CD146 Recombinant Monoclonal Antibody

What is CD146 and why is it important in research?

CD146 (also known as MCAM or MUC18) is an adhesion molecule expressed on various cell types including vascular endothelial cells, smooth muscle cells, and multiple tumor types such as melanoma, renal carcinoma, pancreatic cancer, and breast cancer . It plays crucial roles in cell migration, cell polarity establishment, and signal transduction. The importance of CD146 in research stems from its dual role in normal physiological processes and pathological conditions, particularly cancer progression and metastasis . CD146 antibodies serve as invaluable tools for studying these processes, enabling researchers to detect, quantify, and functionally characterize CD146 expression in various experimental contexts.

What applications are CD146 monoclonal antibodies typically used for?

CD146 monoclonal antibodies are utilized across multiple experimental platforms including:

• Flow cytometry - For detecting CD146-positive cell populations, such as in splenocytes and other immune cells

• Immunohistochemistry (IHC) - For analyzing CD146 expression in tissue sections, particularly in tumor biopsies and normal tissues

• Western blotting - For quantifying CD146 protein expression levels

• Immunocytochemistry/immunofluorescence - For visualizing the subcellular localization of CD146 in cultured cells

• In vivo imaging - Specialized antibodies can be used for PET imaging of CD146-positive tumors in animal models

• Therapeutic applications - Certain anti-CD146 antibodies have demonstrated potential in reducing tumor growth in preclinical models

The appropriate application depends on your specific research question, with recommended dilutions of 1:200 for IHC and 1:2000 for Western blotting when using certain antibody clones such as UMAB154 .

How do I optimize immunostaining protocols for CD146 detection in different tissue types?

Optimizing CD146 immunostaining requires consideration of several factors:

• Tissue fixation: For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is critical. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is commonly effective.

• Antibody dilution: Start with the manufacturer's recommended dilution (e.g., 1:200 for IHC with UMAB154 ) and adjust as necessary based on signal-to-noise ratio.

• Incubation conditions: Primary antibody incubation at 4°C overnight often yields better results than shorter incubations at room temperature.

• Detection system: For visualizing tumor CD146 while distinguishing from vascular CD146, consider dual immunofluorescence approaches with CD31 as an endothelial marker, as demonstrated in published research .

• Controls: Always include positive controls (tissues known to express CD146 such as melanoma) and negative controls (tissues without CD146 expression or primary antibody omission).

For challenging tissues, signal amplification systems or tyramide signal amplification may improve detection sensitivity while maintaining specificity.

What are the best methods for validating CD146 antibody specificity?

Validating antibody specificity is crucial for research reliability. Recommended approaches include:

• Genetic validation: Compare antibody staining between wild-type cells/tissues and CD146 knockout models. In proper validation, staining should be absent in knockout samples, as shown in MCAM knockout C164 cells .

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide or recombinant CD146 protein should abolish specific staining.

• Multiple antibody approach: Use at least two different antibody clones targeting different CD146 epitopes and compare staining patterns.

• Orthogonal methods: Correlate protein detection with mRNA expression using techniques like RT-qPCR or in situ hybridization .

• Cross-reactivity testing: Assess potential cross-reactivity with similar proteins. For example, some anti-MCAM antibodies show less than 5% cross-reactivity with mouse MAdCAM-1 in direct ELISAs .

• Multiple detection methods: Confirm findings using independent techniques such as flow cytometry, Western blotting, and immunohistochemistry to provide converging evidence of specificity.

How can CD146 antibodies be used to study cell polarity in differentiation processes?

CD146 plays a crucial role in establishing cell polarity, particularly during cellular differentiation. Advanced applications include:

• Myogenic differentiation: In elongating myotubes, CD146 localizes asymmetrically at the distal end of growing wild-type myotubes, co-localizing with SCRIB. Researchers can use anti-CD146 antibodies to track this polarized distribution during differentiation .

• Comparative analysis with polarity markers: Co-staining for CD146 alongside polarity markers like VANGL2, MSN, SCRIB, and PAR3 reveals the molecular hierarchy of polarity establishment .

