutically relevant cell populations.

• Developmental Biology Applications:

  • Given ALCAM's involvement in embryogenesis, neurogenesis, and osteogenesis , antibodies against this protein are valuable tools for studying these developmental processes.

  • Immunohistochemistry using ALCAM antibodies helps trace the temporal and spatial distribution of this protein during development and tissue formation.

• Tissue Engineering:

  • ALCAM's role in homotypic interactions involved in the development and maintenance of tissue architecture makes ALCAM antibodies relevant to tissue engineering efforts.

  • Both blocking and non-blocking antibodies can be used to manipulate ALCAM-mediated cell adhesion in engineered tissues.

• Monitoring Differentiation Processes:

  • Changes in ALCAM expression during cell differentiation can be tracked using antibodies, providing insights into differentiation trajectories and cellular identities.

  • Flow cytometric analysis with appropriately titrated antibodies allows for quantitative assessment of these expression changes.

These applications highlight the utility of ALCAM antibodies beyond traditional immunology research, positioning them as valuable tools in the expanding field of regenerative medicine.

What methodological considerations are important when using ALCAM antibodies for immune cell functional studies?

When utilizing ALCAM antibodies for immune cell functional studies, several methodological considerations are critical:

• Timing of Antibody Application:

  • ALCAM expression on immune cells is regulated by activation status—activated T and B cells show increased expression compared to resting cells .

  • Experimental design should account for this temporal regulation, with antibody application timed appropriately relative to activation protocols.

• Functional Blocking Studies:

  • When using blocking antibodies like AZN-L50 that inhibit homotypic ALCAM-ALCAM interactions , include appropriate controls to distinguish ALCAM-specific effects from general perturbations of cell surface protein function.

  • For T cell activation studies, consider the dual roles of ALCAM in both homotypic interactions and heterotypic binding to CD6, which may have distinct functional consequences.

• Dendritic Cell Interactions:

  • ALCAM expression is particularly high on dendritic cells , making these cells important subjects for ALCAM functional studies.

  • For dendritic cell-T cell interaction studies, consider using antibodies at concentrations verified to block these interactions (typically in the range used for adhesion blockade assays).

• Migration and Invasion Assays:

  • When studying leukocyte migration in the context of ALCAM function, use established protocols where cells are mixed with antibodies before seeding in migration/invasion chambers .

  • The ratio of migrated to non-migrated cells should be evaluated rather than absolute cell numbers to normalize for potential variations in cell viability or proliferation.

• In Vivo Applications:

  • For animal models like asthma studies using intranasal antibody delivery, consider both antibody format (full-length vs. fragments) and delivery vehicle to optimize targeting efficiency .

  • Use cross-species reactive antibodies when possible to facilitate translation between animal models and human applications .