• Knockout studies: MCAM knockout cell lines demonstrate disrupted polarity, with markers like VANGL2 becoming evenly spread in the cytoplasm rather than asymmetrically localized. This approach allows researchers to establish causal relationships between CD146 and cell polarity .

• Domain-specific functions: Studies using cells with deletion of specific CD146 domains (such as the endocytosis motif) demonstrate that these motifs are critical for maintaining proper polarity. Similar polarity defects are observed with complete CD146 elimination and specific domain deletions .

• Signaling pathway analysis: CD146 impacts ERK1/2 phosphorylation during differentiation processes. In wild-type cells, myogenic and chondrogenic differentiation lead to downregulation of ERK1/2 phosphorylation, whereas in MCAM mutant cell lines, ERK1/2 phosphorylation increases, suggesting a key role for CD146 in modulating this signaling pathway .

These approaches collectively help decode the molecular mechanisms by which CD146 regulates cell polarity during differentiation processes.

What are the current approaches for developing tumor-specific CD146 antibodies that don't cross-react with vascular CD146?

Developing tumor-specific anti-CD146 antibodies requires sophisticated strategies to differentiate between tumor-associated and normal vascular CD146:

• Expression system selection: Generating the extracellular domain of CD146 in cancer cell lines rather than normal cells may preserve cancer-specific post-translational modifications that can serve as unique epitopes .

• Differential screening: After antibody generation, implementing a rigorous screening cascade that selects for clones binding to cancer cell-expressed CD146 but not to CD146 on endothelial or smooth muscle cells .

• Validation in complex tissues: Validating candidate antibodies on human tumor biopsies with dual staining for endothelial markers (CD31) to confirm selective binding to tumor cells but not to tumor vasculature .

• In vivo imaging validation: Using techniques like PET imaging in xenograft models to confirm that the antibody localizes specifically to CD146-positive tumors in a living system .

• Functional characterization: Assessing the antibody's ability to induce internalization and degradation of tumor CD146, which may differ mechanistically from interactions with vascular CD146 .

This approach has successfully yielded antibodies like TsCD146 mAb that demonstrate remarkable specificity for tumor CD146, opening new avenues for both diagnostic and therapeutic applications .

How can CD146 antibodies be optimized for in vivo tumor imaging applications?

Optimizing CD146 antibodies for in vivo tumor imaging involves several critical considerations:

• Radioisotope labeling: For PET imaging, antibodies can be conjugated with radioisotopes such as 89Zr, 64Cu, or 124I, with selection based on the desired half-life and imaging characteristics. The TsCD146 mAb has been successfully employed for PET imaging of CD146-positive tumors in murine xenograft models .

• Fragment optimization: Full IgG antibodies have long circulation times (~3 weeks) which can create background issues. Consider engineering Fab, F(ab')2, or single-chain variable fragments (scFv) to improve tumor penetration and accelerate blood clearance.

• Pharmacokinetic modifications: Site-specific conjugation techniques preserve antibody binding while controlling the drug-to-antibody ratio, improving consistency and performance.

• Target validation: Before in vivo studies, confirm CD146 expression levels in target tumors and potential cross-reactive tissues using immunohistochemistry or flow cytometry on representative samples .

• Control experiments: Include both positive controls (CD146-positive tumors) and negative controls (CD146-negative tumors or tissues) to confirm specificity in the in vivo setting.

• Quantitative analysis: Develop standardized protocols for signal quantification, considering parameters such as tumor-to-background ratio, standardized uptake values (SUVs), and pharmacokinetic modeling.

• Multimodal imaging: Consider dual-labeled antibodies (e.g., radioisotope plus fluorescent tag) to enable both in vivo imaging and subsequent ex vivo validation through microscopy .

What mechanisms explain the therapeutic effects of certain CD146 antibodies on tumor growth?

The therapeutic effects of certain CD146 antibodies on tumor growth involve multiple cellular mechanisms:

• Antibody-mediated internalization: TsCD146 mAb induces rapid internalization of cell surface CD146 into acidic intracellular compartments (endosomes or lysosomes) in CD146-positive cancer cells. This process begins as quickly as three hours after antibody treatment and occurs at 37°C but not at 4°C, confirming it as an active cellular process rather than passive binding .

• Downregulation of surface CD146: Internalization leads to a reduction in cell surface CD146 levels, disrupting its function in cell adhesion, migration, and signaling pathways critical for tumor growth and metastasis .

• Decreased cellular proliferation: Treatment with TsCD146 mAb results in reduced proliferation of CD146-positive cancer cells, suggesting that CD146 signaling plays a role in promoting tumor cell division .

• Enhanced apoptosis: CD146 antibody treatment increases apoptosis in tumor cells, potentially by interfering with survival signaling pathways or by inducing immunogenic cell death mechanisms .

• Altered signaling pathways: CD146 impacts ERK1/2 phosphorylation, and its targeting by antibodies may disrupt this signaling pathway which is often dysregulated in cancer. In CD146 mutant cell lines, ERK1/2 phosphorylation increases abnormally during differentiation processes, suggesting a role for CD146 in modulating this critical cancer-associated pathway .

These mechanisms collectively contribute to the observed reduction in the growth of human CD146-positive cancer cells xenografted in nude mice after treatment with antibodies like TsCD146 mAb .

How can CD146 antibodies be used to detect circulating tumor cells or cancer-derived microparticles in liquid biopsies?

CD146 antibodies offer powerful tools for liquid biopsy applications through several methodological approaches:

• Flow cytometry-based detection: Anti-CD146 antibodies can be used to identify and enumerate CD146-positive circulating tumor cells (CTCs) in peripheral blood samples. This typically involves:

  • Red blood cell lysis

  • Enrichment of nucleated cells

  • Multicolor flow cytometry with CD146 antibodies alongside other markers to distinguish CTCs from normal cells

• Microparticle analysis: Cancer-derived microparticles (MPs) expressing CD146 can be detected in patient plasma samples using antibodies like TsCD146 mAb that specifically recognize tumor-associated CD146 . The methodology involves:

  • Plasma isolation through differential centrifugation

  • Incubation with fluorescently labeled anti-CD146 antibodies

  • Analysis by flow cytometry or specialized nanoparticle analysis platforms

• Microfluidic capture: CD146 antibodies can be immobilized on microfluidic chips to capture CTCs from blood samples with high efficiency while maintaining cell viability for downstream analysis.

• Immunomagnetic separation: Magnetic beads coated with anti-CD146 antibodies enable positive selection of CD146-expressing cells from blood samples, which can then be enumerated and characterized.

• Combined approach: For comprehensive liquid biopsy analysis, CD146 antibodies can be used alongside antibodies against other tumor markers in multiplexed detection systems.

The tumor-specific antibody TsCD146 mAb has demonstrated particular utility in detecting CD146-positive cancer microparticles in the plasma of patients, offering a potential minimally invasive biomarker for diagnosis, prognosis, and treatment monitoring .

What are common pitfalls in CD146 antibody-based experiments and how can they be addressed?

Researchers frequently encounter several challenges when working with CD146 antibodies:

• False negative results due to epitope masking:

  • Problem: Certain fixation methods may mask CD146 epitopes.

  • Solution: Optimize antigen retrieval methods; consider multiple antibody clones targeting different epitopes.

• Non-specific binding in certain tissues:

  • Problem: Some tissues exhibit high background staining.

  • Solution: Increase blocking time/concentration, optimize antibody dilution, consider alternative blocking reagents (e.g., mouse-on-mouse blocking for mouse tissues).

• Cross-reactivity with related proteins:

  • Problem: Some anti-CD146 antibodies may cross-react with structurally similar proteins.

  • Solution: Validate specificity using knockout controls; note that some anti-MCAM antibodies show minimal (<5%) cross-reactivity with mouse MAdCAM-1 in direct ELISAs .

• Signal intensity variation across sample types:

  • Problem: Inconsistent staining between different tissues or cell types.

  • Solution: Normalize protocols for each sample type; consider using automated staining platforms for consistency.

• Internalization affecting detection:

  • Problem: CD146 undergoes antibody-induced internalization, potentially reducing surface detection.

  • Solution: For surface staining, maintain cells at 4°C to prevent internalization; for internalization studies, compare 37°C and 4°C conditions as control .

• Discrepancies between protein and mRNA expression:

  • Problem: CD146 protein levels may not correlate with mRNA expression.

  • Solution: Use multiple detection methods (protein and mRNA) for comprehensive analysis; consider post-transcriptional regulation .

How should researchers interpret conflicting CD146 staining patterns between different antibody clones?

When facing discrepancies between different anti-CD146 antibody clones, follow this systematic approach:

• Epitope differences analysis:

  • Different antibody clones recognize distinct epitopes that may be differentially accessible in certain contexts.

  • Map the epitopes recognized by each antibody using epitope prediction tools or experimental approaches.

  • Consider that some epitopes may be masked by protein-protein interactions or post-translational modifications.

• Isotype-specific effects:

  • Different antibody isotypes (IgG1, IgG2a, etc.) may exhibit varying levels of non-specific binding.

  • Compare the performance of antibodies with matched isotype controls.

• Clone-specific validation:

  • Verify each clone's specificity using CD146 knockout models .

  • For clone UMAB154, validation included using full-length human recombinant MCAM protein produced in HEK293T cells as the immunogen .

• Context-dependent expression:

  • CD146 may undergo conformational changes or post-translational modifications in different cellular contexts.

  • The tumor-specific antibody TsCD146 mAb demonstrates that tumor CD146 has distinct characteristics from vascular CD146, explaining why some antibodies detect CD146 in tumor cells but not in normal vascular cells .

• Resolution through multiple approaches:

  • Use orthogonal methods (Western blotting, flow cytometry, mass spectrometry) to resolve conflicts.

  • Consider subcellular localization differences – some antibodies may preferentially detect nuclear, cytoplasmic, or membrane-bound CD146.

• Comprehensive reporting:

  • When publishing, report the specific clone, dilution, and staining conditions used.

  • Document any discrepancies between antibodies to inform the broader research community.

What is the optimal methodology for quantifying changes in CD146 expression levels in response to experimental treatments?

Accurate quantification of CD146 expression changes requires rigorous methodology:

How are CD146 antibodies being integrated into comprehensive multi-omics research approaches?

CD146 antibodies are increasingly incorporated into multi-omics research through several innovative approaches:

• Antibody-based proteomics integration:

  • Immunoprecipitation with anti-CD146 antibodies followed by mass spectrometry identifies CD146 interaction partners and post-translational modifications.

  • This approach has revealed relationships between CD146 and polarity-associated proteins like VANGL2, SCRIB, and PAR3 .

• Spatial transcriptomics correlation:

  • CD146 immunohistochemistry combined with spatial transcriptomics provides insights into the relationship between CD146 protein expression and local transcriptional profiles.

  • This helps identify genes co-regulated with CD146 in specific tissue microenvironments.

• Single-cell multi-omics:

  • Flow cytometry with CD146 antibodies can isolate specific cell populations for subsequent single-cell RNA-seq or ATAC-seq.

  • This approach reveals how CD146-positive cells differ transcriptionally and epigenetically from CD146-negative cells within heterogeneous populations.

• Functional genomics validation:

  • CD146 antibodies are used to validate phenotypic effects of genetic manipulations in CRISPR screens.

  • This has been demonstrated in studies examining the impact of CD146 knockout on cell polarity, where immunostaining with various markers revealed distributional changes in polarity proteins .

• Systems biology modeling:

  • Quantitative data from CD146 antibody-based experiments feed into computational models of cellular signaling networks.

  • These models predict the impact of targeting CD146 on downstream pathways, such as ERK1/2 phosphorylation during differentiation processes .

• Translational multi-omics:

  • Tumor-specific antibodies like TsCD146 mAb enable correlation between imaging findings (PET scans), liquid biopsy results (circulating tumor cells/microparticles), and tissue pathology, creating a comprehensive disease signature .

What emerging technologies are enhancing the sensitivity and specificity of CD146 detection in clinical samples?

Several cutting-edge technologies are revolutionizing CD146 detection in clinical samples:

• Digital pathology and artificial intelligence:

  • Whole slide imaging combined with AI algorithms enhances quantification of CD146 expression in tumor samples.

  • Machine learning approaches can distinguish subtle patterns of CD146 staining that correlate with clinical outcomes.

• Highly multiplexed imaging:

  • Cyclic immunofluorescence and imaging mass cytometry allow simultaneous detection of CD146 alongside dozens of other markers in tissue sections.

  • This enables complex phenotyping of the tumor microenvironment and identification of rare CD146-positive cell subpopulations.

• Super-resolution microscopy:

  • Techniques like STORM and PALM achieve nanometer-scale resolution of CD146 localization.

  • This reveals previously undetectable details of CD146 distribution and co-localization with interaction partners at the cell membrane, as has been shown for proteins like SCRIB at the distal end of growing myotubes .

• Ultrasensitive liquid biopsy platforms:

  • Digital PCR and next-generation flow cytometry enhance detection of rare CD146-positive circulating tumor cells.

  • Advanced microfluidic technologies improve capture efficiency and purity of CD146-expressing cells from blood samples.

• Bispecific and multimodal antibodies:

  • Antibodies engineered to simultaneously bind CD146 and another target increase specificity.

  • Multimodal antibodies carrying both imaging agents and therapeutic payloads enable theranostic applications.

• Tumor-specific CD146 detection:

  • Antibodies like TsCD146 mAb that recognize tumor-specific conformations or modifications of CD146 improve specificity in clinical samples.

  • These antibodies can detect CD146-positive cancer microparticles in patient plasma and CD146-positive tumors in biopsies while avoiding cross-reactivity with normal vascular CD146 .

• Proximity ligation assays:

  • These techniques detect protein-protein interactions involving CD146 in situ, providing functional information beyond mere expression levels.

  • This has applications in studying CD146's interactions with polarity complex proteins in various cellular contexts .

What is the comparative performance of different CD146 antibody clones across various applications?

This table highlights the distinct properties of three well-characterized CD146 antibody clones. Each antibody has unique characteristics that make it suitable for specific research applications. The UMAB154 clone is optimized for detection of human CD146 in traditional applications like IHC and Western blotting . The MAB7718 clone offers excellent specificity for mouse CD146 with minimal cross-reactivity to similar proteins, making it valuable for murine studies . The TsCD146 mAb represents an innovative approach with its ability to distinguish between tumor and normal CD146, opening new avenues for cancer-specific detection and therapy .

How does CD146 knockdown/knockout affect key cellular processes based on published experimental data?

This comprehensive data table demonstrates the far-reaching impact of CD146 on cellular processes, particularly those involving cell polarity and differentiation. The consistent finding across multiple experimental systems is that CD146 is required for establishing proper cellular polarity, as evidenced by the mislocalization of polarity markers like VANGL2, SCRIB, and PAR3 in CD146 knockout cells . Additionally, CD146 appears to regulate ERK1/2 signaling during differentiation processes, with knockout cells showing paradoxical increases in phosphorylation . These findings highlight CD146's role not just as a surface marker but as an active participant in fundamental cellular processes.

What is the tissue distribution and relative expression levels of CD146 across normal and disease states?

This comprehensive tissue distribution profile highlights the dual nature of CD146 as both a normal vascular marker and a cancer-associated antigen. In normal tissues, CD146 expression is predominantly restricted to vascular structures (endothelial and smooth muscle cells), while epithelial components are typically negative . In contrast, various carcinomas show aberrant CD146 expression in tumor cells, making it a potential diagnostic and therapeutic target . This differential expression pattern is the basis for the development of tumor-specific antibodies like TsCD146 mAb that can distinguish between normal vascular CD146 and tumor CD146 . The table also demonstrates CD146's role in specialized cell types such as immune cells and differentiating myotubes, where it exhibits distinct expression patterns and subcellular localizations .